UNIVERSITY OF CALIFORNIA, MERCED Effects of Ethanol and ...

UNIVERSITY OF CALIFORNIA, MERCED

Effects of Ethanol and Reactive Species on Hepatitis C Virus

DISSERTATION

submitted in partial satisfaction of the requirements for the degree of

Doctor of Philosophy

with an emphasis in Quantitative Systems Biology

By

Scott E. Seronello

Committee:

Professor Jinah Choi, Advisor Professor Miriam Barlow, Chair Professor Henry J. Forman Professor David Ojcius

2010

iii

The Dissertation of Scott E. Seronello is approved, and it is acceptable in

quality and form for publication on microfilm and electronically:

_____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________

Chair

University of California, Merced

2010

iv

DEDICATION

I would like to dedicate this dissertation to my lovely wife and my indefatiguable son. My wife, Christina, has been my steadfast and supportive companion throughout my Ph.D. experience. My son, Caeden, always knows how to bring a smile to my face.

v

TABLE OF CONTENTS

Page Signature Page ........................................................................................ iii

Dedication............................................................................................... iv

Table of Contents.................................................................................... v

List of Abbreviations .............................................................................. vi

List of Figures......................................................................................... vii

List of Tables .......................................................................................... ix

Acknowledgments .................................................................................. x

Vita ......................................................................................................... xi

Abstract of the Dissertation .................................................................... xii

Chapter 1: Introduction........................................................................ 1

Chapter 2: Materials & Methods ......................................................... 28

Chapter 3: Ethanol enhances hepatitis C virus replication through

lipid metabolism and elevated NADH/NAD+ .................. 44

Chapter 4: Chemical mutagenesis of hepatitis C viral RNA by

ethanol and reactive species............................................... 73

Chapter 5: Summary and Conclusions ................................................ 202

References............................................................................................... 224

vi

LIST OF ABBREVIATIONS

ADH, alcohol dehydrogenase; BSO, L-buthionine S,R-sulfoximine; COX-2,

cyclooxygenase-2; CYP2E1, cytochrome P450 2E1; DADS, diallyl disulfide; DEPC,

diethylpyrocarbonate; DETAPAC, 1,1,4,7,7-diethylenetriaminepentaacetic acid; DPI,

diphenylene iodonium; ER, endoplasmic reticulum; GAPDH, glyceraldehyde 3-

phosphate dehydrogenase; GO, glucose oxidase; GSH, glutathione; HCV, hepatitis C

virus; HIV, human immunodeficiency virus; HNE, 4-hydroxynonenal; HO-1,

hemeoxygenase-1; iNOS, inducible nitric oxide synthase; IRES, internal ribosomal

entry site; L-NMA, NG-methyl-L-arginine acetate; MDA, malondialdehyde; MnSOD,

manganese superoxide dismutase; 4MP, 4-methylpyrazole; NAC, N-acetylcysteine;

Nox, NAD(P)H oxidase; NF-B, nuclear factor kappa B; nt., nucleotides; 8-OHdG, 8-

hydroxydeoxyguanosine; 8-OHG, 8-hydroxyguanosine; PIH, pyridoxal isonicotinoyl

hydrazine; qRT-PCR, quantitative reverse transcriptase-polymerase chain reaction;

RNS, reactive nitrogen species; ROS, reactive oxygen species; siRNA, small

interfering RNA; SNAP, synonymous nonsynonomous analysis program; TCA,

tricarboxylic acid; TGF transforming growth factor beta; TOFA, 5-(tetradecyloxy)-

2-furoic acid; UTR, untranslated region

vii

LIST OF FIGURES

Page

Figure 1.1: Role of HCV proteins in oxidative/nitrosative stress ....... 24

Figure 1.2: Life Cycle of HCV............................................................ 25

Figure 1.3: HCV and alcohol metabolism in chronic hepatitis C ....... 26

Figure 1.4: Proposed mechanisms of changes to HCV replication/

viral titer by alcohol and reactive species......................... 27 Figure 2.1: JFH1 replication in Huh7 cells ......................................... 42 Figure 2.2: HCV RNA transfection and infection............................... 43 Figure 2.3: Flowchart of steps to clone and sequence HCV RNA...... 43 Figure 3.1: Ethanol increases JFH1 replication................................... 65 Figure 3.2: Ethanol increases the replication of subgenomic JFH1

and Con1 replicon RNAs.................................................. 66 Figure 3.3: CYP2E1 Expression in Huh7 cells ................................... 67 Figure 3.4: Endogenous and exogenous ROS suppresses HCV

replication.......................................................................... 68 Figure 3.5: Acetaldehyde increases intracellular HCV RNA ............. 69 Figure 3.6: Role of NADH/NAD+ in the potentiation of

HCV replication by ethanol, acetaldehyde, acetate,

isopropanol, and acetone................................................... 70

viii

Figure 3.7: ADH enhances the elevation of HCV replication by

ethanol, acetaldehyde, acetate, isopropanol, and

acetone .............................................................................. 71 Figure 3.8: Role of lipogenesis in the enhancement of HCV

replication by ethanol, acetaldehyde, acetate,

isopropanol, and acetone................................................... 72 Figure 4.1: Ethanol, reactive species, and oxidative RNA damage .... 94 Figure 4.2: Effects of dipyridyl and GSH ester on oxidative RNA

damage and HCV RNA sequence..................................... 95 Figure 4.3: Inhibiting the RNA damage .............................................. 96 Figure 4.4: Oxidative RNA damage by HCV ..................................... 96 Figure 4.5: Synonymous and nonsynonymous substitutions in

NS5A coding region ......................................................... 97 Figure 4.6: Sites undergoing positive selection................................... 98 Figure 4.7: Mutations involving serine, threonine, and tyrosine by

Position.............................................................................. 99 Figure 4.8: Amino acid variations by position in genotype 1b

NS5A sequences from the HCV database ........................ 102

ix

LIST OF TABLES

Page

Table 4.1: Nucleotide and amino acid substitution rates in

control and treatment groups ............................................. 93 Table 4.2: Nucleotide substitution trends in control and treatment

groups................................................................................. 93 Table 4.3: Frequent mutations involving serine/threonine/tyrosine

residues in the NS5A region .............................................. 100 Table 4.4: Amino acid variations in the HCV database...................... 103 Table 5.1: Mutations that altered function found in literature that

occurred in our study ......................................................... 222

x

ACKNOWLEDGEMENTS

I would like to acknowledge Dr. Jinah Choi, my advisor, for all of her hard

work, for an opportunity to work on exciting and important research projects, and for

believing in me. I would also like to acknowledge Drs. Henry Jay Forman, David

Ojcius, and Miriam Barlow, members of my dissertation committee for their solid

advice and guidance.

I would also like to thank the members of Choi lab, in particular Jessica

Montañez, Kristen Presleigh, Shi Jia Cathy Li, Chieri Ito, Anna Nandipati, Josh

Finley, and Asa Thompsom whose assistance has been invaluable.

I would like to thank Drs. Hongqiao Zhang, David Gravano, T.S. Benedict

Yen, Arthur Cederbaum, Manel Camps, and Jim Bull for discussion. For reagents, I

would like to thank Drs. Takiji Wakita, Jerome Lasker, Stanley Lemon, and Charles

Rice/Apath, LLC. For technical assistance, I also would like to thank Tak Ishikida,

Jennifer Kuehl, David Ardell, Katherine Harris, Monica Medina, and Shinichi

Sunagawa.

Chapter 1, in part, is a reprint of the material as it appears in Free Radical

Biology and Medicine, 2007. Seronello, Scott; Sheikh, Muhammad; Choi, Jinah,

Elsevier, 2007. The dissertation author was the primary author of this paper.

Chapter 3, in part, is a reprint of the material as it appears in Journal of

Biological Chemistry, 2010. Seronello, Scott; Ito, Chieri; Wakita, Takaji; Choi, Jinah,

American Society for Biochemistry and Molecular Biology, 2010. The dissertation

author was the primary investigator and author of this paper.

Chapter 4 is currently being prepared for submission for publication.

Seronello, Scott; Montanez, Jessica; Presleigh, Kristen; Barlow, Miriam; Medina,

Monica; Bull, Jim; Choi, Jinah. The dissertation author was the primary investigator

and author of this paper.

This research was supported by start up funds and faculty research funds to

Jinah Choi from University of California, Merced.

xi

VITA

Scott E. Seronello

2001 Bachelor of Arts, Whittier College, Whittier, CA

2001-2002 Quality Control Technician, Scientific Pharmaceuticals, Pomona, CA 2002-2005 Chemistry Teacher, Golden Valley High School, Merced, CA 2005-2010 Teaching Assistant/Teaching Fellow, School of Natural Sciences,

University of California, Merced 2006-2008 Research Assistant, University of California, Merced 2010 Doctor of Philosophy, University of California, Merced (pending)

FIELDS OF STUDY

Virology and Biochemistry

PUBLICATIONS

Seronello, S.; Presleigh, K.; Barlow, M.; Choi, J. (2010) Ethanol and both endogenous and exogenous reactive species accelerate the mutation rate of hepatitis C virus RNA. Manuscript in preparation. de Mochel, N. S., Seronello, S., Wang, S. H., Ito, C., Zheng, J. X., Liang, T. J., Lambeth, J. D., and Choi, J. (2010) Hepatocyte NAD(P)H oxidases as an endogenous source of reactive oxygen species during hepatitis C virus infection. Hepatology 52, 47-59. Seronello, S., Ito, C., Wakita, T., and Choi, J. (2010) Ethanol enhances hepatitis C virus replication through lipid metabolism and elevated NADH/NAD+. J Biol Chem 285, 845-854. Seronello, S., Sheikh, M. Y., and Choi, J. (2007) Redox regulation of hepatitis C in nonalcoholic and alcoholic liver. Free Radic Biol Med 43, 869-882. Choi, J., Forman, H. J., Ou, J. H., Lai, M. M., Seronello, S., and Nandipati, A. (2006) Redox modulation of the hepatitis C virus replication complex is calcium dependent. Free Radic Biol Med 41, 1488-1498.

xii

ABSTRACT OF THE DISSERTATION

Effects of Ethanol and Reactive Species on Hepatitis C Virus

by

Scott E. Seronello

Doctor of Philosophy with an emphasis in Quantitative Systems Biology

University of California, Merced, 2010

Professor Jinah Choi, Advisor Professor Miriam Barlow, Chair

Hepatitis C virus (HCV) is an RNA virus of the Flaviviridae family that is

estimated to have infected 170 million people worldwide. HCV infection can lead to

serious liver disease such as cirrhosis, steatosis, and hepatocellular carcinoma. In

patients, HCV induces an altered redox status and exhibits a high level of genetic

variability, existing as quasispecies. Ethanol consumption has been associated with

increased HCV pathogenesis, elevated HCV titer, increased sequence heterogeneity of

HCV RNA, and decreased efficacy of antiviral treatment, suggesting multiple modes

of interaction between ethanol and HCV. The goal of my studies was to elucidate the

mechanisms of these multiple modes of interaction between alcohol, oxidative stress,

and hepatitis C.

By treating Huh7 human hepatoma cells that naturally express CYP2E1 with

physiological relevant concentrations of ethanol or its metabolites, we determined that

HCV replication was elevated in the context of the complete virus life cycle. This

xiii

potentiation of HCV replication was dependent on CYP2E1 activity the host

mevalonate pathway, fatty acid synthesis, and an increased NADH/NAD+ ratio. In

contrast, reactive oxygen species rapidly suppressed HCV replication as previously

shown using HCV replicons. These results suggested that lipid metabolism and

alteration of the NADH/NAD+ ratio played an important role in the enhancement of

HCV replication by ethanol.

Finally, we were able to demonstrate that exposing HCV-replicating cells to

ethanol and reactive species elevated both the nucleotide and amino acid substitution

rates of HCV RNA, which were accompanied by oxidative RNA damage. The overall

dN/dS ratio and percentage of sites undergoing positive selection increased with

pronounces changes in the serine/threonine/tyrosine sites. In addition, HCV itself

induced oxidative RNA damage that was exacerbated by ethanol and BSO. Agents

that decreased the RNA damage also reduced the sequence heterogeneity of the HCV

RNA.

Conclusions/Significance: Therefore, ethanol potentiates HCV replication

through the alteration of lipid metabolism and the NADH/NAD+ ratio, which is likely

to contribute to the higher viral titer and pronounced pathogenesis in patients who

drink alcohol. Ethanol may also synergize with virus-induced oxidative/nitrosative

stress to induce RNA damage and likely amplify the error rate of the HCV replicase in

the development of quasispecies, iincreasing the probability of immune evasion and

antiviral resistance.

1

CHAPTER 1: INTRODUCTION

Pathological Interactions between HCV, Ethanol, and Reactive Oxygen Species

2

Hepatitis C virus (HCV) was discovered in 1989 as an etiologic agent of non-A

non-B hepatitis (1). HCV is transmitted through blood and blood-derived products.

Currently, it is estimated that there are 170 million people worldwide who have been

infected with HCV, including 4 million in the U.S. alone, of whom 2.7 million are

viremic (2-4). About 80% of HCV infections result in a persistent infection that can

lead to serious health complications, such as cirrhosis and hepatocellular carcinoma

(HCC) (5,6). HCC is the eighth most frequent cancer worldwide, and HCV-associated

chronic liver disease is the leading cause for liver transplantation in the U.S (5-7).

HCV is responsible for at least 8,000 deaths and more than $600 million spent

annually in health care and work-loss in the U.S. It has been estimated that over the

next twenty years, the proportion of infected patients with cirrhosis will increase from

16 to 32%, and other complications will also increase dramatically, including hepatic

decompensation (increase by 106%), HCC (by 81%), and liver-related deaths (by

180%) (8). Current anti-HCV therapy, which consists of pegylated interferon alpha

and ribavirin, achieves sustained virological response (SVR) in only 50 – 60 % of

individuals undergoing treatment (9,10). The SVR is as low as 30% in patients with

HCV genotype 1 and a high viral load (10). Antiviral drug resistance, undesirable side

effects, the cost of these drugs, and incomplete understanding of the mechanism of

pathogenesis continue to pose considerable challenges to the management of hepatitis

C. There is currently no vaccine against HCV.

HCV is a single, positive-stranded RNA virus of the Flaviviridae Family (11).

The HCV genome is about 9.6 kb in length and consists of the 5’ untranslated region

3

(UTR), the structural (C, E1, E2) and nonstructural (p7, NS2, NS3, NS4A/B,

NS5A/B) protein-coding regions, and the 3’ UTR (Fig. 1.1). HCV enters the cell

through clathrin-mediated endocytosis, and the virion is then uncoated, releasing the

genome into the cytosol (Fig. 1.2). The translation of the positive-stranded RNA

genome, which occurs in a cap-independent manner, is mediated by an internal

ribosomal entry site (IRES), located at the 5’ end of the genome, and produces a

polyprotein, that is cleavage by host and viral proteases to generate the individual viral

proteins. The structural proteins are cleaved by host signal peptidases, whereas the

nonstructural proteins are cleaved by the viral cysteine protease activity of NS2 and

the serine protease activity of NS3/4 (12). Some of the HCV proteins can also be

synthesized from the alternate reading frames, potentially through multiple

mechanisms that include translational frameshifting and internal initiation of

translation (13-16). HCV RNA replication, which occurs in the cytosol, is mediated

by NS5B, an RNA-dependent RNA polymerase, and other proteins that comprise the

replication complex. HCV NS4B alters host membranes to produce a structure called

the membranous web, where the HCV replication complex is located (17,18). HCV

core recruits replication complexes to lipid droplet-associated membranes where

virions are assembled by the formation of the capsid by the core protein and the

internalization of the genome, which most likely then buds into the endoplasmic

reticulum (ER) or an ER-derived compartment (19). Then, the virions, which are

enveloped by lipid membranes plus viral proteins, are exported from the cell via

normal host secretory pathway (12). Recent reviews that summarize the HCV

4

lifecycle (Fig. 1.2) and functions of the virally-encoded proteins are found in

references (12) and (20).

Despite continued studies, the mechanism by which HCV induces these

pathogenic changes in the liver remains largely unresolved. However, HCV-induced

pathogenesis is affected by various host and environmental factors, suggesting

complex interactions between HCV and these factors (21-23). These host and

environmental factors include age, gender, co-infection with other viruses, and alcohol

consumption (24-27). In addition, HCV infection leads to severe oxidative/nitrosative

stress in patients (28-38), and this has been recognized as an important component of

HCV-induced liver disease. This chapter presents a comprehensive summary of

current understanding of redox regulation of HCV in hepatopathogenesis, focusing on

the interactions between HCV, reactive species, and alcohol.

REDOX REGULATION OF HEPATITIS C VIRUS IN THE PATHOGENESIS

OF LIVER DISEASE

HCV infection is associated with elevated levels of reactive oxygen/nitrogen

species (ROS/RNS) and decreased antioxidant levels in patients (22,28-36,38,39).

HCV patients have increased lipid peroxidation product levels in their serum,

peripheral blood mononuclear cells, and liver specimens (28,30,32,33,35,37,40).

Other evidence of oxidative damage include elevated levels of 8-

hydroxydeoxyguanosine (8-OHdG) and 4-hydroxynonenal (HNE) (31,33,39,41,42).

Glutathione (GSH) content is decreased in the liver, blood, and lymphatic system

5

while the percentage of glutathione disulfide (GSSG) is increased, suggesting

increased GSH turnover (28-30,36,38,39,43).

Interestingly, oxidative/nitrosative stress is more pronounced with HCV than

hepatitis B virus (HBV) (30), which also causes viral hepatitis. Possible mechanisms

for severe increases in oxidative/nitrosative stress during HCV infection include

chronic inflammation (i.e., activation of phagocytic NAD(P)H oxidase), which is

rather non-specific, and iron overload, which appears to be more specific to HCV

(29,30,33,44-56). In addition, ROS production within the hepatocytes can further

result in the activation of neighboring Kupffer cells (57). These cells express many

pro-inflammatory cytokines and, when ROS level increases, they begin to swell, until

they burst and release their cellular content into the extracellular matrix of the liver.

Cytokines like tumor necrosis factor-alpha and transforming growth factor-beta

(TGF) can increase ROS levels and play key roles in the mediation of liver diseases

during states of increased oxidative stress (23,58,59)}, such as by promoting insulin

resistance and fatty liver by inhibiting lipoprotein lipase and adiponectin, as well as

promoting fibrosis by activating hepatic stellate cells (60-62).

Furthermore, HCV proteins may specifically increase oxidative/nitrosative

stress in the infected cell (Fig. 1.1). For example, HCV core protein has been shown

to increase the levels of ROS/RNS, oxidized thioredoxin, lipid peroxidation products,

and antioxidant gene expression, such as that of manganese superoxide dismutase

(MnSOD) and metallothionein family proteins, and to enhance the sensitivity to toxins

such as ethanol and CCl4 (41,63-68). In terms of a mechanism, HCV core gene

6

expression decreases the intracellular/mitochondrial GSH levels and the mitochondrial

NADPH levels, which are accompanied by increased Ca2+ uptake as well as ROS

generation at Complex I in mitochondria (63-65,69). Although whether the

mitochondrial, cytosolic, and/or total intracellular GSH are decreased in a consistent

and predictable manner is still unclear, only the mitochondrial, and not the total liver

GSH, was altered with the HCV core protein in a recent study (63). As GSH is

transported from the cytosol into the mitochondria, and mitochondrial GSH is

relatively resistant to depletion, for example, by L-buthionine S,R-sulfoximine (BSO),

which inhibits GSH biosynthesis, a decreased transport or an increased utilization of

GSH in the mitochondria, rather than decreased synthesis of GSH, are suspected.

Indeed, a parallel decrease in the NADPH level is most consistent with an increased

consumption of GSH, as NADPH participates in a reaction that recycles GSSG back

to GSH. In addition, core protein has also been shown to modulate the production of

cytokines and host enzymes that can increase ROS/RNS, such as inducible nitric oxide

synthase (iNOS) and cyclooxygenase-2 (COX-2) (70-76). These observations are

consistent with the fact that HCV core protein, in addition to forming viral capsid, has

many other functions in the modulation of host cell functions and pathogenesis (77-

79).

In addition to the core protein, nonstructural proteins may play a role in the

modulation of host redox status by HCV (Fig. 1.1). For example, host antioxidant

defense (e.g., MnSOD, heme oxygenase-1 (HO-1), catalase, and GSH) is elevated in

the HCV subgenomic replicon cells, which only express the nonstructural genes of

7

HCV, suggesting adaptation to oxidative/nitrosative stress (64,80,81). These effects

may be explained in part by increased synthesis of nitric oxide and increased gene

expression of COX-2 with NS5A and/or NS3 (72,73,75,76). NS5A has also been

suggested to induce ER stress and Ca2+ release from the ER, resulting in increased

Ca2+ uptake and ROS generation by mitochondria (80). Furthermore, HCV NS3

protein can stimulate ROS generation by activating NAD(P)H oxidase (Nox) of

phagocytes (82,83). Importantly, HCV core and nonstructural proteins both induce

oxidative stress; however, they may induce different antioxidant responses, indicating

different mechanisms (64). For instance, core has been reported to up-regulate

MnSOD expression without elevating HO-1 and GSH levels (64) whereas

nonstructural genes increase MnSOD, HO-1, and GSH (64,80,81). HO-1 is also

elevated in cells expressing HCV genes from core up to the N-terminal domain of NS3

(84). These multiple interactions between HCV proteins and the host immune system

may thus act together to generate pro-oxidative environment in the HCV-infected

liver. However, the biological significance of some of these seemingly opposing

effects that the core and the nonstructural proteins have on the host antioxidant status

is unclear. It should be noted that these proteins are produced simultaneously during

the HCV lifecycle (Fig. 1.2). Therefore, whether the concurrent expression of the core

and the nonstructural proteins up-regulates, down-regulates, or cancels out the effects

of one another, in the natural context (i.e., with the entire HCV genome and the

complete viral lifecycle) remains to be shown.

8

Oxidative stress plays critical roles in various liver diseases (21,23,49-51,85-

88). Even among symptom-free HCV carriers, redox perturbation was correlated with

increase in flare-ups of alanine aminotransferase (ALT) (89). Therefore, increased

ROS/RNS have been proposed to play an important role in the HCV-induced

pathogenesis (21,22,49-51,85-87). Note that there are many factors that determine the

biological effects of reactive species, which include the type of reactive species, their

concentrations, proximity to cellular macromolecules/metals, and the cell's capacity to

eliminate these molecules and/or reverse their effects. Both redox signaling and

irreversible damage to cellular macromolecules have been implicated in pathogenesis

(23,90). Therefore, ROS/RNS-induced pathogenesis during hepatitis C is likely to

involve both irreversible cell damage and some components of redox signaling.

In particular, oxidative/nitrosative stress has been strongly implicated in the

HCV-induced carcinogenesis. For example, iron overload may increase oxidative

DNA damage (56) and has been suggested to increase the risk of HCC in transgenic

mice expressing the HCV polyprotein (91). In addition, HCV core-induced iNOS

generates RNS that, like ROS, can cause DNA damage and increase mutations within

the immunoglobulin and tumor suppressor genes (73,92). These genotoxic effects of

oxidative/nitrosative stress are expected to contribute towards the development of

HCC and B-cell lymphoma during HCV infection, and such association has also been

documented in vivo, in HCV core-transgenic mice (65,93). Other mechanisms by

which core protein enhances HCC include the modulation of tumor suppressor genes

and proto-oncogenes as well as inhibition of apoptosis, although there is some

9

evidence that core protein actually initiates apoptosis (41,94-104)[71]. In this regard,

it should be noted that ROS/RNS can have diverse effects on cell growth and

apoptosis (105,106). Reactive species may also facilitate the development of HCC by

progressively damaging the liver and inducing cirrhosis that increases the risk of HCC

(107,108). Consequently, antioxidants have been proposed as an adjunct therapy for

chronic hepatitis C (109).

SYNERGISTIC EFFECTS OF ALCOHOL AND HCV IN LIVER DISEASE Chronic alcohol consumption is a well-known risk factor for liver disease

(26,110,111). Over 12,000 deaths per year in the U.S. are attributed to alcohol-related

liver disease (112). The relationship between alcohol and HCV infection is not well

understood, although the prevalence of HCV infection in patients with a history of

alcohol abuse is significantly higher than in the general population, leading to the

identification of alcohol abuse as a risk factor for hepatitis C (113-116). Specifically,

almost one–third of alcoholics with clinical symptoms of liver disease have been

infected with HCV, which is four times the rate of HCV infection found in alcoholics

who do not have liver disease (117-119). HCV-induced pathogenesis is exacerbated

by alcohol consumption, suggesting a synergy between the virus and ethanol (27,114-

116,120-137). For instance, pro-inflammatory NF-κB signaling is induced by both

HCV core protein and ethanol via acetaldehyde (138), and the core protein increases

the secretion of TGF in the presence of alcohol (68). Alcohol intake is associated

with increased progression of fibrosis, higher likelihood of cirrhosis, and an elevated

10

risk for HCC in individuals with chronic HCV infection (139,140). Likewise, HCV

infection exacerbates alcohol-related liver damage (123,141-145).

One obvious mechanism of synergy between ethanol and HCV infection in

chronic liver disease involves the modulation of the host immune response by ethanol

and HCV. The functions of antigen-presenting dendritic cells and other key immune

cells are disrupted by both ethanol and HCV proteins (146). Geissler and colleagues

noted that chronic alcohol feeding of mice inhibited T–helper cell and cytotoxic T–

lymphocyte functions that play a pivotal role in clearance of HCV from the body

(147). In addition, alcohol consumption is known to significantly decrease the

efficacy of interferon therapy in chronic hepatitis C patients (148). Oxidative stress,

alcohol, and HCV core protein have each been proposed to inhibit the cellular

interferon response by interfering with the JAK-STAT signaling pathway (149-151).

This inhibition of the immune response would allow for viral persistence and

exacerbated pathogenesis via oxidative stress.

Ethanol also increases the generation of ROS, decreases GSH content, causes

lipid peroxidation, and leads to selenium deficiency through multiple mechanisms

(68,111,152-164). The production of ROS/RNS and highly toxic by-products during

ethanol metabolism has been recognized as a key mechanism by which ethanol is

hepatotoxic (86,152,153,156,158,165,166). Hepatocytes have three oxidative

mechanisms of metabolizing ethanol (Fig. 1.3). In the cytosol, ethanol is metabolized

to acetaldehyde by alcohol dehydrogenase (ADH) which leads to the production of

ROS (7). The corresponding build up of NADH can also interfere with the electron

11

transfer system in the mitochondria, facilitating ROS generation (167-169).

Additionally, NADH promotes the synthesis of fatty acids and inhibits their oxidation,

enhancing the development of steatosis (170). The microsomal ethanol oxidizing

system, located on the endoplasmic reticulum, which primarily consists of ethanol-

inducible cytochrome P450 2E1 (CYP2E1), converts ethanol to acetaldehyde and

generates ROS (154,169,171,172). Ethanol may also be metabolized by catalase in

peroxisomes (173). Once ethanol is metabolized to acetaldehyde, aldehyde

dehydrogenase quickly converts it to acetate (173). Acetaldehyde can react with

mitochondrial GSH causing a decrease in the antioxidant defense of the mitochondria

to increase susceptibility to oxidative stress (7,170). Ethanol can also be non-

oxidatively metabolized into fatty acid ethyl esters, which can interfere with

mitochondrial function and alter the proliferation and lipid dynamics of the cell (174).

Therefore, alcohol-induced oxidative stress is likely to synergize with HCV-

induced alterations of the host redox status to enhance the pathogenesis of liver

disease in hepatitis C patients who drink alcohol. Indeed, ethanol has been reported to

exacerbate oxidative stress in HCV core-transgenic mice and chronic hepatitis C

patients (65,68,175). Increased CYP2E1 expression during hepatitis C infection may

also increase the ROS levels and increase the susceptibility to alcohol toxicity

(70,154,176-178), as ethanol has been shown to induce significantly higher ROS/RNS

levels in CYP2E1-overexpressing hepatoma cells that also express HCV core protein

when compared to cells expressing either CYP2E1 or core gene alone (176). It may

be speculated that such combined oxidative/nitrosative stress would accelerate DNA

12

mutations and facilitate transformation during chronic hepatitis C (41,65,73,92-104).

Unfortunately, despite the well-established role of oxidative/nitrosative stress in the

pathogenesis of human diseases, not many studies have directly demonstrated the

pathological effects of oxidative/nitrosative stress in the HCV-infected alcoholic and

non-alcoholic liver to this date, and much work will be necessary to precisely

delineate the molecular interactions between HCV, alcohol, and the redox status in the

pathogenesis of HCV infection.

Besides oxidative/nitrosative stress, acetaldehyde can damage cells and

potentially enhance pathogenesis, for example, by rapidly forming adducts with cell

constituents, including DNA (Fig. 1.3) (7,170,179-181). Chemical modification of

DNA by metabolite(s) of ethanol will increase mutations and thereby increase the risk

of HCC (7). Acetaldehyde also enhances the transcription of collagen genes and

interferes with DNA repair mechanisms (168,170,180,182). Moreover, ethanol

metabolism may increase the expression of pro-carcinogenic genes, such as galectin-1

(7,183). Therefore, it may be hypothesized that alcohol synergizes with HCV through

combined oxidative/nitrosative stress as well as acetaldehyde formation, both of which

can be pathogenic to liver.

ALCOHOL, REACTIVE SPECIES, AND HCV REPLICATION

Alternatively, alcohol and reactive species may affect the virus itself and exert

its pathogenic effects indirectly (Fig. 1.4). In fact, hepatitis C patients who drink

alcohol typically show a pattern of hepatic injury that is more characteristic of chronic

13

viral hepatitis than alcohol-induced injury (184,185). These findings suggest that

alcohol enhances the pathogenic effects of HCV rather than exerting its independent

effects on the liver. Interactions between alcohol, reactive species, and HCV itself

involve at least three separate mechanisms: modulation of viral replication,

modulation of the host antiviral response that in turn affects the viral titer, and

modification of the viral genome (Fig. 1.4).

Alcohol, reactive species, and HCV replication

Several clinical studies have correlated increased serum HCV titer with the

amount of alcohol consumed (144,186-190). HCV titer is significantly greater in

patients consuming greater than 10g of alcohol per day (191). Habitual drinkers also

showed higher levels of HCV RNA than non-habitual drinkers (186). Abstinence or

moderation of alcohol consumption could result in a substantial drop in the HCV RNA

levels in some patients (144,190,191). Whether heavy, moderate, and light alcohol

intake leads to consistent elevations in serum as well as intrahepatic HCV RNA is still

unclear (192-195). Nevertheless, alcohol can increase intrahepatic HCV RNA titer in

patients (189,196) and subgenomic HCV RNA replication in vitro, in Huh7 human

hepatoma cells, suggesting that alcohol may affect the viral life cycle itself (197).

Possible mechanisms include activation of the endogenous opioid system and nuclear

factor kappa B (NF-B) (197). Products of ethanol metabolism, most likely

downstream of aldehyde dehydrogenase, were implicated (197). Notice that these

subgenomic replicon cells support continuous HCV RNA genome replication without

14

producing virus particles. Whether alcohol is able to increase the production of

infectious virus particles and/or its infectivity has not yet been determined.

Another possible mediator in the modulation of HCV replication by alcohol

would be reactive species, generated during hepatic metabolism of alcohol (Fig. 1.3).

Indeed, the common location of HCV replication and CYP2E1 on the endoplasmic

reticulum increases the likelihood of interaction between ROS and HCV during

alcohol metabolism. Previously, high concentrations of iron (50 and 100 M) were

reported to enhance HCV replication (198). Increased mitochondrial generation of

ROS with HCV, secondary to the ER stress, has also been suggested to promote HCV

replication through Stat-3 and antioxidant/antioxidant genes, to have the opposite

effects (199-201). These studies have since been cited as evidence that oxidative

stress favors HCV replication, assuming that iron enhances HCV replication through

oxidative stress.

However, a recent study that quantitatively monitored the enzyme kinetics of

HCV NS5B, the RNA-dependent RNA polymerase, extensively demonstrated that

Fe2+ and Fe3+ can directly bind to NS5B and inhibit its replicase activity (202). The

results are consistent with previous reports that, too, have demonstrated suppression of

HCV replication by other divalent cations such as zinc (202-205). In addition, Stat-3

participates in the anti-HCV activity of interferon (201,206,207), and the HCV-

induced oxidative stress has recently been suggested to inhibit HCV RNA replication

instead, by activating the gene expression of COX-2 and increasing the production of

prostaglandin E2 (76).

15

Using subgenomic and genomic replicons that continuously replicate in Huh7

human hepatoma cells, we also found that H2O2, at concentrations that did not deplete

intracellular GSH or induce cell death, rapidly suppressed HCV RNA replication in a

dose-dependent manner (208,209). The suppressive effects of peroxide were

comparable to those of interferon gamma and cyclosporine A, which are potent

inhibitors of HCV replication (208,210-213) (unpublished observation). The

subgenomic replicon of Con1 sequence (genotype 1b) and a genomic hybrid derived

from Con1/H77c sequences (genotype 1a/1b) were likewise affected, and the

suppression could be partially reversed with N-acetylcysteine and completely blocked

by buffering intracellular calcium, suggesting a signaling event (208). Furthermore,

the mechanism most likely involved a disruption of the HCV replication complex on

cell membranes, and other agents that elevated cytosolic calcium concentration had

similar suppressive effects on HCV replication, as reported by others (212). Our

findings were recently corroborated by Yano et al. who showed that vitamins A and E

elevated HCV RNA content in a different HCV replicon model of genotype 1b (214).

These results suggest that the activation of Nox2 protein during inflammation, such as

during chronic hepatitis C, functions to help decrease HCV replication. This

conclusion is in line with clinical observations that individuals who have chronic

granulomatous disease and do not have functional Nox2, tend to suffer from chronic

infection (215). In addition, H2O2, in contrast to its pro-viral effects on human

immunodeficiency virus (HIV), has been shown to negatively regulate hepatitis B

virus replication without affecting cell metabolism (216,217). These findings are

16

consistent with the fact that reactive species can act as second messengers that

participate in and modify signaling (90) and suggest that, during inflammation,

reactive species do not have to reach high enough concentrations to directly damage

viruses and virus-infected cells if they can induce antiviral redox signaling.

Furthermore, it may be concluded that whether reactive species up-regulate or down-

regulate the replication of a particular virus would depend on the mechanics of viral

replication and their interplay with the components of redox signaling.

Therefore, oxidative stress is more likely to suppress than promote HCV RNA

replication. Although it is difficult to reconcile the differences in these studies, part of

the differences could have resulted from the choice of antioxidants/antioxidant genes

that were used in the earlier studies. For example, antioxidants/antioxidant genes can

display pro-oxidant as well as antioxidant functions (218-222), and such dual and, at

times, conflicting findings are unfortunately not uncommon in the study of

antioxidants. To clearly define the relationship between oxidative stress and HCV

replication, one will need to take into consideration not only the kinetics, dosage-

effects, and the mechanism of action of chemical/biological agents being studied but

also, the cellular adaptive response to oxidative stress and its kinetics. In this regard,

the oxidative suppression of HCV replication complex we found is highly consistent,

occurring in response to H2O2, tertiary butyl hydroperoxide, extracellular generation

of H2O2 with glucose oxidase plus glucose, tert-butylhydroquinone, decreasing

intracellular GSH content with BSO, as well as HNE (208,209) (unpublished

observation). The suppression starts at concentrations as low as 0.1 M H2O2 and

17

starts within 15 – 30 min of exposure, progressively worsening by 6 hrs, and resulting

in significant decreases in both the rate of HCV replication and the total viral RNA

content over time. Therefore, oxidative stress is clearly capable of suppressing HCV,

at least at the level of genome replication, through calcium elevation in cell culture.

Antioxidants, alcohol, reactive species, and the host immune response

The question of whether oxidants suppress HCV replication complex naturally

raises another related question, which is whether antioxidants would then favor viral

replication and have adverse effects on patients. Unfortunately, whether oxidants

increase or inhibit the activity of the HCV replication complex in vivo, in patients, and

whether antioxidants modulate this process has remained unclear. In one study, serum

viral load was identified as a negative predictor of plasma antioxidant levels (223).

On the other hand, serum HCV RNA was positively correlated with erythrocyte

malondialdehyde (MDA) but not with plasma MDA levels in another study (224).

Intrahepatic oxidative stress and HCV RNA levels were not measured. These studies

demonstrate inherent difficulties in utilizing simple correlations to define the effect of

oxidative stress on HCV replication, as HCV titer itself will affect the host redox

status (65,66,73,80,82,83) (Fig. 1.4). A recent study, which examined the effects of an

antioxidant cocktail, saw a reduction in the HCV RNA titer in 25% of patients.

However, the study did not include a control group, and it is impossible to distinguish

the effects of antioxidants from random fluctuations in the viral titer and other

miscellaneous effects (109,225).

18

Therefore, it remains to be clearly determined how oxidative stress and

antioxidants affect HCV titer in patients and whether alcohol modulates HCV titer

through oxidative stress. Nevertheless, antioxidants and removal of iron have now

been shown in several studies to improve the clinical symptoms of hepatitis C and/or

the outcome of anti-HCV therapy, albeit not without some controversies; importantly,

however, no adverse effects have been reported (109,226-230). These results are

consistent with the well-known effects of oxidative stress on liver disease. If

antioxidants do in fact help lower the viral titer in vivo, in hepatitis C patients, it is

possible that this would be explained by the modulation of host immune response by

reactive species and alcohol (Fig. 1.4). As mentioned earlier, alcohol and reactive

species can inhibit the antiviral activity of interferon alpha (7,40,43,149,151). In one

study, heavy drinkers who did not abstain from drinking before interferon treatment

showed a total lack of HCV RNA clearance, whereas those who normally drank

heavily but abstained from drinking before the interferon treatment showed some

improvement in the HCV RNA clearance, and the virus completely disappeared in

about 16 percent of heavy drinkers who abstained before treatment (148). In this

scenario, antioxidants, regardless of their independent effects on HCV genome

replication, would help clear HCV by improving the host immune response.

Antioxidants have also been proposed to reduce hemolytic anemia, a major side effect

associated with oxidative membrane damage during ribavirin therapy, and thereby

improve the tolerability of ribavirin therapy (231,232). Therefore, whether

antioxidants/iron reduction therapy is beneficial to patients would depend on multiple

19

variables, including effects on the HCV RNA replication, other steps of the viral

replication cycle, oxidative stress-induced pathogenesis, and response to therapy.

Again, whether antioxidants have beneficial or adverse effects on the HCV titer, in the

absence of concurrent antiviral therapy, such as in the immune suppressed individuals,

remains unclear. Choice of antioxidants is likely to be important as some antioxidants

can have pro-oxidant effects, particularly in the presence of an iron overload (233).

Modification of viral genome

Another potential variable in the complex interactions between alcohol and

HCV titer involves the mutagenic potential of alcohol and reactive species. RNA

viruses, such as HCV, exist as a population of closely related, but distinct genetic

variants, referred to as “quasispecies” (234). High rates of mutation allow these

viruses to maximize adaptability, while conserving essential genetic information

(235). The high mutation rates are generally attributed to the lack of proofreading

activity of viral replicases as well as genetic recombination, and these have been

suggested to provide the viruses with a greater repertoire of genetic sequences to

escape the host immune surveillance and to facilitate the development of antiviral

resistance (236).

Alcohol intake is associated with increased heterogeneity of HCV RNA in

patients (237,238). Some of these changes to the viral genetic sequences are likely to

stem from the many effects that alcohol has on the host immune system. It is also

possible; however, that alcohol or reactive species induce chemical modifications of

20

the viral RNA which would lead to an increased mutation rate of the HCV genome. In

fact, although chemical modification of nucleic acids is mostly studied in the context

of DNA damage in chemically-induced carcinogenesis, these agents can also modify

RNA (239), which would be important to RNA viruses that rely on RNA genomes.

Here, alcohol and ROS/RNS-induced chemical modifications of the viral RNA would

amplify the error rates of the already error-prone viral replicase and enhance the

antigenic drift, promoting the generation of quasispecies, immune escape, and the

development of antiviral drug resistance by HCV. Thus, alcohol may also affect the

HCV titer indirectly, by driving viral evolution (Fig. 1.4).

ALCOHOL, OXIDATIVE STRESS, AND HCV INFECTION – HYPOTHESIS

Therefore, HCV is likely to interact with alcohol, reactive species, and

antioxidants through multiple mechanisms. As oxidative stress is a key factor in HCV

and alcohol-induced liver disease, alcohol is likely to synergize with HCV-induced

oxidative/nitrosative stress to enhance the pathogenesis of liver disease. How alcohol,

reactive species, and antioxidants ultimately affect the production of infectious HCV

particles as well as the infectivity, persistence, and transmissibility of HCV in vivo

remains to be fully characterized. It should be noted that HCV produces hepatic as

well as extrahepatic complications (240). Many hepatitis C patients are also co-

infected with HIV and other viruses, showing accelerated disease progression.

Furthermore, whether all HCV sequences produce oxidative/nitrosative stress in a

consistent and predictable manner remains to be determined (28,29,241). The

21

synergistic hepatopathogenesis and high prevalence of alcohol abuse and HCV

infection signify the importance of research into the mechanisms behind their

combined effects.

As previously mentioned, these effects fall into three general categories:

modulation of host immune response, direct effects on HCV replication cycle

including potential effects on RNA genome replication, packaging, secretion, or

infectivity, and mutation of HCV genome via chemical modification of viral RNA

(Fig. 1.4). Host immune modulation by ethanol and reactive species is well

characterized, although its physiological relevance as the primary cause of the adverse

effects on HCV and antiviral therapy is questionable since HCV already impairs the

host immune response (22,149,242). With respect to the latter two categories, the

focus of this thesis, lack of effective in vitro and in vivo models of the complete HCV

life cycle has, until recently, severely limited research on the effects of ethanol and

reactive species on HCV (151,197,201,208,209,214,243,244).

The advent of the JFH1 genotype 2a infectious HCV cell culture system in

2005 has allowed HCV to be studied in the context of the complete virus life cycle

(245-247). Using this and existing replicon HCV cell culture systems, the work in this

thesis was aimed at answering the following three questions about the interaction

between ethanol and HCV infection:

1. How do ethanol and its metabolites affect HCV replication in the context of the

entire life cycle?

2. By what mechanism does ethanol exert these effects?

22

3. Can ethanol and HCV-induced oxidative stress synergize to increase the

mutation rate of the virus and thus enhance the development of quasispecies?

In the first study, we examined the direct effect of ethanol and reactive species

on HCV replication in the infectious JFH1 cell culture system. Since ethanol can

increase HCV RNA levels in patients and subgenomic HCV replicon cells

(189,196,197), we therefore hypothesized that ethanol would potentiate HCV

replication in the context of the complete viral life cycle. On the other hand, we

hypothesized that ROS is likely to rapidly suppress HCV replication in the JFH1-

transfected cells, just as it did in our previous studies with the HCV replicon system

(208,209). In terms of mechanism, we initially hypothesized that one or more

downstream products of ethanol metabolism, possibly acetaldehyde, would be

responsible for the proposed increase in HCV replication by ethanol.

In the second study, we examine how ethanol and reactive species affect both

oxidative RNA damage and the mutation rate of HCV. In patients, alcohol intake is

associated with increased complexity of HCV quasispecies (237,238), therefore we

hypothesized that ethanol and reactive species would increase the mutation frequency

via oxidative modification of HCV genomic RNA. Our goal in examining the

mechanisms behind how ethanol interacts with HCV is to discover novel therapeutic

options that could improve treatment outcomes in patients who drink alcohol.

Therefore, we also hypothesized that pharmacological agents that target the genotoxic

effects of ethanol and reactive species can decrease the sequence heterogeneity of the

HCV RNA. It should also be noted that studying how ethanol may affect the mutation

23

rate of HCV is likely to reveal new insights into the mechanisms underlying the

development of quasispecies and treatment resistance in patients.

24

5’UTR 3’UTRC 4B 5A 5B

4A

E1 E2

NS2

p7

NS35’UTR 3’UTR

C 4B 5A 5B

4A

E1 E2

NS2

p7

NS3

HCV IRES

5’UTR 3’UTRC 4B 5A 5B

4A

E1 E2

NS2

p7

NS35’UTR 3’UTR

C 4B 5A 5B

4A

E1 E2

NS2

p7

NS3

HCV IRES

ROS/RNS, lipid peroxidation 63-67,69

Oxidized Trx, cytokines that ROS 64,68,70

Antioxidant gene expression 66,67

ER stress 80,200

Mitochondrial Ca2+ uptake/ROS 63,69

Sensitivity to CCl4 66

iNOS, COX-2 ? 71-76

GSH, mitochondrial NADPH 63-65

Phagocytic NADPH oxidase 82,83

iNOS 73

ROS/RNS 80

iNOS, COX-2 ? 72,75

ER stress/mitochondrial Ca2+

uptake ROS 80

ROS/RNS & Oxidized Trx 64

MnSOD, HO-1, catalase 64

Δ GSH 64

HO-1 84

Figure 1.1. Role of HCV proteins in oxidative/nitrosative stress. Genome organization of HCV showing the role of individual HCV proteins in the induction of oxidative/nitrosative stress. Superscript numbers correspond to references.

25

Figure 1.2. Life Cycle of HCV.

26

Figure 1.3. HCV and alcohol metabolism in chronic hepatitis C *Refer to Figure 1.1 and the accompanying text for the mechanism of increased oxidative/nitrosative stress during HCV infection.

Chronic hepatitisCirrhosisSteatosisHCC

ADH(Cytosol)

CYP2E1(Microsomes

from ER)

ALDH(Mitochondria &

Cytosol)

AcetaldehydeEthanol Acetate

NADH + H+NAD+ NADH + H+NAD+

NADPH + H+ NADP+

ADH(Cytosol)

CYP2E1(Microsomes

from ER)

ALDH(Mitochondria &

Cytosol)

AcetaldehydeEthanol Acetate

NADH + H+NAD+ NADH + H+NAD+

NADPH + H+ NADP+

ROS/RNS ROS/RNS

IFN response Immune function

Collagen Adducts to cellular macromolecules GSH DNA repairHCV

ROS/RNS* ROS/RNS*

ROS/RNS Fatty acid synthesis & oxidation Collagen

ROS/RNS Fatty acid synthesis & oxidation Collagen

27

Figure 1.4. Proposed mechanisms of changes to HCV replication/viral titer by alcohol and reactive species.

Alcohol

Reactive Species

Modulation ofimmune response

(e.g.– IFN response)

Direct effect onHCV replication

complex and/or hostfactors

HCV genomemutation

HCV Titer

Alcohol

Reactive Species

Modulation ofimmune response

(e.g.– IFN response)

Direct effect onHCV replication

complex and/or hostfactors

HCV genomemutation

HCV Titer

Other effects

OxidativeStress

28

CHAPTER 2: MATERIALS AND METHODS

29

HCV CONSTRUCTS, CELLS, AND REAGENTS

The HCV constructs used in this dissertation were previously constructed. We

used three genotype 2a HCV constructs; pJFH1 (produces infectious virus particles,

accession #AB047639), replicative-null pJFH1-GND, and subgenomic pSgJFH1-Luc

(accession #AB114136 that had the neomycin resistance gene replaced with a

luciferase reporter gene) (246,248). The functioning of the JFH1 HCV system was

verified by measuring intracellular HCV RNA by Northern Blot (Fig. 2.1A), RNase-

protected extracellular HCV RNA by quantitative real time reverse transcriptase-

polymerase chain reaction (qRT-PCR) (Fig. 2.1B), negative sense HCV RNA by qRT-

PCR (Fig. 2.1C), infectivity of naïve Huh7 cells by qRT-PCR (Fig. 2.1D), and HCV

core protein by immunofluorescence (Fig. 2.1E) versus the replicative-null JFH1-

GND construct or mock transfection. Two genotype 1b Huh7 cell clones (SgPC2 and

Clone B) supporting continuous replication of a subgenomic HCV replicon of Con1

S1179I (accession #AJ242652) sequence, containing a neomycin resistance gene, were

used as well (209,249). These subgenomic replicons support HCV RNA replication

but no virus is formed.

The statin drugs, lovastatin and fluvastatin, were obtained from Calbiochem,

EMD Chemicals Inc. Pyridoxal isonicotinoyl hydrazone (PIH) was synthesized

according to published methods by first dissolving 0.01 moles of isonicotinic acid

hydrazide in 100 mL of water by heating, then adding 0.01 moles of pyridoxal

hydrochloride and 0.011 moles of anhydrous sodium acetate, and finally boiled under

reflux for 30 minutes. The mixture was cooled, filtered, washed with water, and then

30

dried in a vacuum dessicator overnight (250-253). All other biochemical reagents

including ethanol (molecular biology grade), acetaldehyde, BSO, H2O2, isopropanol,

sodium acetate, acetone, tert-butanol, Trolox, N-acetylcysteine, diallyl disulfide,

cerulenin, 4-methylpyrazole, dipyridyl, and GSH ethyl ester were obtained from

Sigma Aldrich.

CELL CULTURE, HCV RNA TRANSFECTION, AND INFECTION

Huh7 hepatoma cells were cultured in Dulbecco’s modified Eagle medium

(Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/mL of

penicillin, and 100 µg/mL streptomycin in an incubator at 37°C and 5 % CO2

(208,209). Additionally, the HCV Huh7 cell clones were maintained in the above

medium supplemented with 0.5 mg/mL G418 (Invitrogen), though the G418 was

removed 24 h prior to an experiment.

For in vitro HCV RNA synthesis, 15 µg of JFH1 or Con1 DNA was linearized

with XBaI or ScaI, respectively. After phenol extraction and ethanol precipitation, in

vitro transcription is prepared using Promega T7 RNA polymerase system by adding

40 µL of 5x transcription buffer, 10 µL of 100mM DTT, 40 µL of 2.5 mM rNTP mix,

5 µL of 1 µg/µL linearized DNA in diethylpyrocarbonate (DEPC) water, 96 µL of

DEPC-treated water, 4 µL of RNasin, and 5 µL of 80 U/µL T7 RNA polymerase to a

reaction tube and incubating at 37ºC for 1.5 hrs. Then the reactions are treated with 12

U of RNAse-free DNase I (Ambion or GE Healthcare) for 1 hr at 37ºC to remove the

template DNA. The resulting HCV RNA is phenol/chloroform extracted twice,

31

chloroform extracted once, ethanol-precipitated twice, and resuspended in DEPC-

treated water (208,209).

Transfection of HCV RNA was performed as described (Fig. 2.2A) (208,209).

Approximately 5 x 106 Huh7 cells were trypsinized, rinsed with serum-free medium,

pelleted then resuspended in 0.4 mL of serum-free medium, and briefly mixed with 10

µg of in vitro-transcribed HCV RNA, then electroporated at 200 V and 950 µF. The

electroporated cells were plated and the medium was changed 3 hrs after transfection.

For the in vitro infectivity assays, 2 ml of the extracellular medium from JFH1 RNA-

transfected cells was cleared of cell debris by centrifugation at 5000 rpm and 4°C for 5

min and used to inoculate naïve Huh7 or Huh7.5 cells with 3 ml of fresh medium, as

shown in Fig. 2.2B (246,254). Treatments were initiated 24 hours after infection, and

the cells were harvested after another 24 or 48 hrs.

RNA EXTRACTION AND QUANTITATION

Total intracellular RNA was extracted, using Trizol reagent (Invitrogen)

according to the manufacturer’s protocol. Cells were scraped in Trizol and placed in

tubes. To the tubes, 0.2 mL of chloroform is added per 1 mL of trizol used and then

the tubes are shaken for 15 sec and incubated at room temperature for 2 minutes.

Then the tubes are centrifuged for 15 min at 10,000 rpm and 4°C. The top phase is

placed into a new tube, to which 0.5 mL of isopropanol per 1 mL of Trizol used is

added. The tubes are shaken for 15 sec, left at room temperature for 10 min, and then

centrifuged for 10 min at 12,500 rpm and 4°C. The pellets are rinsed with chilled 70

32

% ethanol in DEPC-treated water, air dried, and resuspended in DEPC-treated water.

To extract extracellular HCV RNA, 0.25 mL of cell culture medium was first treated

with RNase A (100 g/ml) for 30 min at room temperature, then RNA was extracted

using 0.75 mL Trizol LS, 0.2 mL of chloroform, 0.5 mL of isopropanol, and 20 µg of

glycogen as a carrier, and resuspended in 10 µL of DEPC-treated water.

Intracellular RNA was quantified by Northern blot or qRT-PCR (208,209,246).

For Northern blot analysis, 5-8 µg of total RNA was run on a formaldehyde gel at 80

V for 2-2.5 hrs and then transferred overnight to a nylon membrane. The membranes

were UV-crosslinked and probed with 32P-labeled DNA probes prepared from

nucleotides (nt.) 4128 – 8273 or 358 – 2816 of JFH1, generated with ScaI and ApaL I,

respectively, or 3669 to 6016 of the Con1 subgenomic replicons. Images were

quantified by densitometry, using Optiquant Cyclone 4.00 (Perkin Elmer), and data

were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA

content.

For qRT-PCR, a stock mix was made with Applied Biosystems real-time PCR

reagents, containing 12.5 µL of 2x Mastermix, 0.05 µL of each primer and probe (if

needed), 0.625 µL reverse transcriptase, and filled the volume to 23 µL with PCR

water for each well. From the stock, 23 µL was added to each well, followed by 2 µL

of 50 ng/µL RNA sample. The plate was run in an Applied Biosystems 7300 Real-

time PCR machine for 30 min at 48°C for reverse transcription, 10 min at 95°C to

deactivate the reverse transcriptase and activate the DNA polymerase, and then 45

cycles of 95°C for 15 sec and 60°C for 45 sec with data collection occurring at this

33

last step. The primer sequences for JFH1 were 5’ TCTGCGGAACCGGTGAGTA 3’

(nt. 146 to 164; forward), and 5’ TCAGGCAGTACCACAAGGC 3’ (nt. 277 to 295;

reverse), and the sequence of the fluorogenic probe, labeled with 6-FAM and TAMRA

(Biosearch Technologies, Inc.), was 5’ CCAGTCTTCCCGGCAATTCCG 3’ (nt. 168

to 188). For Con1, the primer sequences were as previously described(208). Standard

curves were generated using in vitro-transcribed HCV RNA's. Intracellular HCV

RNA levels were normalized by 18S rRNA or GAPDH mRNA. Extracellular HCV

RNA was quantified by qRT-PCR as described above, except RNA samples were not

diluted before adding to the wells.

PROTEIN ANALYSIS

Relative protein levels were analyzed for HCV NS5A, human CYP2E1, and -

actin by Western blot, as previously described (208), or for HCV Core by

immunofluorescence. For Western Blot, cells were sonicated in 2x Laemmli buffer,

and an aliquot was removed for acetone precipitation and protein assay with a

bicinchoninic acid assay kit (Pierce) to determine protein concentrations for loading

normalization. The remainder of the protein sample was boiled for 5 min at 95-100°C.

Tris-glycine 10-20% protein gels (Invitrogen) were loaded with 5-10 µg of the protein

sample per well and run in 1% SDS running buffer at 120 V for 2-3 hrs. Gels were

transferred onto PVDF membranes overnight at 4°C, and then probed with

monoclonal anti-NS5A, polyclonal anti-CYP2E1, or monoclonal anti--actin

antibodies and their corresponding horseradish peroxidase-conjugated secondary

34

antibodies. Quantification of the Western blots was performed by densitometry using

the Kodak IS2000R software.

For immunofluorescence, cells transfected with JFH1 HCV RNA were plated

on coverslips. Cells were fixed with 3.5 % formaldehyde for 5 min and incubated

with phosphate buffered saline containing 1 % (w/v) bovine serum albumin, 0.05 %

(w/v) NaN3, and 0.02 % (w/v) saponin. Samples were subsequently incubated for 1 hr

with primary and then another 1 hr with fluorophore-conjugated secondary antibodies

at 37°C. The coverslips were then mounted on microscope slides, and imaged via

confocal laser scanning microscopy (Nikon).

LUCIFERASE REPORTER ASSAY

Relative luciferase activity was determined using a Luciferase Reporter Assay

Kit (Promega Corp.) (248). After various treatments, SgJFH1Luc RNA-transfected

cells were collected in PBS then centrifuged at 5000 rpm for 5 minutes. The PBS was

removed and the cells were lysed with 100 µL of 1x Reporter Lysis Buffer. After a

freeze-thaw cycle to ensure lysis, the cell lysates were vortexed for 15-20 seconds and

centrifuged at 12,500 rpm and 4°C for 5 min. The supernatant was transferred to a

new tube and stored until needed at -80°C. Luciferase activity was determined by

adding 50 µL of Luciferase Assay Reagent into luminometer tubes allowing it to

equilibriate for 10-20 seconds. Then to the luminometer tubes, 1-5 µL of the

supernatant from the cell lysates was added, gently mixed for 6 seconds, and placed in

the luminometer. The luminescence reading was determined with a 10 second delay

35

and a 10 second read time. Luciferase activities were normalized by total protein

content, determined with a bicinchoninic acid assay kit (Pierce).

IN VITRO HCV REPLICATION ASSAY

In vitro replication assay was carried out, as previously described

(208,209,255). JFH1-transfected or SgPC2 cells were grown to approximately 60-70

% confluent in 60 mm dishes. After 5 hrs of ethanol treatment, the cells were washed

for 2 minutes with 1 mL of lysolecithin-containing wash solution (150 mM sucrose;

30 mM HEPES, pH 7.4; 33 mM ammonium chloride; 7 mM KCl; 4.5 mM magnesium

acetate; 250 µg/mL lysolecithin). An additional 1.5 mL is then added and all of the

liquid is removed from the dishes by aspiration. Cells are collected in 120 µL of

incomplete replication buffer (100 mM HEPES, pH 7.4; 50 mM ammonium chloride;

7 mM KCl; 1 mM spermidine; 1 mM each of ATP, GTP, and UTP; and 10 µM CTP in

DEPC-treated water) and placed in eppendorf tubes. The cytosolc lysates are lysed by

pipetting up and down more than 100 times and briefly vortexing. Then the tubes are

centrifuged at 14,000 rpm and 4°C for 5 min. The supernatant (cytoplasmic fraction)

is aliquotted 3-4 tubes containing about 70 µL each and frozen in the -80°C freezer for

later use. The replication reaction was prepared by thawing the cytoplasmic fractions,

followed adding 30 µCi of α-32P-CTP (5000 µCi/mmol) and 1 µg of actinomycin D.

The replication was allowed to proceed for 1 hr at 30°C, until 70 µL of replication

termination buffer (10 mM Tris, pH 7.5; 1 mM EDTA; 150 mM NaCl; and 0.5 % SDS

in DEPC-treated water) was added . Then, RNA products were phenol-chloroform

36

extracted, ethanol precipitated, resuspended in 8 µL of DEPC-treated water, and run

on a 1 % formaldehyde agarose gel, which was subsequently analyzed, using

Optiquant Cyclone 4.00.

CYP2E1 SMALL INTERFERING RNA (siRNA)

Huh7 and SgPC2 cells were transfected with 50 nM CYP2E1 (Santa Cruz

Biotech.) or non-targeting control (Dharmacon) siRNAs, using RNAiMax

(Invitrogen). Cells were first plated in 6-well plates to be approximately 50 %

confluent at the time of transfection. One hour prior to the transfection, the cells were

rinsed with PBS and 2 mL of antibiotic-free medium was added to each well. Then

two tubes were set up for each type of siRNA. The first tube contained 5 µL of 20 µM

stock siRNA, 45 µL of PBS, and 50 µL of Opti-MEM I (Invitrogen) per well. The

second tube contained 1 µL of Lipofectamine RNAiMax and 99 µL Opti-MEM I per

well. Both tubes were incubated at room temperature separately for 5 minutes, then

the second tube was added to the first, mixed well, and incubated at room temperature

for 30 minutes. To each well of cells, 200 µL of RNAi mixture was added, and the

plates were incubated at 37°C and 5 % CO2 for 4 hrs before changing to normal

medium.

CYP2E1 ACTIVITY ASSAY

Cells were treated with and without 0.2% ethanol for 48 hrs and lysed.

CYP2E1 activity was determined by measuring hydroxylation of p-nitrophenol as

37

described, except NADPH, instead of the NADPH-generating system, was used (256).

Incubation tubes containing 440 µL of 50 mM potassium phosphate, pH 7.4; 10 µL of

5 mM p-nitrophenol; and 30 µL of 20 mM NADPH were prewarmed to 37ºC. Then

20 µL of diluted enzymes (100 µg protein) was added to each tube to initiate the

enzymatic reactions and the tubes were incubated at 37ºC for 30 min in a water bath.

Addition of 100 µL of 20 % (v/v) trichloroacetic acid was done to stop the reactions

before placing them on ice. The acidic reaction mixture was centrifuged at 10,000 x g

and 4°C for 5 min and had 500 µL of the supernatant placed in a new tube containing

250 µL of 2 M NaOH, then vortexed. The absorbance was measured at 535 nm on a

Perkin Elmer LS-5 spectrophotometer and CYP2E1 activity was calculated using a

standard curve of p-nitrocatechol (0-20 nmol) added to tubes of heat-inactivated

enzymes. The specificity was demonstrated by inhibiting the reaction with 100 µM

CYP2E1 antibodies, and portion of the activity that is inhibited by CYP2E1 antibodies

was then calculated and reported.

NADH/NAD+, CHOLESTEROL, AND ATP ASSAYS

NADH and NAD+ levels were determined by enzymatic NADH recycling

assay, using the NAD+/NADH Quantification Kit from Biovision, per manufacturer's

recommendations. After various treatments, cells were collected in 400 µl of

NADH/NAD+ extraction buffer. Samples were immediately subjected to two

freeze/thaw cycles and filtered using Microcon YM-10 (Millipore). Then, the samples

were split into two sets, one of which was used to carry out the thermal decomposition

38

of NAD+ at 60ºC for 30 min, measuring NADH, whereas the other set measured total

NADH plus NAD+ content. To a 96-well plate, 50 µL of each sample is added to each

well, mixed with 98 µL of NAD Cycling buffer and 2 µL of NAD Cycling enzyme

mix, and incubated at room temperature for 5 min. The reactions had 10 µL of NADH

developer added to each, were incubated at room temperature for 1-4 hrs, and read on

the spectrophotometer at 450 nm. Standard curves were used to calculate the

NADH/NAD+ ratio from the NADH and total NADH and NAD+ amounts.

Total intracellular cholesterol was measured using the Cholesterol/Cholesteryl

Ester Quantitation kit (Biovision) per manufacturer’s instructions. Approximately

60,000 cells were homogenized in 200 µL of chloroform:isopropanol:Triton X-100

(7:11:0.1) and then centrifuged for 10 minutes at 13,000 rpm and room temperature.

The organic (lower) phase was collected, air dried at 50ºC to remove chloroform, and

vacuum dried for 30 min to remove trace organic solvents. The dried lipids were

resuspended in 200 µL of Cholesterol Reaction buffer by vortexing for 5 min. To

each well, 12 µL of the resuspended sample or standard were added and mixed with

44 µL of Cholesterol Reaction buffer, 2 µL of Cholesterol Probe, 2 µL Enzyme mix,

and 2 µL Cholesterol Esterase. The reactions were covered with foil, incubated at

37ºC for 60 min, and then read on the spectrophotometer at 570 nm. Standard curves

were used to calculate cholesterol amounts.

Total ATP content was measured using Somatic Cell ATP Assay Kit (Sigma-

Aldrich). Cells were grown in 6-well plates, treated for 24-48 hrs, and washed twice

with KRPH. The ATP was released with 0.5 mL of ATP releasing solution, and

39

measured by luciferase reaction with a luminometer. The ATP data was normalized

by total protein content, determined with a bicinchoninic acid assay kit (Pierce).

SEQUENCING AND SEQUENCE ANALYSIS

Total RNAs were collected in Trizol and extracted as described in the RNA

Quantification section above. Then, the NS5A region (nt. 4612 to 6013 of Con1

replicon) was reverse transcribed/amplified with AccuScript High Fidelity RT-PCR

System (Agilent). The reverse primer used for reverse transcription and PCR

amplification was 5’-ATCAATCGATTGTCTAGAGCTGAAGAGGCTTCACCAG-

3’, and the forward primer used in PCR amplification was 5’-

GCAATCTTGTACAAGCTTCGCAGCGCATGGCGTGAT-3’. The XbaI and

HindIII restriction enzyme sites are underlined. The cDNA products were cloned into

pUC19 via HindIII and XbaI sites and sequenced at the Joint Genome Institute

(Livermore, CA) and University of California Berkeley Sequencing (Berkeley, CA)

(Fig. 2.3). Sequences were aligned against Con1 (S1179I) sequence, using

BioEdit/MEGA 4.0 Molecular Evolutionary Genetics Analysis software. Cumulative

synonymous and nonsynonymous mutations were computed using Synonymous

Nonsynonomous Analysis Program (SNAP; http://hcv.lanl.gov)(257). The dN/dS

ratio was calculated using SNAP. Amino acid substitution trends were analyzed by

position with MutationCounter, a mutation counting program developed for this

project. The sequences were compared with 1,212 genotype 1b NS5A sequences

pooled from the Los Alamos HCV sequence database which included sequences from

40

the European and Asian databases (257). Sequences harboring gaps, undetermined

nucleotides, or incomplete NS5A sequences were removed prior to the analysis. The

experiment was repeated twice with the first trial (sequenced by the Joint Genome

Institute) and the second trial (sequenced by UC Berkeley Sequencing) yielding the

same conclusions. The compiled data from the two studies are reported in Table 4.1.

DETERMINATION OF 8-OH-G CONTENT

Total RNA was isolated using Trizol (Invitrogen) and analyzed for 8-OHG by

ELISA with an RNA Damage Kit (Cell Biolabs, Inc.) following the manufacturer's

protocol. The RNA was digested with 10 U nuclease P1 (Sigma or US Biological) for

2 hrs at 37°C, followed by incubation with 10 U of alkaline phosphatase (Promega)

for 1 hr at 37°C. The microwell strips, which had been coated with 1 µg/mL 8-

hydroxyguanosine (8-OHG) conjugate the night before, were incubated with 50 µL of

unknown or 8-OHG standard for 10 min and then with 50 µL of anti-8OHG antibody

added for 1 hr on an orbital shaker at room temperature. The microwells were washed

three times with 250 µL of 1x wash buffer, and then incubated with 100 µL of

enzyme-conjugated secondary antibody for 1 hr on the shaker at room temperature.

The wells were washed three more times and then incubated with 100 µL of substrate

solution until the color begins to change, when 100 µL of stop solution is added and

the absorbance is read at 450 nm. The 8-OHG quantities were determined using an 8-

OHG standard curve in the concentration range of 0 to 40 ng/mL of 8-OHG.

41

STATISTICS

Data were analyzed using Student's t test, one-way analysis of variance, and z-

test or Chi-square analysis of proportions using SigmaStat 3.1 (Jandel Scientific). A p

value ≤ 0.05 was considered significant. All experiment repetitions were either shown

as the mean of several independent experiments ± standard error or as representative

Northern or Western blot images.

42

Figure 2.1. JFH1 replication in Huh7 cells. (A) Intracellular JFH1 RNA versus GND over time by Northern, (B) RNase-resistant extracellular JFH1 RNA over time by qRT-PCR, (C) Negative strand HCV RNA by qRT-PCR, (D) Intracellular JFH1 RNA from Huh7 cells infected with virus-containing medium, (E) HCV Core protein detection by immunofluorescence.

Infectivity Assay(Intracellular HCV RNA)

Mock GND JFH1

HC

V R

NA

co

pie

s (x

10

^6)

pe

r m

illio

n c

ells

0

2

4

6

8

10

Negative Strand HCV RNA

GND JFH1

HC

V R

NA

cop

ies

(x10

^6)

per

mill

ion

cells

0

10

20

30

40

50

60

Extracellular HCV RNA

Time (hrs)

16 24 48 96

HC

V R

NA

Cop

ies

(x10

^6)

per

mL

0

5

10

15

20

25

GNDJFH1

(A) (B)

(D)(C)

(E)

JFH1(20x) JFH1 (60x)Mock (20x)

43

Figure 2.2. HCV RNA transfection and infection. (A) HCV RNA is electroporated into Huh7 hepatoma cells. (B) HCV-containing medium is collected, centrifuged to clear debris, and used to infect naïve Huh7 cells.

Figure 2.3. Flowchart of steps to clone and sequence HCV RNA.

+

Vector Insert Ligated Product

Bacteria

Plate on LB agar plates treated with ampicillin

RNA cDNAAccuscript RT

PCR amplification with Pfu polymerase

Many copies of cDNA insert

pUC19 Vector

Digestion with XbaI and HindIII

Miniprep and sequence

Gel purify

44

CHAPTER 3: ETHANOL ENHANCES HEPATITIS C VIRUS REPLICATION THROUGH LIPID METABOLISM AND

ELEVATED NADH/NAD+

45

ABSTRACT

Ethanol has been suggested to elevate HCV titer in patients and to increase

HCV RNA in replicon cells, suggesting that HCV replication is increased in the

presence and absence of the complete viral replication cycle, but the mechanisms

remain unclear. In this study, we use Huh7 human hepatoma cells that naturally

express comparable levels of CYP2E1 as human liver to demonstrate that ethanol, at

subtoxic and physiologically relevant concentrations, enhances complete HCV

replication. The viral RNA genome replication is affected for both genotypes 2a and

1b. Acetaldehyde, a major product of ethanol metabolism, likewise enhances HCV

replication at physiological concentrations. The potentiation of HCV replication by

ethanol is suppressed by inhibiting CYP2E1 or aldehyde dehydrogenase and requires

elevated NADH/NAD+ ratio. In addition, acetate, isopropanol, and concentrations of

acetone that occur in diabetics enhance HCV replication with corresponding increases

in the NADH/NAD+. Furthermore, inhibiting the host mevalonate pathway with

lovastatin or fluvastatin and fatty acid synthesis with 5-(tetradecyloxy)-2-furoic acid

or cerulenin significantly attenuates the enhancement of HCV replication by ethanol,

acetaldehyde, acetone, as well as acetate, while inhibiting -oxidation with -

mercaptopropionic acid increases HCV replication. Ethanol, acetaldehyde, acetone,

and acetate increase the total intracellular cholesterol content, which is attenuated with

lovastatin. In contrast, both endogenous and exogenous ROS suppress the replication

of HCV genotype 2a, as previously shown with genotype 1b.

46

Conclusions/Significance: Therefore, lipid metabolism and alteration of

cellular NADH/NAD+ ratio are likely to play a critical role in the potentiation of HCV

replication by ethanol rather than oxidative stress.

INTRODUCTION

Ethanol consumption is a well-known risk factor for chronic liver diseases.

Ethanol is also a key cofactor in the pathogenesis induced by HCV, and it decreases

the efficacy of anti-HCV treatments (115,144). Likewise, HCV infection exacerbates

liver damage caused by prolonged alcohol abuse (144). It has also been reported that

patients with a history of alcohol abuse are more likely to be infected with HCV than

the rest of the population (115).

In addition, ethanol has been suggested to exacerbate HCV-induced liver

diseases in part, by affecting the viral titer (144,191,197,258). Hepatitis C patients

who drink alcohol typically show a pattern of hepatic injury that is more characteristic

of chronic viral hepatitis than alcohol-induced injury, suggesting that alcohol enhances

the pathogenic effects of HCV rather than exerting its independent effects on liver

(184). Several clinical studies have correlated increased serum and intrahepatic HCV

titer with the amount of alcohol consumed (144,191,258). Abstinence or moderation

of alcohol consumption could reduce the HCV titer in some patients (144,191).

Furthermore, in vitro studies suggest that ethanol increases HCV RNA level in Huh7

human hepatoma replicon cell lines that continuously support the HCV RNA

replication without virus production (197,244,259). These studies suggest that ethanol

47

enhances HCV replication both in the presence and absence of the complete viral

replication cycle. HCV replicon systems and more recent virus-producing cell culture

models have increased our understanding of HCV and provide us with tools for

studying potential interactions between HCV and pathological cofactors, such as

ethanol (20).

Nevertheless, whether ethanol directly enhances HCV production in the

context of the complete viral replication cycle has not been demonstrated.

Furthermore, the mechanism by which ethanol modulates HCV RNA replication

remains controversial as ROS and lipid peroxidation products, which can be generated

during ethanol metabolism, can suppress, rather than increase, HCV RNA replication

in cells, suggesting the involvement of other metabolites of ethanol

(208,209,214,260,261). Oxidative hepatic ethanol metabolism is a multi-step process

(258). Alcohol dehydrogenase, the predominant ethanol-metabolizing enzyme, is

found in the cytosol and produces acetaldehyde and NADH. Ethanol-inducible

cytochrome P450 (CYP2E1), which is induced during extended ethanol exposure, is

another major ethanol-metabolizing enzyme located in the endoplasmic reticulum and

generates NADP+ and ROS in addition to acetaldehyde. Catalase, which is found in

peroxisomes, is thought to not contribute significantly to ethanol metabolism under

normal conditions. Once ethanol is metabolized into acetaldehyde, it is rapidly

converted into acetate and NADH by aldehyde dehydrogenase. Acetaldehyde and

other products of ethanol metabolism have been implicated in many pathogenic effects

48

of ethanol. Whether these metabolites also participate in the modulation of HCV

replication by ethanol, however, has not yet been tested.

Therefore, the goal of this study was to determine the effects of ethanol

exposure on HCV replication in the context of the complete HCV replication cycle

and the mechanisms, comparing the effects of other metabolites of ethanol with those

of ROS. Our data show that ethanol and acetaldehyde, at subtoxic and physiologically

relevant concentrations, elevate complete HCV replication, as opposed to the

suppression caused by endogenous and exogenous ROS. Our data further suggest that

elevation of the ratio of NADH/NAD+ and modulation of lipid metabolism are likely

to play critical roles in the modulation of HCV replication by ethanol. Possible

implications on in vivo HCV replication, patient education, and disease management

are also discussed.

RESULTS

Ethanol increases the complete replication of HCV at physiological

concentrations

To examine whether ethanol increased the complete replication of HCV,

positive-sense genomic JFH1 RNA was produced by in vitro transcription, using T7

RNA polymerase, and transfected into Huh7 human hepatoma cells. Then, the

transfected cells were exposed to 0 – 1.0 % (v/v; 0 - 172 mM) ethanol once daily for

48 hrs. Then, the cells and the cell culture medium were harvested and analyzed for

intracellular and RNase A-resistant extracellular HCV RNA's by a combination of

49

Northern blots and qRT-PCR. Ethanol significantly increased the intracellular JFH1

HCV RNA levels to 237 ± 40 and 305 ± 61 % of untreated controls at 0.2 and 0.5 %

concentrations, respectively (P < 0.05) (Fig. 3.1A and 3.1B, top panel). HCV NS5A

protein level similarly increased with ethanol treatments (Fig. 3.1B, bottom panel).

Extracellular HCV RNA was also significantly elevated with the 0.2% ethanol

treatment, indicating increased virus secretion (Fig. 3.1C). Next, we examined

whether virus-infected cells responded similarly to ethanol treatment with elevated

HCV RNA. We found that 0.2% ethanol also increased HCV RNA in Huh7 cells

infected with cell culture-generated JFH1 virions (Fig. 3.1D). JFH1 GND mutant,

which harbors a critical mutation (GDD:GND) in NS5B, the viral polymerase, did not

replicate or generate infectious virus particles, as expected (Fig. 2.2A). These

concentrations of ethanol did not induce any cytotoxicity, as assessed by cell

morphology and measuring the ATP content (Fig. 3.1E). The 0.2 % ethanol,

equivalent to blood alcohol concentration of 34.4 mM, that significantly enhanced

HCV replication, is approximately twice the legal limit for driving under the influence

in many countries, including the U.S. The 0.5 % ethanol lies in the toxic range but can

also be achieved physiologically, particularly in chronic alcohol users. In addition,

ethanol is volatile and the amount that remains would be significantly less than what

was added to cell culture medium (262). These data, therefore, suggest that ethanol

can enhance complete HCV replication, at physiologically attainable concentrations.

50

Ethanol enhances HCV RNA replication of genotypes 2a and 1b

Previously, ethanol was shown to elevate HCV RNA content in Huh7 cells that

supported subgenomic HCV RNA replication without virus production (197,244,259).

To test whether the JFH1 RNA replication was also affected by ethanol, we

transfected Huh7 cells with JFH1 SgJFH1-Luc RNA and exposed the cells to ethanol

for 48 hours. Then, HCV replication was monitored by measuring the firefly

luciferase activity (248). Ethanol increased the luciferase activity in these cells,

suggesting that the JFH1 RNA genome replication was affected (Fig. 3.2A).

Genotype 2a HCV infection is found globally, with the prevalence ranging

from less than 2 to about 30 % depending on the geographical region (263,264).

However, as the most prevalent HCV genotype is genotype 1, we also repeated these

experiments, using Con1 subgenomic replicon (SgPC2) cells (209,249). Again,

significant increases in the genotype 1b HCV RNA could be demonstrated with 0.1 to

1 % ethanol (Fig. 3.2B, top panel). Similar increases in the HCV NS5A protein

content was demonstrated by Western blots (Fig. 3.2B, bottom panel).

To confirm that the rate of the HCV RNA genome replication is accelerated by

ethanol, we measured the activity of the HCV RNA replication complex. JFH1-

transfected cells were exposed to ethanol for 5 hrs and then, the cytoplasmic lysates,

containing the HCV replication complex, were isolated. Then, the in vitro RNA

replication assay was performed in the presence of -32P-labeled CTP and

actinomycin D, as previously described (209). JFH1 cell extracts produced a single

band that corresponded to the expected size of the HCV RNA, indicating active viral

51

RNA replication, whereas the JFH1 GND extracts did not (Fig. 3.2C). Ethanol

significantly increased the rate of HCV RNA replication (Fig. 3.2C). Ethanol also

accelerated the in vitro replication rate of Con1 strain (Fig. 3.2D). On the other hand,

ethanol did not increase the HCV IRES activity, as assessed by the HCV IRES activity

assay, using pRL-HL (data not shown) (265). The data suggest that ethanol increases

the rate of HCV RNA replication without directly enhancing its translation rate, at

least when these processes are evaluated separately. Therefore, increases in the NS5A

protein content with ethanol (Fig. 3.2B) are likely to have resulted from increased

levels of the viral RNA template available for translation.

CYP2E1 is present in Huh7 cells at significant levels as in human liver

Next, we started examining the mechanism by which ethanol increased HCV

replication, first, by identifying key steps of ethanol metabolism that mediated this

effect (Fig. 3.3A). Alcohol dehydrogenase I was not detected in significant levels in

our Huh7 cells (data not shown). To confirm that ethanol metabolism is occurring in

our cells, we then analyzed our Huh7 cells for the expression of CYP2E1. Our Huh7

cells expressed significant levels of CYP2E1 protein, which was about 2.2 ± 0.5 fold

less than human liver (Fig. 3.3B). CYP2E1 expression could also be enhanced by 1.5

± 0.2 fold with daily treatment with 0.2 % (v/v) ethanol for 48 hrs (Fig. 3.3C). This

enhanced expression of CYP2E1 could be maintained for at least two weeks. The

CYP2E1 activities (Fig. 3.3D) were within the expected range for human liver, which

is 0.25 to 3.3 nmol/min/mg protein, and paralleled the CYP2E1 expression levels (Fig.

52

3.3C) (256). CYP2E1 activity of human liver shown in Fig. 3.3B was 1.83 ± 0.01

nmol/min/mg. Con1 SgPC2 cells had similar expression and activity levels of

CYP2E1 as Huh7 cells (0.81 ± 0.02 nmol/min/mg without ethanol; 1.01 ± 0.06

nmol/min/mg with 0.2 % ethanol).

The ethanol-induced potentiation of HCV replication could be abrogated with

25 µM diallyl disulfide (DADS), an inhibitor of CYP2E1 (Fig. 3.3E). In addition,

CYP2E1 siRNA, which decreased CYP2E1 protein level to 35 ± 9 % (P < 0.05) of the

controls transfected with non-targeting control siRNA, also significantly blunted the

potentiation of HCV replication by ethanol (Fig. 3.3E). These data suggest that

ethanol is being metabolized by these cells, and that CYP2E1 activity is critical for the

potentiation of HCV replication by ethanol in our system.

ROS suppresses JFH1 replication

Hepatic ethanol, particularly CYP2E1-mediated, metabolism generates ROS in

addition to acetaldehyde (153) (Fig. 3.3A) and previously, we showed that ROS could

suppress subgenomic Con1 and H77c/Con1 hybrid HCV RNA replication in these

cells (208,209). To resolve these seemingly conflicting observations, we continued to

examine how ROS affected JFH1. To examine the effects of endogenously generated

ROS, we first used BSO. BSO depletes GSH, a major endogenous antioxidant, by

inhibiting its de novo synthesis. Therefore, BSO would amplify the effects of

endogenous ROS, generated during normal cellular metabolism and in response to

HCV (258). BSO decreased intracellular GSH content by approximately 80 ± 12 % in

53

Huh7 cells (P < 0.05). In addition, BSO decreased both intracellular and extracellular

JFH1 RNA levels (Fig. 3.4A and 3.4B). To confirm that BSO was acting specifically

by decreasing GSH, cells were treated with BSO and GSH ethyl ester, which enters

cells and is cleaved by cellular esterases to restore GSH inside cells, bypassing the

inhibition of GSH biosynthesis by BSO. GSH ethyl ester partially restored both

intracellular and extracellular HCV RNA (Fig. 3.4A and 3.4B). Adding extracellular

GSH, which is broken down into its constituents then taken up for intracellular de

novo GSH synthesis and does not bypass the BSO-inhibited step, could not restore the

HCV RNA level in these cells, as expected. The data suggest that BSO decreases

HCV replication specifically by decreasing GSH.

To examine the effects of the exogenous ROS, JFH1 RNA-transfected cells

were incubated with 0.25 mU/mL of glucose oxidase (GO), which produces H2O2

extracellularly through an enzymatic reaction in the presence of glucose, mimicking

ROS generation during inflammation. GO decreased the intracellular JFH1 RNA by

30 + 8 % (P < 0.05) and exacerbated the suppression of HCV RNA by BSO (Fig.

3.4C). In addition, JFH1 RNA levels decreased with 25, 50, and 100 M H2O2 (Fig.

3.4D). Treating cells with BSO plus GO or either agent alone likewise suppressed the

subgenomic JFH1 RNA replication (Fig. 3.4E). BSO and H2O2 also countered the

enhancement of HCV replication by ethanol (Fig. 3.4F). Furthermore, N-

acetylcysteine (NAC) and Trolox, a water soluble vitamin E, either increased or had

no significant effect on the ethanol-induced enhancement of HCV replication (Fig.

3.4G). These cell treatments did not induce cytotoxicity, as determined by the ATP

54

assay (data not shown). Thus, ROS were not likely to be responsible for the

potentiation of HCV RNA replication by ethanol. These data are consistent with the

suppression of HCV RNA replication previously observed with HCV genotype 1

(208,209).

Acetaldehyde increases the replication of HCV

We next evaluated whether another major product of ethanol metabolism,

acetaldehyde, had similar effects on HCV as ethanol. Acetaldehyde, at

physiologically relevant concentrations (266), significantly increased the HCV RNA

content in both non-virus producing and virus-producing JFH1 cells (Fig. 3.5A and

3.5B). Infecting naïve cells with virus-containing medium and then treating with 5

µM acetaldehyde also led to significant increases in HCV replication (Fig. 3.5C). To

examine whether acetaldehyde had similar effects on genotype 1b HCV, SgPC2 cells

were also incubated with acetaldehyde and analyzed for changes in HCV replication.

Acetaldehyde likewise elevated the HCV RNA level in these cells (Fig. 3.5D).

Another Con1 HCV subgenomic replicon cell clone, Clone B, derived in another

laboratory (249), responded similarly to ethanol and acetaldehyde, indicating that the

response is not specific to our cell clone (Fig. 3.5D). Thus, acetaldehyde is sufficient

to potentiate HCV replication of both genotypes 1b and 2a.

55

Isopropanol and acetone also potentiate HCV replication – the role of

NADH/NAD+

We continued to investigate whether acetaldehyde itself or products of

acetaldehyde metabolism are critical for the potentiation of HCV replication by

ethanol by inhibiting aldehyde dehydrogenase with cyanamide (see Fig. 3.3A).

Cyanamide suppressed the potentiation of HCV replication by ethanol just as

inhibiting the first step of ethanol metabolism with 4-methylpyrazole (4MP) and

DADS did, suggesting that it is not acetaldehyde itself but a downstream product of

acetaldehyde metabolism that increases HCV replication (Fig. 3.6A, left panel).

Acetaldehyde metabolism by aldehyde dehydrogenase generates NADH and

acetate (Fig. 3.3A). To determine the potential role of NADH, we first evaluated the

effects of isopropanol. Isopropanol (0.2 %, v/v) increases the levels of NADH like

ethanol but generates acetone instead of acetaldehyde. To our surprise, isopropanol

also increased the HCV RNA level (Fig. 3.6B, left panel) (267). Both isopropanol and

ethanol increased NADH/NAD+ ratio in these cells, as expected (Fig. 3.6B, right

panel). In contrast, tert-butanol did not elevate HCV replication or the NADH/NAD+

ratio (Fig. 3.6B).

Moreover, we found that acetate itself increased the level of HCV RNA as

treating cells with acetone also did. In addition, ethanol, acetaldehyde, acetate,

isopropanol, and acetone all showed corresponding increases in NADH/NAD+ ratios

(Fig. 3.6B, right panel) (258,268). The NADH/NAD+ ratios were positively

correlated with HCV RNA content in all of these treatments (r = 0.948, p < 0.001)

56

(Fig. 3.6B). The suppression of HCV replication by cyanamide, 4MP and DADS in

Fig. 3.6A (left panel) was also associated with corresponding decreases in the

NADH/NAD+ ratios (Fig. 3.6A, right panel). Therefore, changes in HCV replication

paralleled the changes in the NADH/NAD+ ratio, produced by these treatments.

Then, we examined whether increased NADH/NAD+ ratio was required for

the potentiation of HCV replication by ethanol and these other agents. Pyruvate,

which re-oxidizes cytosolic NADH to NAD+, completely abrogated the increases in

HCV replication and NADH levels during ethanol, acetaldehyde, acetate, isopropanol,

and acetone treatments (Fig. 3.6C). Methylene blue, which also oxidizes NADH, had

similar effects on HCV as pyruvate (data not shown). In contrast, lactate, which

produces NADH in the cytosol independent of ethanol, increased NADH levels to

235.9 ± 11.9 % (P < 0.05) of the control level but had little to no effect on HCV

replication (Fig. 3.6D). Together, these data indicate that while an alteration of

cellular NADH/NAD+ levels seems necessary for the ethanol-induced increases in

HCV replication, elevated NADH/NAD+ may not be sufficient to increase HCV

replication.

Additionally, to confirm that these increases would likely occur in patients

with functional ADH, we transfected Huh7 cells with ADH I using Lipofectamine

(Invitrogen) (Fig. 3.7A), then transfected the cells with JFH1 RNA, and repeated the

ethanol, acetaldehyde, acetate, isopropanol, and acetone treatments. Ethanol increased

the level of HCV RNA which was abrogated by the ADH inhibitor, 4MP (Fig. 3.7B).

Ethanol and the other treatments elevated the HCV RNA levels in the ADH-

57

transfected cells an averge of 216 ± 6 % higher than respective treatments in PCDNA

3.1 vector-transfected control cells (Fig. 3.7C). This enhancement of the treatment

effects suggests that functional ADH in patients would only exacerbate the effects of

ethanol and its metabolites on HCV titer.

The potentiation of HCV replication by ethanol requires lipogenesis

NADH has diverse functions in the cell, and one of these functions includes

modulation of lipid metabolism. For example, NADH can inhibit mitochondrial -

oxidation and increase fatty acid synthesis (170). It is well-established that ethanol

modulates fatty acid metabolism in part through NADH, and that this plays an

important role in the development of steatosis in the alcoholic liver (170). Acetate and

acetone would generate acetyl-CoA, which also drives lipogenesis (170,268).

Furthermore, cholesterol metabolism and fatty acid biosynthesis are important in HCV

RNA replication (269). Lovastatin and fluvastatin, which are competitive inhibitors of

3-hydroxy-3-methyl-glutaryl-CoA reductase, and 5-(tetradecyloxy)-2-furoic acid

(TOFA) and cerulenin, which inhibits fatty acid biosynthesis, have been shown to

suppress the basal level of HCV replication (269,270). Therefore, we next examined

whether the potentiation of HCV RNA replication by above agents might be inhibited

by modulators of lipid metabolism.

Lovastatin, fluvastatin, TOFA, and cerulenin almost completely inhibited the

potentiation of HCV RNA replication by ethanol, acetaldehyde, isopropanol, acetone,

and acetate (Fig. 3.8A and 3.8B). In addition, inhibiting β-oxidation of fatty acids

58

with β-mercaptopropionic acid caused a 15.2 ± 1.7-fold (p<0.01) increase in HCV

replication in these cells (Fig. 3.8C). Furthermore, ethanol, acetaldehyde, acetone, and

acetate treatments increased the total intracellular cholesterol content, which was

attenuated by lovastatin (Fig. 3.8D). Lactate, which increased NADH/NAD+ without

increasing HCV replication, had no significant effect on cholesterol levels (Fig. 3.8D).

The data suggest that the elevation of HCV replication by ethanol, acetaldehyde,

acetone, and acetate is mediated by increases in intracellular cholesterol and can be

abrogated by the inhibition of cholesterol or fatty acid biosynthetic pathways.

DISCUSSION

High HCV titer is associated with the development and progression of liver

diseases (271). In addition, ethanol consumption, high BMI, and high viral titer are

strongly associated with poor response to anti-HCV therapy (272). Therefore, the

increased HCV replication we saw with physiological levels of ethanol and

acetaldehyde is likely to contribute to the pathogenesis and at least partly explain the

negative effects that ethanol has on interferon-α therapy. Ethanol has been shown to

suppress the antiviral function of interferon-α by interfering with the JAK-STAT

signaling pathway (151); however, this is not likely to explain the potentiation of HCV

replication we saw with ethanol because HCV effectively suppresses the type I

interferon response in Huh7 cells. Additionally, ethanol and acetaldehyde could

increase HCV replication in RIG-I-defective Huh7.5 cells (Fig. 3.5C; also, data not

shown) (254,273). Importantly, some ethanol treatments in this study were performed

59

while wrapping cell culture dishes with parafilm to decrease loss of ethanol due to

evaporation. However, we observed similar potentiation of HCV replication by

ethanol, with and without the parafilm. The use of the parafilm also did not induce

hypoxia as no significant change in the expression of hypoxia-inducible factor-1α

could be found (data not shown).

Previously, it has been suggested that some of the key ethanol metabolizing

enzymes might not be expressed in Huh7 cells (151). Indeed, we also found that

alcohol dehydrogenase I is decreased in our Huh7 cells compared to human liver.

However, CYP2E1 activity of our cells were within the normal range for human liver,

and CYP2E1 expression could be enhanced by ethanol (Fig. 3.3). In addition, ethanol

and acetaldehyde elevated the NADH/NAD+ ratio, indicating that ethanol is being

metabolized by our cells. Furthermore, transfection of ADH I into our cells

exacerbated the increase of HCV replication by ethanol and its metabolites, which

could be abrogated by an ADH inhibitor (Fig. 3.7), suggesting that functional ADH

leads to more dramatic elevation of HCV RNA levels. Also, note that even though our

cells do not have all of the normal ethanol metabolizing enzymes, our discovery that

acetaldehyde and acetate can enhance HCV replication is significant, as they bypass

these reactions.

Previous study by Zhang et al., using various chemical inhibitors of ethanol

metabolism, suggested that some downstream metabolites of ethanol were involved in

the potentiation of subgenomic HCV RNA replication by ethanol (197). Our data are

in agreement with this study and suggest that ethanol and acetaldehyde also directly

60

enhance HCV replication in the context of the complete viral replication cycle. In

terms of the mechanism, we found that isopropanol, acetone, and acetate also increase

HCV replication, and increased NADH/NAD+ ratio was required for the potentiation

of HCV replication by ethanol, acetaldehyde, as well as isopropanol, acetone, and

acetate. In contrast, t-butanol, a tertiary alcohol that is poorly metabolized by humans

and does not increase the NADH/NAD+ ratio, did not elevate HCV replication, as

predicted by our model (Fig. 3.6B). The NADH/NAD+ ratio in ethanol-treated cells

was decreased by cyanamide (Fig. 3.6A), suggesting that NADH is generated

downstream of acetaldehyde (Fig. 3.3A). Acetate, the downstream metabolite of

acetaldehyde, was previously considered inert but there is evidence that it can be

converted to acetyl-CoA and other metabolic intermediates by mammalian cells

(153,170). Isopropanol is known to be metabolized into acetone and possibly other

ketone bodies that can also be converted to acetyl-CoA (268). The mechanism by

which isopropanol increases the NADH/NAD+ ratio in our system is unclear and may

involve residual ADH or hitherto uncharacterized enzyme activity that is induced by

HCV.

In terms of how NADH increases HCV replication, NADH plays key roles in

cellular bioenergetics and can modulate fatty acid synthesis as well as suppress -

oxidation (153,170). We were interested in the potential involvement of lipids because

HCV replicates in cholesterol-rich compartments in the cell, and cholesterol and fatty

acid metabolism have been shown to be important for HCV replication (269).

Specifically, cholesterol metabolism increases basal HCV replication by the

61

geranylgeranylation of FBL2 (269). We found that inhibiting the host mevalonate

pathway with statins and fatty acid synthesis with TOFA or cerulenin blunted the

potentiation of HCV replication by ethanol, acetaldehyde, isopropanol, acetone, and

acetate, while inhibiting β-oxidation dramatically increased HCV replication (Fig.

3.8). In addition, the potentiation of HCV replication by these agents was

accompanied by an increase in the intracellular cholesterol content, which was

attenuated by lovastatin (Fig. 3.8D). Regarding potential effects of NADH on the

ATP, overall ATP levels were not significantly perturbed in these cells by ethanol or

other treatments (data not shown), suggesting that ATP is not likely to explain the

effects that ethanol had on HCV. In fact, ethanol also increased the rate of HCV

replication in the in vitro replication assay (Fig. 3.2C and 3.2D) which was performed

in the presence of excess ATP. Taken together, these data indicate that the

potentiation of HCV replication by ethanol, acetaldehyde, acetate, isopropanol, and

acetone ultimately requires host lipid metabolism and is sensitive to lipid modulators,

which points to potential targets for therapy. The concentrations of lovastatin and

fluvastatin used here are higher than the doses used clinically to treat

hypercholesterolemia. However, it is possible that statins, if used in combination with

antivirals or other lipid modulators, will help control HCV replication, particularly in

chronic alcoholics who show resistance to standard anti-HCV therapy (274). It is also

interesting to note that the concentrations of acetone that enhanced HCV replication in

this study are physiological levels that can be attained during metabolic dysfunction

such as diabetes and during starvation (268), and HCV infection can lead to insulin

62

resistance (275). In addition, acetate, which increased HCV replication at µM to mM

concentrations in this study (Fig. 3.6B and data not shown), is used in hemodialysis.

Interestingly, increasing the NADH/NAD+ ratio with lactate was not sufficient

to increase HCV replication, suggesting that other factors may also play a role (Fig.

3.8D). Lactate also did not increase the intracellular cholesterol level. These results

are consistent with an important role of cholesterol in the regulation of HCV

replication. The data also indicate that even though ethanol and lactate both increase

the NADH/NAD+ ratio, ethanol is more lipogenic than lactate in these cells. The

reason for these differences is unclear but it might be explained at least in part by the

fact that ethanol can inhibit citric acid cycle as well as gluconeogenesis, which may

cause acetate/acetyl CoA produced by ethanol metabolism to be shunted more towards

the lipogenic pathways, whereas these processes are likely to be stimulated by lactate

(276). Ethanol can also decrease the total oxidation of fatty acids to CO2,,and increase

the breakdown of glycogen which may further drive lipogenesis in these cells (276-

278). Further investigation into these effects will be beneficial to understanding how

different metabolic conditions would affect HCV replication in hepatocytes.

Recently, McCartney et al. reported an elevation of HCV RNA by ethanol in

Huh7 replicon cells, transfected with CYP2E1; the effect could be suppressed by

NAC, leading to the conclusion that the increase was due to ROS generation by

CYP2E1 (244). In contrast, we have consistently found that ROS suppresses HCV

replication while antioxidants tend to counter this suppression (208,209,214,260,261)

(Fig. 3.4). In particular, our BSO studies clearly demonstrate that endogenous ROS

63

are sufficient to suppress HCV replication in cell culture (208,209). Also, NAC and

vitamin E either enhanced or had no significant effect on the potentiation of HCV

replication by ethanol (Fig. 3.4G) as well as acetaldehyde, isopropanol, acetone,

acetate (data not shown). The reason for this discrepancy is unclear. However,

CYP2E1 generates acetaldehyde as well as ROS, both of which can react with thiols,

such as cysteine and GSH, which are generated from NAC, and the study by

McCartney et al. did not differentiate whether the potentiation of HCV replication by

ethanol was due to ROS, acetaldehyde, or other variables (170,244). NAC can also

have other effects on cells, including alteration of the pH and acting as a pro-oxidant,

and careful monitoring of the pH and comparison with other antioxidants and pro-

oxidants, therefore, are necessary. Indeed, our findings have been recently

corroborated by other studies that show that HCV RNA replication is enhanced by

antioxidants (e.g., vitamins E and C) and suppressed by lipid peroxidation products

and ROS (214,260,261,279). The mechanism by which ROS suppress HCV

replication is still not completely clear but it is likely to involve calcium and the

dissociation of HCV replication complex from the membranes (208,209). Detailed

understanding of the mechanism by which ROS suppress HCV replication and how

acetaldehyde, NADH, acetyl-CoA, and ROS affect HCV in vivo will require

additional in vitro and animal studies.

Therefore, we show that physiological levels of ethanol, acetaldehyde, and

acetone promote HCV replication in the context of the complete HCV replication, and

that the response is likely mediated by the modulation of host lipid metabolism

64

requiring elevated NADH/NAD+. Further study into the precise mechanisms of this

regulation may lead to the development of novel treatments that target both the virus

and its pathogenic interactions with ethanol in chronic hepatitis C patients.

65

Figure 3.1. Ethanol increases JFH1 replication. Huh7 cells transfected with JFH1 RNA were analyzed for intracellular HCV RNA by (A) qRT-PCR (n = 6) or (B) Northern blots (n = 4) and for HCV NS5A protein content by Western blot (n = 3) (B, bottom panel) after 48 hrs of ethanol treatments. (C) Extracellular HCV RNA levels were analyzed by qRT-PCR for 0.2 % ethanol treatments (n = 4). (D) Naïve Huh7 cells were inoculated with virus-containing medium and analyzed for HCV RNA after 48 hrs of 0.2% ethanol treatment (n = 4). (E) Ethanol concentrations used showed no significant toxicity measured by ATP content (n=3). *Indicates statistically significant difference for indicated sample sizes (P < 0.05).

66

Figure 3.2. Ethanol increases the replication of subgenomic JFH1 and Con1 replicon RNAs. (A) Huh7 cells transfected with SgJFH1-Luc RNA were analyzed for luciferase activity after 48 hr ethanol treatments (n = 3). (B) Stable Huh7 clones expressing SgCon1-Neo (SgPC2) were incubated with ethanol for 24 hrs and analyzed for HCV RNA, GAPDH mRNA, and NS5A and β-actin proteins (n=3) by Northern and Western blots, respectively (n = 3). (C-D) Cytosolic lysates were prepared from (C) JFH1 and JFH1-GND RNA-transfected cells and (D) SgPC2 cells, after 5 hrs of ethanol treatment, these lysates were used to carry out in vitro replication assays (n = 3). Bottom panels show ethidium bromide staining of rRNA as the loading control. *Indicates statistically significant difference for indicated sample sizes (P < 0.05).

67

Figure 3.3. CYP2E1 expression in Huh7 cells. (A) CYP2E1-dependent ethanol metabolism. (B) Human liver tissue, Huh7 cells transfected with 50 M non-targeting control or CYP2E1 siRNA, and skeletal muscle tissue were analyzed for CYP2E1 protein content by Western blot (n = 3). (C – D) Mock- or JFH1-transfected Huh7 cells were incubated with or without 0.2 % (v/v) ethanol for 48 hrs and analyzed for (C) CYP2E1 expression by Western blot (n=3) and (D) CYP2E1-dependent p-nitrophenol hydroxylation activity (n = 3). (E) SgPC2 cells were exposed to 0.2 % ethanol ± 25 M DADS for 24 hours or transfected with 50 nM control or CYP2E1 siRNA for 24 hours and then incubated with ethanol for 24 hrs and analyzed for HCV RNA by Northern blot (n = 3). *Indicates statistically significant difference for indicated sample sizes (P < 0.05).

68

F

igu

re 3

.4. E

nd

ogen

ous

and

exo

gen

ous

RO

S s

upp

ress

HC

V r

epli

cati

on.

JFH

1-tr

ansf

ecte

d H

uh7

cell

s w

ere

trea

ted

wit

h B

SO

wit

h an

d w

itho

ut 2

mM

GS

H o

r G

SH

est

er (

A &

B)

(n =

3),

GO

+ g

luco

se w

ith

and

wit

hout

16

hr p

re-t

reat

men

t wit

h 20

M

BS

O (

C)

(n =

4),

or

bolu

s H

2O2

(D)

(n =

4)

for

24 h

rs.

The

n, J

FH1

intr

acel

lula

r (A

, C, D

) an

d ex

trac

ellu

lar

(B)

HC

V R

NA

leve

ls w

ere

anal

yzed

by

qRT

-PC

R.

(E)

Huh

7 ce

lls

tran

sfec

ted

wit

h S

gJFH

1-L

uc R

NA

wer

e as

saye

d fo

r lu

cife

rase

act

ivit

y af

ter

24 h

r tr

eatm

ent w

ith

0.25

mU

/mL

glu

cose

oxi

dase

+ g

luco

se w

ith

and

wit

hout

the

BS

O p

retr

eatm

ent

(n =

3).

(F)

SgP

C2

cell

s w

ere

trea

ted

wit

h 0.

2 %

eth

anol

± H

2O2

plus

BS

O f

or 2

4 hr

s, a

nd a

naly

zed

for

HC

V R

NA

and

G

AP

DH

mR

NA

by

Nor

ther

n bl

ot.

(G)

SgP

C2

cell

s w

ere

trea

ted

for

24 h

rs w

ith

etha

nol ±

5 m

M N

AC

or

0.5

µM

Tro

lox

(wat

er-s

olub

le v

itam

in E

). T

hen,

HC

V R

NA

and

GA

PD

H m

RN

A le

vels

wer

e m

onito

red

by N

orth

ern

blot

and

qua

ntif

ied

by d

ensi

tom

etry

(n

= 3

). *

Indi

cate

s st

atis

tica

lly

sign

ific

ant d

iffe

renc

e fo

r in

dica

ted

sam

ple

size

s (P

< 0

.05)

.

69

Figure 3.5. Acetaldehyde increases intracellular HCV RNA. SgJFH1-Luc (A) and JFH1 RNA-transfected cells (B), Huh7.5 cells inoculated with JFH1 virions (C), SgPC2 and Clone B cells (D) were incubated with acetaldehyde for 24 hrs and analyzed for HCV RNA by Northern blot or qRT-PCR (n = 3). *Indicates statistically significant difference for indicated sample size (P < 0.05).

70

Fig

ure

3.6

. Rol

e of

NA

DH

/NA

D+

in t

he

pot

enti

atio

n o

f H

CV

rep

lica

tion

by

eth

anol

, ace

tald

ehyd

e, a

ceta

te,

isop

rop

anol

, an

d a

ceto

ne.

SgP

C2

cell

s, s

uppo

rtin

g C

on1

subg

enom

ic H

CV

RN

A r

epli

cati

on, w

ere

trea

ted

with

(A

) 0.

2%

etha

nol ±

0.1

mM

4M

P p

lus

25 µ

M D

AD

S or

0.1

mM

cya

nam

ide

(n =

3);

(B

) 0.

2 %

eth

anol

, 5 µ

M a

ceta

ldeh

yde,

5 µ

M

acet

ate,

0.2

% is

opro

pano

l, 2

mM

ace

tone

, or

25 m

M te

rt-b

utan

ol (

n =

4);

(C

) 0.

2 %

eth

anol

, 5 µ

M a

ceta

ldeh

yde,

5 µ

M

acet

ate,

0.2

% is

opro

pano

l, an

d 2

mM

ace

tone

, wit

h an

d w

itho

ut 5

mM

pyr

uvat

e (n

= 3

); o

r (D

) 0.

2% e

than

ol o

r 5

mM

lact

ate

for

3 hr

s fo

r N

AD

H/N

AD

+ r

atio

mea

sure

men

t or

24 h

rs f

or H

CV

RN

A le

vels

. H

CV

RN

A le

vels

wer

e m

onit

ored

by

Nor

ther

n bl

ot (

A-D

, lef

t pan

els)

. N

AD

H/N

AD

+ r

atio

s w

ere

mea

sure

d by

an

enzy

mat

ic N

AD

H r

ecyc

ling

ass

ay, a

s de

scri

bed

in E

xper

imen

tal P

roce

dure

s (A

-D, r

ight

pan

els)

. N

orth

ern

blot

s w

ere

quan

tifi

ed b

y de

nsit

omet

ry.

*Ind

icat

es s

tati

stic

ally

si

gnif

ican

t dif

fere

nce

for

indi

cate

d sa

mpl

e si

zes

(P <

0.0

5).

71

Co

ntr

ol

Eth

ano

l

Ace

tald

ehyd

e

Aceta

te

Iso

pro

pa

no

l

Aceto

ne

HC

V R

NA

(%

Co

ntr

ol)

0

100

200

300

400

500ADH-transfected Vector Control

a,b

a,ba,b

a,b a,b

a a a a a

Control Ethanol

HC

V R

NA

(%

Co

ntr

ol)

0

50

100

150

200

250

300

350

No Inhibitor4MP

a

(A) (B)

(C)

Hu

man

Liv

er

Vec

tor

Co

ntr

ol

AD

H-t

ran

sfec

ted

Figure 3.7. ADH enhances the elevation of HCV replication by ethanol, acetaldehyde, acetate, isopropanol, and acetone. (A) ADH expression in Huh7 cells transfected with ADH or PCDNA 3.1 vector control compared to human liver. These ADH-transfected and vector control cells were then transfected with JFH1 RNA, treated with (B) 0.2% ethanol ± 4MP or (C) 0.2% ethanol, 5 µM acetaldehyde, 5 µM acetate, 0.2% isopropanol, or 2 mM acetone. Intracellular HCV RNA was measured by qRT-PCR. Letter a indicates statistically significant difference from its respective control (P < 0.05). Letter b indicates a statistically significant different its respective vector control treatment (ethanol vector control, acetaldehyde vector control, acetate vector control, isopropanol vector control, and acetone vector control) (P < 0.05).

72

Figure 3.8. Role of lipogenesis in the enhancement of HCV replication by ethanol, acetaldehyde, acetate, isopropanol, and acetone. SgPC2 cells were treated for 24 hrs with (A-B) 0.2 % ethanol, 5 µM acetaldehyde, 0.2% isopropanol, 2 mM acetone, 5 µM acetate ± 30 min pretreatment with (A) 5 µM lovastatin, 5 µM fluvastatin, (B) 5 µg/ml TOFA, 5 µg/ml cerulenin, or with (C) 2 mM -mercaptopropionic acid (-MPA). Then, HCV RNA levels were monitored by Northern blot and quantified by densitometry (n = 3). (D) SgPC2 cells, treated for 24 hrs with ethanol, acetaldehyde, acetone, and acetate ± lovastatin, were monitored for cholesterol levels (n = 3). Lovastatin was activated, as described, before use (269). *Indicates statistically significant difference for indicated sample sizes (P < 0.05).

73

CHAPTER 4: CHEMICAL MUTAGENESIS OF HEPATITIS C VIRAL RNA BY ETHANOL AND REACTIVE SPECIES

74

ABSTRACT

HCV exhibits a high level of genetic variability and exists as quasispecies.

Alcohol use has been associated with increased sequence heterogeneity of HCV RNA

and decreased efficacy of antiviral therapy but how ethanol affects the HCV RNA

sequence has remained unclear. Ethanol metabolism generates genotoxins that are

likely to damage not only the host DNA but also RNA. HCV also induces

oxidative/nitrosative stress with oxidative DNA damage. Therefore, we investigated

whether ethanol and reactive species could act as mutagen to the viral RNA increasing

the mutation rate of HCV. Human hepatoma cells supporting a continuous replication

of genotype 1b HCV RNA (Con1, AJ242652) were exposed to ethanol, acetaldehyde,

hydrogen peroxide, or L-buthionine-S,R-sulfoximine (BSO). Then, the HCV NS5A

region was sequenced and compared with genotype 1b HCV sequences found in the

database. Ethanol and BSO elevated both nucleotide and amino acid substitution rates

of HCV RNA that were accompanied by oxidative RNA damage. Dipyridyl, a cell-

permeable iron chelator, and GSH ethyl ester decreased the RNA damage and reduced

the sequence heterogeneity of the HCV RNA. In addition, HCV (JFH1, AB047639)

itself induced oxidative RNA damage that was exacerbated by ethanol and BSO. The

overall dN/dS ratio and the percentage of sites undergoing positive selection increased

with ethanol and reactive species with pronounced changes in the

serine/threonine/tyrosine sites coded by the NS5A region.

Conclusions/Significance: Therefore, ethanol may synergize with virus-

induced oxidative/nitrosative stress to induce RNA damage, which is likely to amplify

75

the error rate of the HCV replicase in the development of quasispecies. Agents that

decrease the RNA damage may help reduce the mutation rate of HCV.

INTRODUCTION

RNA viruses are ubiquitous intracellular parasites with compacted genomes.

RNA viruses are characterized by rapid replication and a particularly high level of

genetic variability, existing as a population of closely related but distinct genetic

variants, referred to as quasispecies. High rate of mutation allows these viruses to

operate near the error threshold, for maximal adaptability to the changing environment

while conserving essential genetic information, and poses significant challenge to

antiviral therapy and the development of vaccines (235). The high mutation rate of

RNA viruses has been attributed to the lack of proofreading activity of viral replicases

among other factors although additional, previously unidentified mechanisms might

also play a role (280).

HCV is a positive-sense RNA virus of Flaviviridae family. The HCV genome

is about 9.6 kb in length and consists of the 5’ UTR, structural protein coding regions

(C, E1, E2), nonstructural (p7, NS2, NS3, NS4A/B, NS5A/B) protein regions, and 3’

UTR. HCV is estimated to infect about 170 million people worldwide, including at

least four million people in the U.S. alone. Current anti-HCV therapy, which

combines the use of pegylated interferon alpha and a nucleoside analog, ribavirin,

results in a sustained virological response in only about 50 - 60 % of patients

undergoing treatment (9,10,281-283). Both the efficacy of antiviral therapy and

76

success of re-treating the resistant population with antiviral compounds are strongly

affected by HCV sequence (283-287). Specific regions in the HCV genome have also

been identified that can affect the sensitivity of the virus to interferon, such as the

NS5A region (283,288-290). HCV, like other RNA viruses, also exists as a

quasispecies (234,291-294). Reports have also indicated a correlation between

drinking ethanol and increased heterogeneity of HCV RNA in patients, although the

underlying mechanisms are unknown (237,238).

Ethanol metabolism generates acetaldehyde which can react with the host

DNA (7). Ethanol also increases the generation of ROS and causes lipid peroxidation

while decreasing GSH and selenium through multiple mechanisms (68,111,152-164).

ROS and lipid peroxidation products can attack the bases and deoxyribose backbone

of DNA, which can result in single- and double-strand breaks plus other mutagenic

effects (295,296). RNS can also induce DNA damage by nitrosative deamination and

other modifications of bases and sugar residues (297). Similarly, HCV itself increases

oxidative/nitrosative stress which is exacerbated by ethanol, and GSH is depleted in

patients infected with HCV (22,28,30-36,38,65,66,73,80,82,83,298). Increased levels

of 8-OHdG, which can cause DNA mispairing, have been documented in patients as

well as in several in vitro models of HCV (31,92,241,295,298,299).

The presence of 8-OHdG in hepatitis C patients suggests similar damage is

occurring to cellular and viral RNAs. In fact, although chemical modification of

nucleic acids is most extensively studied in the context of the DNA damage in

chemical carcinogenesis, reactive species can chemically modify RNA as well as the

77

DNA (239,300-304). The RNA damage that ensues is less likely to be repaired than

the corresponding changes to the host DNA, which would be particularly important for

viruses that rely on RNA genomes. RNA damage is likely to amplify the error rate of

already error-prone viral replicases and increase the mutability of the viral RNA,

providing the virus with a greater genetic repertoire to adapt to changes in the

environment and develop resistance to therapy. In fact, alcohol, genotype 1, iron

overload, age, co-infection with HIV, steatosis, fibrosis, and male gender, all of which

are associated with increased ROS/RNS and/or decreased antioxidant capacity, have

each been linked with interferon resistance (115,148,241,289,305-309). Therefore,

one mechanism whereby ethanol promotes the heterogeneity of HCV RNA could

involve its genotoxic effects.

The goal of this study, therefore, is to test the effects of genotoxic substances

on HCV RNA. We hypothesize that ethanol and reactive species increase the

mutation rate of HCV, and that pharmacological agents targeting their genotoxic

effects can decrease the sequence heterogeneity of the viral RNA

(7,65,68,175,294,310). Using in vitro HCV cell culture systems that are well-

characterized, we hereby demonstrate that ethanol and reactive species increase

oxidative RNA damage and sequence heterogeneity of the HCV RNA genome. Both

the RNA damage and sequence heterogeneity of HCV RNA could be decreased by

agents that interfered with the genotoxic effects of ethanol and reactive species. In

addition, the percentage of amino acid sites with a dN/dS ratio >1 and overall dN/dS

ratios increased with ethanol and reactive species, including pronounced changes in

78

the serine/threonine/tyrosine residues coded by the HCV NS5A region, indicating

increased positive selection. Mutation trends and possible biological consequences of

these mutations are discussed.

RESULTS

Ethanol and reactive species increase the mutation frequency of HCV RNA

To test whether ethanol can affect the HCV RNA sequence, Con1 replicon

cells that support genotype 1b subgenomic HCV RNA replication (209,249) were

incubated with ethanol (0.5 % v/v), BSO (20 M), H2O2 (100 M), or acetaldehyde

(10 M) once daily for 48 hrs with a change of the cell culture medium at the time of

each treatment. Acetaldehyde and H2O2 are generated during hepatic metabolism of

ethanol. BSO, by inhibiting GSH biosynthesis, mimics GSH depletion seen in

hepatitis C patients that would amplify the effects of endogenous ROS and RNS

(30,306,311). Then, the RNA was collected, and the NS5A region corresponding to

nt. 4612 to 6013 of the Con1 replicon was reverse transcribed using AccuScript

reverse transcriptase and amplified with PfuUltra (Agilent). PfuUltra is a high fidelity

DNA polymerase with an error rate of 4.3 x 10-9; AccuScript reverse transcriptase has

an error rate of 1.61 x 10-7. Then, the cDNA products were cloned into pUC19 and

sequenced by the Sanger method. The T7 HCV RNA transcript, initially used to

transfect Huh7 human hepatoma cells to generate the replicon cells, was also

sequenced to assess the basal level of RNA heterogeneity potentially rising from the

heterogeneity of HCV DNA used in the in vitro transcription reaction as well as errors

79

generated during transcription and/or RT-PCR cloning (312-314). We focused on

NS5A because of its moderate level of diversity and its role in viral replication cycle

and interferon resistance (281,315-321). Sequences from our experiment and the

HCV database were aligned against the Con1(S1179I) and Con1(wt) sequences,

respectively, using BioEdit and MEGA 4.0.

After subtracting the T7 baseline values, the nucleotide substitution rate of the

control untreated cells was determined to be 2.88 x 10-4 nucleotide changes per site

(Table 4.1). Considering the cell passage number, this represents about 1.1 x 10-3

nucleotide substitutions per genome site per year. The ethanol group showed an

increased nucleotide substitution rate, with 1.71 x 10-3 nucleotide changes per site and

4.97 x 10-3 amino acid changes per codon, which represent about 6 and 18 fold

increases over the control, respectively (P < 0.001). BSO group also displayed a

significant increase in the sequence heterogeneity of HCV RNA, showing 1.20 x 10-3

nucleotide changes per site and 3.24 x 10-3 amino acid substitutions per codon, which

were about 4 and 12 fold higher than the control, respectively (P < 0.05). On the other

hand, H2O2 and acetaldehyde treatments did not increase the nucleotide substitution

rates significantly over the control (5.28 x 10-4 and 4.25 x 10-4 nt. change per site,

respectively, P > 0.05). The amino acid substitution rate did not increase with

acetaldehyde but increased by about 10 fold with H2O2. Therefore, ethanol and

reactive species could increase the nucleotide and amino acid substitution rates of

HCV RNA over 48 hr.

80

HCV tends to show more transitions (Ts) than transversions (Tv) (317,322).

Analysis of 1,212 genotype 1b sequences in the HCV sequence database

(http://hcv.lanl.gov/content/hcv-db/index) revealed that transitions account for over

83.5 % of all nt. substitutions (Table 4.2) (257). The Ts/Tv ratio of genotype 1b HCV

sequences in the database was determined to be 5.05 (Table 4.1). Likewise, transitions

comprised the majority of the nt. substitutions in both control and treatment groups

(Tables 4.1 and 4.2). Ethanol increased transitions more than transversions so that the

Ts/Tv ratio rose from 3.3 to 6.8 whereas it remained at 3.3 in the BSO group. The

most common nt. substitutions were U → C transitions in ethanol and BSO groups as

well as the database (Table 4.2).

Ethanol, reactive species, and oxidative RNA damage

The observation that GSH depletion with BSO had a greater effect on the HCV

RNA sequence than exogenous H2O2 or acetaldehyde suggested that there was a

substantial level of endogenous oxidative/nitrosative stress in these cells and that

endogenous ROS/RNS might have a greater effect on the HCV RNA than

acetaldehyde. Thus, we next examined whether the above increases in the sequence

variability of HCV RNA were associated with parallel increases in the oxidative RNA

damage. Huh7 cells were treated with ethanol or BSO for 48 hrs and analyzed for 8-

hydroxyguanosine (8-OHG) content (323). 8-OHG is the best-characterized and

abundant form of oxidatively modified RNA species (295,297,324). NiCl2 was

included in these assays as a positive control. NiCl2 induced a 43.7 ± 5.8 fold increase

81

in 8-OHG content over the control (P < 0.05) (Fig. 4.1A). In addition, ethanol

increased 8-OHG content by 32.8 ± 2.3 fold (P < 0.05). 8-OHG also increased

significantly, by 21.0 ± 2.0 fold in the BSO group (P < 0.05) (Fig. 4.1A). Therefore,

ethanol and reactive species could damage the RNA under the same conditions that

elevated the sequence heterogeneity of HCV RNA. Daily treatment of Con1 replicon

cells with 0.1 % ethanol (v/v) or 20 M BSO for 2 weeks likewise led to significant

increases in 8-OHG in these cells (Fig. 4.1B). Elevation of 8-OHG with H2O2 and

ethanol were confirmed by Northwestern blot analysis (data not shown) (323).

Effect of decreasing the RNA damage

Then, we examined whether these increases in 8-OHG could be decreased by

agents that decrease the level of ROS/RNS. HCV infection is associated with

significant iron overload (30). Therefore, we used an iron chelator to decrease the

RNA damage induced potentially by the Fenton reaction (48). '-Dipyridyl, a cell

permeable iron chelator, significantly decreased the level of 8-OHG in both ethanol

and BSO groups (Fig. 4.2A). We also used GSH ester to supply GSH exogenously.

GSH participates in detoxification reactions that remove reactive species such as H2O2

and acetaldehyde (170,325). GSH ester also effectively decreased the level of 8-OHG

in ethanol and BSO groups (Fig. 4.2A). Similarly, the RNA damage could be

decreased by pyridoxal isonicotinoyl hydrazone (PIH, another cell-permeable iron

chelator), 1,1,4,7,7-diethylenetriaminepentaacetic acid (DETAPAC, an iron chelator),

diphenylene iodonium (DPI, an inhibitor of flavoproteins such as NAD(P)H oxidases),

82

NG-methyl-L-arginine acetate (L-NMA, an inhibitor of nitric oxide synthase), and

diallyl disulfide (DADS; an inhibitor of CYP2E1) (Fig. 4.3).

Then, we tested whether dipyridyl and GSH ester could also reduce the

sequence variability of HCV RNA by treating control, ethanol, and BSO-treated cells

with or without dipyridal or GSH ester and sequencing the viral RNA. Dipyridyl

decreased the nt. substitution rates of both ethanol and BSO groups so that there was

no longer any significant increase over the control (Fig. 4.2B). In addition, dipyridyl

effectively abolished any increase in the amino acid substitution rate caused by BSO

and significantly attenuated the amino acid substitution rate in the ethanol group (P <

0.05) (Fig. 4.2C). Similarly, GSH ester decreased nt. and amino acid substitution rates

in both ethanol and BSO groups to levels not significantly above the control (Fig. 4.2B

and 3C). These data strongly suggest that the HCV RNA sequence is affected by

RNA genotoxins, and agents that decrease the RNA damage can attenuate the

mutagenic effects of ethanol and reactive species.

HCV induces oxidative RNA damage

HCV induces severe oxidative/nitrosative stress in the host by increasing the

generation of reactive species (69,73,298,326-328). Thus, JFH1 strain of genotype 2a

that supports complete replication cycle of HCV and produces infectious HCV in cell

culture was used to determine whether HCV was sufficient to induce oxidative RNA

damage even in the absence of ethanol and BSO. Huh7 cells were transfected with

JFH1 RNA and either exposed to 0.5 % ethanol (v/v) or 20 M BSO daily for 48 hrs,

83

or to 0.1% ethanol (v/v) or 20 M BSO with and without 5 M dipyridyl or 1 mM

GSH ester daily with a change of medium prior to each treatment for 2 weeks. As

shown in Fig. 4.4, HCV by itself significantly increased 8-OHG with 48 hr and 2 week

treatments, both of which were exacerbated by ethanol and BSO. These data,

together with the literature describing oxidative DNA damage in various cell culture

models of HCV and in patients, suggest that, in addition to DNA damage, oxidative

RNA damage occurs as a usual consequence of HCV infection, even in the absence of

ethanol and other sources of genotoxins.

Sequence analysis of NS5A region suggests increased positive selection

To further examine whether HCV sequences were also subject to positive

selection, we computed the dn/ds ratio, using SNAP (329). The dN/dS ratio of our

control group was 0.31 but it increased to 1.05 and 0.72 in the ethanol and BSO

groups, respectively (Table 4.1). Percentage of sites showing positive selection in our

study increased from 4.25 % in control group to 12.3 % in ethanol and 12.3 % in BSO

groups (Fig. 4.5A). These results indicated that ethanol and BSO also increased

positive selection within 48 hrs. Average comparisons of 1,212 genotype 1b

sequences in the database revealed that 19.5 % of the NS5A-coding region

experiences positive selection while about 79 % shows purifying selection. In

addition, 96.5 % of nonsynonymous mutations, compared to only 24.6 % of

synonymous mutations, occurred at sites that underwent positive selection in our

84

study, suggesting that most of the nonsynonymous mutation in our study would lead to

a functional change.

Next, we evaluated the effect of dipyridal and GSH ester on the dn/ds ratio.

Dipyridal and GSH ester co-treatments reduced the dn/ds ratio in the ethanol group

from 1.05 to 0.46 and 0.43, respectively. Dipyridal and GSH ester also decreased the

dn/ds ratio in the BSO group from 0.72 to 0.46 and 0.21, respectively. Dipyridal did

not affect the dn/ds ratio of the control group whereas GSH ester decreased it slightly

from 0.31 to 0.24. The percentage of sites undergoing positive selection decreased

with dipyridal and GSH ester co-treatments (Fig. 4.5A).

To determine whether the mutations clustered at any specific loci, cumulative

synonymous and non-synonymous amino acid mutations were plotted against codons

using SNAP (Fig. 4.5B). Vertical steps represent either synonymous or

nonsynonymous mutations occurring at that codon. Mutations in the control group

tended to occur throughout the NS5A sequence. In contrast, synonymous and

nonsynonymous mutations clustered in the C-terminal half of NS5A in ethanol and

BSO groups (Fig. 4.5B). H2O2 and acetaldehyde groups showed similar clustering of

mutations as ethanol and BSO groups (data not shown). Sites undergoing positive

selection in the control, ethanol, and BSO groups with and without dipyridyl and GSH

ester, as well as the database are mapped in Fig. 4.6. In this study, 36.4 % of the sites

in the ethanol group and 36.4 % of the sites in the BSO group that underwent positive

selection corresponded to sites in the database that underwent positive selection,

85

compared to 21.1 % of the sites in the control group. Additionally, 55.2 % of all the

sites in the database that underwent positive selection showed up in our study.

The C-terminal half of NS5A contains basal phosphorylation sites (Fig. 4.7A).

Interestingly, we found that 50.3 % of all nonsynonymous mutations either added or

removed serine, threonine, or tyrosine residues in the treatment groups, versus only

9.5 % in the control group (P < 0.05). Mutations that either generated or removed

serines accounted for 26.3 % of total amino acid substitutions in the treatment groups,

compared to 0 % in the control group (P < 0.05). Mutations that either generated or

removed threonines accounted for 21.0 % of total amino acid substitutions in the

treatment groups versus 4.8 % in the control group (P < 0.05). Mutations involving

tyrosine occurred less frequently, representing only 3.1 % of all amino acid changes.

The most frequent changes from serine were to proline, constituting 59 % of all amino

acid changes from serine in our study, while most frequent changes to serine were

from phenylalanine, comprising 29 % of all amino acid substitutions to serine.

Interestingly, the majority of mutations involving serine in the domain 3 of NS5A

removed rather than generated serine, whereas mutations that generated serine were

found more frequently in domains 1 and 2 (Fig. 4.7A and 4.7B). Mutations that

involved threonine or tyrosine did not show such obvious clustering except certain

mutations occurred more frequently at specific sites (Fig. 4.7C) (Table 4.3, also see

below). In comparison, mutations that involve serine, threonine, and tyrosine

represented 40.1 % of all genotype 1b NS5A sequence variations in the database (Fig.

4.8). Of particular interest were F127S, T122A, Y321C, N69T, S383P, S401P,

86

S408P, and T379A, which occurred frequently in our study; these substitutions were

also found in naturally occurring genotype 1b HCV sequences in the database (Table

4.3). In particular, 91.8 % (79 out of 86) of all amino acid changes at codon 127 in the

database involved F to S substitutions, and 78.2 % (79 out of 101) of all F to S

substitutions occurred at codon 127 (Table 4.3). All of the sites at which the

aforementioned mutations occurred underwent positive selection during treatment in

our study. Furthermore, positive selection also occurred at T122, F127, T379, and

S401 in the database. A comprehensive list of all amino acid variations compiled

from 1,212 genotype 1b HCV sequences in the database is listed by position and by

the type of amino acid substitutions in Table 4.4.

DISCUSSION

Virus infections are associated with perturbation of the host redox status and

increased generation of ROS/RNS (30,330-334). There is also increasing evidence

that reactive species increases the mutability and/or virulence of RNA viruses (335-

339). Mechanisms are thought to include modulation of the host immune system as

well as modification of the viral sequences. The role of UV radiation and chemical

carcinogens in DNA damage and carcinogenesis is well-established. Nevertheless,

how such genotoxins affect the RNA genome and mutation rates of viruses is poorly

understood. In this study, we use HCV, an important hepatotropic human pathogen,

plus ethanol, which is consumed in relatively high quantities and metabolized to

generate genotoxic substances in the liver, to examine how genotoxins affect the

87

genetic variability of viral RNA. Ethanol and reactive species directly increased the

sequence heterogeneity of HCV RNA in the context of hepatocytes alone, in the

absence of the immune cells, within 48 hrs. Increased nt. and amino acid substitution

rates of HCV RNA were associated with oxidative RNA damage. Iron chelators, GSH

ester, and inhibitors of ROS/RNS generating enzymes decreased this damage,

suggesting a role of reactive species in the RNA damage triggered by ethanol and

BSO. Most of all, the same agents could decrease the nt. and amino acid substitutions

induced by ethanol and BSO. These findings raise an important possibility that

endogenous and exogenous genotoxins affect the mutation rate of viruses by

chemically modifying the viral RNA, amplifying the error rate of an already error-

prone viral replicase. Our findings also suggest a role of human behavior in the

evolution of viruses. To our knowledge, this is the first study pointing to a diverse yet

targeted biochemical approach to attenuate the mutation rate of a virus. It will be

important to determine whether iron chelators, GSH ester, and other inhibitors of

reactive species can decrease the mutation rate of HCV in vitro and in vivo, to enhance

the efficacy of the existing antiviral therapy and facilitate the development of future

antivirals and vaccines. As reactive species can modify RNAs as well as DNAs in

general, rather than HCV RNA specifically, and oxidative/nitrosative stress is

documented in many different virus infections (332,340,341), findings of this study

may apply to other viruses and their interactions with the physicochemical

environment affecting viral evolution.

88

During alcohol intake, alcohol concentration in the blood can exceed

concentrations of 100 mM (342,343). The 0.5 % (v/v) ethanol we used is equivalent

to 85.6 mM and lies in the toxic range but can be achieved physiologically,

particularly in chronic alcohol users. In addition, ethanol is volatile and the

concentration that remains will be significantly less than what is added to cell culture

medium (262). The 0.1 % ethanol (v/v), which also inflicted RNA damage with

longer exposure, is equivalent to blood alcohol concentration of 17.2 mM, which is

approximately the legal limit for driving under the influence in many countries. Note

that our cells express CYP2E1 and, therefore, can metabolize ethanol (Fig. 3.3) (344).

In addition, acetaldehyde levels used in our study are physiologically attained

concentrations (343). As mentioned, GSH is suppressed in hepatitis C patients but

GSH is elevated by infectious HCV in cell culture (30,258,306,326). The reason for

this discrepancy is unclear and can involve a number of factors including the status of

p53 and extent of oxidative stress, potentially exceeding the host antioxidant capacity

in vivo (345). To mimic GSH depletion seen in vivo in our in vitro cell culture system,

we used BSO, which inhibits GSH de novo synthesis. The oxidative RNA damage

and increased sequence heterogeneity of HCV RNA seen in the replicon cells treated

with BSO (Fig. 4.1), therefore, suggests that HCV RNA sequence is likely to be

affected by the genotoxic effects of reactive species in vivo, in patients. Moreover,

infectious JFH1 induced RNA damage even in the absence of ethanol or BSO, and this

was exacerbated by co-incubation with ethanol or BSO (Fig. 4.4). These data suggest

that the proposed genotoxic effects are a usual consequence of HCV infection and that

89

HCV is capable of increasing the generation of reactive species in the infected cell,

which would be amplified by GSH depletion occurring in vivo in patients and by

exogenous sources of genotoxins such as ethanol. Possible endogenous sources of

genotoxins activated by HCV include the mitochondria, hepatocyte NAD(P)H

oxidases, inducible nitric oxidase synthase, and CYP2E1, most of which can be

targeted pharmacologically (69,73,298,326-328,344). Indeed, it may be speculated

that increased mutability would be one of the major consequences of chronic

oxidative/nitrosative stress induced by the virus in an infected cell. Importantly, the

nucleotide substitution rates of HCV RNA in our study are close to those observed in

patients (i.e., control value is about half of in vivo nt. substitution rate while ethanol

and BSO groups have two to three times this value) (346). Differences found might

be attributed to variable exposure to exogenous genotoxins, diurnal/hormonal

fluctuation of GSH as well as fluctuations in the level of oxidative/nitrosative stress

with viral titer and stage of liver disease in vivo.

Oxidative modification of the RNA, like corresponding lesions on the DNA,

can be mutagenic, for example, causing mispairing of the bases (347,348). The role of

such mutagenic events, which would be expected to be more or less random, occurring

at any exposed regions of the viral RNA, is supported by increases in transition

mutations. Indeed, increased occurrence of oxidative chemical modification of bases

such as 8-OHG, would be expected to increase the rate of mutations. However,

reactive species can also modify amino acids. In addition, although errors generated

by the HCV viral polymerase is more likely to create transition mutations, similar to

90

that of poliovirus, oxidative/nitrosative stress can induce either transitions or

transversions depending on the types of reactive species and polymerase involved

(295,297,349-351). Thus, the possibility of reactive substances chemically modifying

the viral polymerase itself, leading to amplification of its error rate, has not been ruled

out, and the relative contribution of the oxidative modification of the RNA versus

polymerase remains to be determined. Differentiating these possibilities will require

the development of in vitro assays where specific components (e.g., replicase vs. viral

RNA template) are oxidatively modified without affecting other components in the

assay, and without completely destroying the function of the replicase (202,352,353).

Ability to selectively sequence nascent viral transcripts versus the oxidized viral RNA

precursor, without affecting the natural base pairing of the RNAs which would lead to

sequencing artifacts, will also be necessary (236,354,355). Furthermore, the dN/dS

increased with ethanol and BSO treatments in our study, indicating there is also

positive selection. Relative contribution of actual mutations versus selection remains

to be determined, though our data suggests that positive selection plays an important

role, since 55.2 % of sites that undergo positive selection in the database were also

found to undergo positive selection in our study.

In terms of biological effects of the mutations, resulting increases in the

sequence heterogeneity of viral RNA genomes is likely to facilitate antiviral resistance

and immune escape of the virus. Indeed, alcohol is a major cofactor in HCV-induced

pathogenesis. Alcohol consumption decreases the efficacy of the antiviral therapy

(115,148,305) while antioxidants generally improve the outcome of anti-HCV therapy

91

(356). Whether the mutations induced by ethanol affect the virulence of HCV remains

to be tested. Interestingly, alcohol is also suggested to affect the viral replication, and

previously we showed that alcohol and acetaldehyde potentiated HCV RNA

replication through lipid metabolism and by elevating NADH/NAD+ ratio in the cell

(197,344). NS5A has diverse functions ranging from participation in viral RNA

replication to alteration of cell metabolism (315,357-359). Phosphorylation of NS5A

can alter the replication fitness of HCV and act as a molecular switch that regulates

viral RNA genome replication, translation, and secretion of virion. Increased

detection of NS5A sequences with modified serine, threonine, and tyrosine residues in

our study suggests that ethanol and reactive species altered cell signaling and

mutations and in turn altered the ability of NS5A to be phosphorylated. Increased

removal of serine residues within the basal phosphorylation region of NS5A in ethanol

and BSO groups further suggests that the basal phosphorylation of NS5A has a

significant effect on HCV so that the removal of these sites allowed those sequences to

persist in the cell. In fact, ethanol and reactive species, in addition to damaging

cellular macromolecules, can modulate cell signaling (90,360). In a recent study,

CKII-induced basal phosphorylation of NS5A decreased the activity of NS5B and its

association with viral RNA templates (249,315,361-364). On the other hand,

previously, it was reported that removing NS5A basal phosphorylation sites besides

S457 of genotype 2a JFH1 had no effect on HCV replication (321,365,366). These

studies were conducted in the absence of an inducer of phosphorylation, however, and

whether ethanol and reactive species alter the phosphorylation state of NS5A at any

92

specific regions and how the observed mutations in turn affect HCV replication

remain to be tested (321,367-369). We also found other nonsynonymous amino acid

substitutions in various structural and functional domains of NS5A, including the

double-stranded RNA-activated interferon induced protein kinase (PKR) and the

putative interferon sensitivity determining regions (ISDR); however, no single

sequence had more than two amino acid substitutions within the ISDR. S437

(equivalent to S457 of JFH1), hyperphosphorylation sites, and zinc coordination motif

were not changed (209,249,321).

Therefore, in this study, we showed that ethanol and oxidants could increase

oxidative RNA damage and increase the mutation frequency of HCV in hepatocytes in

the absence of other components of the host immune system. Oxidative RNA damage

could also be decreased by chemical interventions. Our findings suggest another

potential key mechanism of adverse interaction between ethanol and HCV and another

important consequence of chronic oxidative/nitrosative stress induced by HCV other

than pathogenesis (258). The study also raises a possibility that, by understanding the

nature of these chemical-biological interactions, it may be possible to attenuate the

mutation rate of HCV.

93

Table 4.1. Nucleotide and amino acid substitution rates in control and treatment groups a

Treatment Group

n b Nt. change per sitec

Fold increase

Amino acid change per sited

Fold increase

Ts/Tv dN/dS

Control 21 2.88 x 10-4 - 2.80 x 10-4 - 3.3 0.31 Ethanol 21 1.71 x 10-3

(P< 0.001)e 5.9 4.97 x 10-3

(P< 0.001)e

17.8 6.8 1.05

BSO 28 1.20 x 10-3

(P< 0.001)e 4.2 3.24 x 10-3

(P< 0.001)e

11.6 3.3 0.72

a Nucleotide and amino acid sequences were compared against Con1 sequence used in the study.

b Number of sequences analyzed in each group c,d Data were normalized by the T7 baseline values which were 1.27 x 10-3 nt. change

per site and 1.96 x 10-3 amino acid change per site. e Statistically significant difference from control Table 4.2. Nucleotide substitution trends in control and treatment groupsa

Nt. substitution Control Ethanol BSO Database b U→C 3.57 13.67 10.20 7.86A→G 3.57 11.89 8.43 6.54C→U 5.35 8.32 3.10 5.86G→A 2.97 5.95 5.77 7.57A→C 1.19 0.59 0.44 0.51A→U 0.59 1.19 0.89 0.66G→C 0 2.97 1.33 1.32G→U 0 1.19 1.33 0.69U→A 0 0 0 1.00U→G 0.59 0 0 0.15C→A 1.19 0 1.33 0.58C→G 0.59 0 1.77 0.59

a Data represent relative frequency of each type of nucleotide substitution. Transition mutations are shown in the first four rows.

b Relative frequency of each type of nucleotide substitution in the genotype 1b HCV sequences in the database.

94

Figure 4.1. Ethanol, reactive species, and oxidative RNA damage. (A) Con1 replicon cells were incubated with 0.5 % (v/v) ethanol, 20 M BSO, or 250 M NiCl2 daily for 48 hrs. Then, samples were analyzed for 8-OHG by ELISA using 8-OHG as standard. Data were expressed as fold increase from the control (0.122 ± 0.027 ng/ml). (B) Con1 replicon cells were incubated with 0.1 % (v/v) ethanol, 20 M BSO, or 50 M NiCl2 daily for 2 weeks and analyzed for 8-OHG by ELISA. Data were expressed as fold increase from the control (0.418 ± 0.109 ng/ml). * Indicates statistically significant difference from the control (P < 0.05; n = 4 in panel A, n = 6 in panel B).

Treatment

Control Ethanol BSO

8-O

HG

(F

old

Incre

ase

)

0

10

20

30

40

50

60

NiCl2

*

*

*

Treatment

Control Ethanol BSO

8-O

HG

(F

old

Incr

ea

se)

0

2

4

6

8

10

12

14

NiCl2

**

*

(A) (B)

95

Figure 4.2. Effects of dipyridyl and GSH ester on the oxidative RNA damage and HCV RNA sequence. Con1 replicon cells were incubated with 0.5 % ethanol or 20 M BSO daily for 48 hr with and without 10 M dipyridyl or 2 mM GSH ester. Then, the samples were analyzed for 8-OHG by ELISA using 8-OHG as a standard (A) or sequenced and analyzed for nt. (B) and amino acid (C) substitution rates. Data were expressed as fold increase from the control where the control value in (A) was 0.122 ± 0.027 ng/ml (n = 6 per group). Control values in (B) and (C) were 2.88 ± 0.77 x 10-4 nt. changes per site and 2.80 ± 0.61 x 10-4 amino acid changes per site, respectively. Letter a indicates statistically significant difference from control (no inhibitor, P < 0.05). Letters b and c represent statistically significant change from ethanol (no inhibitor) and BSO (no inhibitor), respectively (P < 0.05).

Treatment

No Inhibitor Dipyridyl GSH Ester

Am

ino

Aci

d S

ub

stit

uti

on

Rat

e(F

old

Incr

ease

)

0

5

10

15

20ControlEthanolBSO

a

a

a,ba,ba,c a,c

Treatment


Nt.

Su

bst

itu

tio

n R

ate

(Fo

ld In

crea

se)

0

2

4

6

8ControlEthanolBSO

a

a

Treatment


8-O

HG

(F

old

Incr

ease

)

0

10

20

30

40ControlEthanolBSO

a

a

a,b

a,ba,c

a,c

(A)

(C)(B)

96

Figure 4.3. Inhibiting the RNA damage. Con1 replicon cells were incubated with 0.5 % ethanol or 20 M BSO daily for 48 hr with and without 0.1 mM DETAPAC, 5 M PIH, 1 mM L-NMA, 40 M DPI, or 25 M DADS. Then, the samples were analyzed for 8-OHG by ELISA, as described. Data were expressed as fold increase from the control where the control value was 0.076 ± 0.026 ng/ml. * Denotes statistically significant difference from the control (P < 0.05; n = 4).

Figure 4.4. Oxidative RNA damage by HCV. Huh7 cells were transfected with JFH1 RNA and incubated with (A) 0.5 % (v/v) ethanol or 20 M BSO daily for 48 hrs, or (B) 0.1% (v/v) ethanol or 20 M BSO with and without 5 M dipyridyl or 1 mM GSH ester daily for 2 weeks. Then, the samples were analyzed for 8-OHG by ELISA using 8-OHG as standard. Data were expressed as fold increase from the control where the control value was 0.106 ± 0.012 ng/ml and 0.291 ± 0.054 ng/ml for the 48 hr and 2 week time points, respectively. Letters a and b indicate statistically significant difference from the –HCV control and +HCV control, respectively, and letter c indicates statistically significant difference from the respective no inhibitor controls (control no inhibitor, ethanol no inhibitor, and BSO no inhibitor) (P < 0.05; n = 4).

Control

Ethanol

BSOControl

Ethanol

BSO

8-O

HG

(F

old

Incr

eas

e)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5No InhibitorDipyridylGSH Ester

- HCV + HCV

aa a

a,b

a,b

a,c

c c c c ccc c

c

Control

Ethanol

BSOControl

Ethanol

BSO

8-O

HG

(F

old

Incr

ease

)

0

2

4

6

8

10

- HCV + HCV

aa

a

a,b

a,b

(A) (B)

Treatment

8-O

HG

(F

old

Incr

ease

)

0

10

20

30

40

50

ControlEthanolBSO

No Inhibitor

DETAPAC

PIH Dipyridal

L-NMAGSH ester

DPIDADS

*

*

*

* *

*

**

**

**

*

**

*

97

Codon0 50 100 150 200 250 300 350 400

Re

lati

ve C

ha

ng

es

0

1

2

3

4

5

6

7

SynonymousNonsynonymous

Ethanol

Treatment

Sit

es U

nd

erg

oin

g

Po

siti

ve S

ele

ctio

n (

%)

0

2

4

6

8

10

12

14ControlEthanolBSO

No Inhibitor Dipyridyl GSH ester

a a

b

a,b a,ba,b

b b

Codon0 50 100 150 200 250 300 350 400

Rel

ativ

e C

han

ges

0

1

2

3

4

5

6

7


BSO

Codon0 50 100 150 200 250 300 350 400

Re

lati

ve C

han

ges

0

1

2

3

4

5

6

7


Control

(A)

(C) (D)

(B)

Figure 4.5. Synonymous and nonsynonymous substitutions in the NS5A coding region. (A) Percentage of sites undergoing positive selection (dN/dS ratio > 1) in control, ethanol, and BSO groups with and without dipyridyl or GSH ester. Letter a indicates statistically significant difference from control (no inhibitor) and letter b indicates statistically significant difference from respective no inhibitor controls (control no inhibitor, ethanol no inhibitor, and BSO no inhibitor) (P < 0.05). Cumulative synonymous and non-synonymous mutations in (B) control, (C) ethanol, and (D) BSO groups were plotted against codons via SNAP. Numbers represent NS5A codon number.

98

Codon

0 100 200 300 400

Codon

0 100 200 300 400

Codon

0 100 200 300 400

Codon

0 100 200 300 400

Codon

0 100 200 300 400

Codon

0 100 200 300 400

Codon

0 100 200 300 400

Codon

0 100 200 300 400

Codon

0 100 200 300 400

Codon

0 100 200 300 400

No Inhibitor

Dipyridyl

GSH Ester

Control Ethanol BSO

Database

Figure 4.6. Sites undergoing positive selection. Location of sites undergoing positive selection in control, ethanol, and BSO groups with and without dipyridyl or GSH ester, and the HCV database. Grey lines indicate sites undergoing positive selection that match between this study and the database. Black lines indicate sites undergoing positive selection that do not match between this study and the database.

99

Codons

0 50 100 150 200 250 300 350 400 450

Am

ino

Aci

d C

hang

e (F

requ

ency

)

0

1

2

3

4

5

6

7

From Thr To Thr From TyrTo Tyr

Codons

0 50 100 150 200 250 300 350 400 450

Am

ino

Aci

d C

hang

e

(Fre

quen

cy)

0

1

2

3

4

5

6

7

From Ser To Ser

(A)

(B) (C)

-helix

1 5 25 237 276 447

Hyperphosphorylation(S225, S229, S232, S235)

ISDR

ConservedPhosphorylation

(S222)

Domain I (1 – 213) Domain II (240 – 342) Domain III (356 – 447)

Basal phosphorylation Basal phosphorylation(2200-2250) (2350 – 2419)

NS4A interaction NLS(354-362)

Zinc-coordination

NS5B interaction2’,5’-OAS binding(1-148)

Figure 4.7. Mutations involving serine, threonine, and tyrosine by position. (A) Structural and functional domains of HCV NS5A. Numbers represent Con1 NS5A codon number. The figure is adapted from (315). (B) Location of amino acid substitutions involving serines. (C) Location of amino acid substitutions involving threonines and tyrosines. Putative basal phosphorylation regions are labeled.

100

Table 4.3. Frequent mutations involving serine/threonine/tyrosine residues in the NS5A region

Mutation Study counta

Database countb

Accession Numbers c Reference d Type of

substitutions, database e

F127S 5 79 AB285036, AF207760, AF265148-50, AF265163-65, AM402055-58, AM402060-78, AM402080-82, AM402084-90, AM402092-110, AM402112-116, AM402118-122, AM402125, AM402127-129, AY808022, DQ491974, DQ492015, EF407482, EF407488, EU482883

(370-377) F127S (79) F127C (1) F127L (4) F127Y (2)

T122A 4 4 DQ491961, DQ491964, DQ491966, DQ492018

(377) T122A (4) T122K (3) T122R (37) T122V (91) T122W (2)

Y321C 4 3 AM401892, AM401896, AM401974

(370) Y321C (3) Y321D (3) Y321N (1)

N69T 3 16 AJ507172, AM402271, DQ491995-97, DQ491999, DQ492000-07, EF407495, HM042050

(370,371,377-379)

N69T (16) N69A (1) N69K (1) N69S (3)

S383P 3 10 AF165061, AF207769, AM401885, AM493449, DQ244140, EF407461, EF407470, EF407482, EU862837, FJ958916

(370,371,373,374,380-383)

S383P (10) S383F (2)

S401P 3 39 AB285042, AF165061-62, AF265158, AF483269, AJ000009, AJ507156, AJ507169, AJ507182, AM493489-507, AY008261, AY808015, AY808026, AY808030-31, AY808043, DQ492060, DQ492152, EU155281, EU256062, EU256080

(372,373,375-377,379,381,382,384-386)

S401P (39) S401A (2) S401F (6) S401L (39) S401T (1)

S408P

3 3 AM401886, AM402364, AM402374

(370) S408P (3) S408F (1) S408T (5) S408Y (1)

T379A 2 64 AB049092, AB285040, AF165045-46, AF207755, AM402055, AM402060,

(370-374,378,381,387,388)

T379A (64) T379S (7)

101

AM402066, AM402070-71, AM402074-75, AM402081, AM402088, AM402090, AM402096, AM402098, AM402101-102, AM402106, AM402108, AM402111-112, AM402115, AM402125, AM402127, AY460204, EF407470, EF407477, EU155218, EU155381, EU862837, EU781832, HM042053

a Frequency (count) of substitutions occurring in the treatment groups. b Frequency (count) of substitutions in the genotype 1b HCV sequences in the

database. c Accession numbers for HCV sequences in column b. d References for HCV sequences in columns b and c. e Type of amino acid substitutions occurring at that position in the genotype 1b HCV

database and frequencies (count), shown in parenthesis.

102

Codon0 100 200 300 400

Nu

mb

er o

f m

uta

tio

ns

1

10

100

1000

Codon0 100 200 300 400

Nu

mb

er o

f m

uta

tio

ns

1

10

100

1000

Codon0 100 200 300 400

Nu

mb

er o

f m

uta

tio

ns

1

10

100

1000

Codon0 100 200 300 400

Nu

mb

er o

f m

uta

tio

ns

1

10

100

1000

Codon0 100 200 300 400

Nu

mb

er o

f m

uta

tio

ns

1

10

100

1000

Codon0 100 200 300 400

Nu

mb

er o

f m

uta

tio

ns

1

10

100

1000

From To

Serine

Threonine

Tyrosine

Figure 4.8. Amino acid variations by position in the genotype 1b NS5A sequences from database. Location of amino acid substitutions involving serines, threonines, and tyrosines.

103

Table 4.4. Amino acid variations in the HCV database

Pos Codon from

Codon to

AA from

AA to

Accession #’s

1 tcc gcc S A AJ133096, EF407470, EF407500, EU155300, EU482833

1 tcc ccc S P AM401884, AM401918, AM402082, AM402309

2 ggc gac G D AJ507158, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402325, AM402326, AM402327, AM402328, EU155263

2 ggc gat G D EU862837

2 ggc aac G N AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402324, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, EU155368

2 ggc agc G S AJ507162, EF407486, EU155318, HM042058, M96362, U16362

3 tcg acg S T AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, EU155376

3 tcg aca S T EU155260, EU155306

5 cta cca L P AM401862

6 aga aag R K AB010249, AB285036, AB285037, AB285054, AB285055, AF034151, AF265163, AJ507163, AM402302, AY240968, AY240969, D11168, D90208, EU155281, FJ958928

6 aga tgg R W AF265161

7 gat gaa D E AF165061, EU155318

7 gat gag D E M96362, U16362

7 gat ggc D G AM402350, AM493498

8 gtt gct V A DQ491949, FJ958920

8 gtt ata V I AB285052

8 gtt atc V I AF483269, AJ507156, AM402286, AY808021, EU155370, EU256089, GU451224

8 gtt att V I AB080299, AB285067, AF165053, AF165054, AJ507172, AM494937, AY460204, AY808014, AY808033, D10934, D85516, DQ492093, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF638081, EU255960, EU255961, EU781830, FN435993, GU451221, GU451222, GU451223, HM042059, M96362, U16362

9 tgg agg W R AM402291

9 tgg cgg W R DQ492021, DQ492023, DQ492078, DQ492088, DQ492173

10 gat gaa D E AJ507163, AJ507182, EU256090, EU482833, GQ913871, GU451224

10 gat ggc D G AM401943, AM402115, DQ492095

10 gat tac D Y AB285048

11 tgg tga W * DQ492151

11 tgg cgg W R AM402080

104

11 tgg agg W R DQ492116

12 ata atg I M AM493444

12 ata aca I T DQ492000

12 ata gta I V AF207761, DQ491944, DQ492141, DQ492180, DQ492194, DQ492195, EU155224

12 ata gtg I V AB285054, AB285055, AM402329, AM402330, AM402331, AM402332, AM402333, AM402334, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, EU256102

13 tgc cgc C R DQ491968

13 tgc tac C Y AM402086

14 acg gcg T A AB049092, AF207761, AM402265, AM402356, DQ491989, DQ492067, DQ492141, DQ492152

14 acg att T I AY808022

14 acg atg T M AB154195, AB154196, AB285078, DQ859970, EF638081, HM042052

14 acg tcg T S AY808009, D10934, DQ492194, DQ492195, EF407471

14 acg gtg T V EU155382

15 gtg gcg V A AB285067, AY240968

15 gtg ata V I EU155381

16 ttg atg L M DQ491946, EF638081, EU155223

16 ttg ccg L P DQ492158

16 ttg tcg L S AM402379

16 ttg gtg L V AJ507198, DQ492145, EU256089

17 act gcc T A AF165063, AF165064, DQ492087, EF407499

17 act gct T A AB010250, AB080299, AB154185, AB154186, AB154190, AB285047, AB285062, AB285066, AB285071, AB442221, AB442222, AF034151, AF165055, AF165056, AF207754, AF207760, AF265156, AJ507162, AJ507172, AJ507178, AJ507186, AJ507191, AM402269, AM402300, AM493503, AY587016, AY808010, AY808017, AY808022, D30613, D45172, D89815, DQ244140, DQ492066, DQ492067, DQ492070, DQ492073, DQ492074, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, EF407469, EF407485, EU155222, EU155223, EU155230, EU155255, EU155301, EU155307, EU155326, EU234061, EU255962, EU256081, EU256091, EU482880, EU857431, GQ913873, M84754

17 act att T I AB049091, AB285045, AM401954, AM401955, DQ492010, EF407467, EU155259, EU155306

17 act atg T M AB285079

17 act aac T N EU155369

17 act agc T S AB049100, AB154195, AB154196, AB285044, AB285052, AB285054, AB285055

17 act agt T S AB285057, AB285059, AB285064, AB285072, AB285075, AB442219, AB442220, AF165047, AF165048, AF165051, AF165052, AF165059, AF207752, AF207753, AF207762, AF207763, AF207769, AF207770, AF207771, AF265142, AF265143, AF265144, AF265145, AF265146, AF265147, AF265153, AF265160, AF265161, AF265162, AF483269, AJ133096, AJ507156, AJ507160, AJ507181, AJ507198, AM401926, AM401939, AM494937, AY240968, AY808013, AY808019, AY808021, AY808026, AY808033, AY808036, D50481, D90208, DQ492094, DQ492095, DQ492096, DQ492097, DQ492098, DQ492099, DQ492100, DQ492101, DQ492102, DQ492103, DQ492104, DQ492105, DQ492106, DQ492107, DQ492108

17 act tca T S DQ492109

105

17 act tcc T S DQ492110, DQ492111, DQ492112, DQ492113, DQ492114, DQ492115, DQ492116, DQ492117

17 act tct T S DQ492118, DQ492119, DQ492120, DQ492121, DQ492122, DQ492123, DQ492158, EF407459, EF407462, EF407468, EF407470, EF407475, EF407476, EF407480, EF407487, EF407488, EF407492, EF407500, EU155219, EU155220, EU155226, EU155232, EU155254, EU155256, EU155260, EU155262, EU155263, EU155303, EU155325, EU155327, EU155330, EU155334, EU155336, EU155361, EU155364, EU234062, EU239714, EU255960, EU256001, EU256059, EU256066, EU256083, EU256090, EU482875, EU482883, EU482885, EU781825, EU781827, EU781830, GQ913870, GU451218, GU451219, HM042053, HM042056, HM042058, HM042059, HM042060, HM042063

17 act gtt T V AB049090

18 gat ggc D G AM402359, DQ492106, DQ492134

18 gat aac D N DQ492014

19 ttc ctc F L AM402262, AM402307, DQ492197

19 ttc tta F L AF034151, DQ492192

19 ttc ttg F L AJ507165, DQ492011

19 ttc tcc F S AM402065

20 aag gag K E AM401859, AM401917, AM401954, AM401955, AM402074, AM493474, AM493495, FJ958914

20 aag atg K M AM402275

20 aag cag K Q DQ492192

20 aag agg K R AM402127, AM402300, AM402333, EF407472, EU155329, HM042058

21 acc gcc T A AF207754, AM401941, DQ492000, EF407485

21 acc atc T I EU155281

21 acc aac T N AY808032

21 acc tcc T S AY808037

22 tgg ggg W G DQ492110

22 tgg cgg W R AM493506

23 ctc atc L I AF207765

23 ctc ccc L P DQ491958, DQ491969, DQ492021

24 cag aag Q K AB049100, AB080299, AB442221, AF207756, D85516

24 cag cga Q R AM401865, EU155361

24 cag cgg Q R AF176573, AY808034, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492168, DQ492174, EF407459, EU155225, EU256088

25 tcc gcc S A EF407461

25 tcc ccc S P DQ491970

25 tcc acc S T AB285039, AB285046, AB285071, AF265148, AF265149, AF265150, AF265163, AF265164, AF265165, AJ507187, DQ491992, EF407477, EU155254, EU155263, EU862837, GQ913872

26 aag gag K E AM401882, AM402261, AM402268, DQ491977, DQ492014, DQ492146

26 aag atg K M AM402364

26 aag cag K Q DQ492162

26 aag aga K R D50481, DQ492057, EF407491

26 aag agg K R AB285045, AJ507157, AM401853, D50485, DQ491961, DQ491964, DQ491966, DQ492154, DQ492155, DQ492156, EF407485, EU256001, EU781832, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931,

106

FJ958932, FJ958933, FJ958934, FJ958935

27 ctc atc L I AF165059, AF165060, EU155255

27 ctc ccc L P AM401851, DQ492132, DQ492135, DQ492139, DQ492148

27 ctc gtc L V AB049090, AF207760, D50481, D50485, DQ492184, FJ958895, FJ958898, FJ958899, FJ958900, FJ958902, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958911, FJ958912, FJ958913, FJ958915, FJ958916, FJ958918, FJ958919, FJ958921, FJ958922, FJ958923, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958932, FJ958933

28 ctg atg L M AB049100, AB080299, AB442221, AF165059, AF165060, AF207763, AJ507172, AJ507197, D85516, EU155372

28 ctg ccg L P DQ492168, DQ492174

28 ctg cag L Q DQ492084

28 ctg gtg L V AF207756, EU256080, EU256084

29 ccg tca P S FJ958897

29 ccg tcg P S AM402324, AM402346

30 cga aaa R K AB010249, D11168, D16435, D89872, EU155219, EU155226

30 cga aag R K AB285067

30 cga ctg R L AB442221, AY808034, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039

30 cga atg R M AF265154, AF265155, AF265156, EU482879

30 cga caa R Q AB080299, AB285069, AJ507183, AY808028, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, EU234062

30 cga cag R Q AB049100, AB285065, AF207756, AF333324, AM402060, D63857, DQ492131, EF407472, EF407487, EF407501, EF407502, EU155228, EU155258, EU155262, EU155361, EU155374, EU256085, EU256088, EU256091, EU862835, GU451220, M58335

30 cga tgg R W AM402377

31 ttg ttt L F AY808037

31 ttg ata L I AY808015, EU155373, EU234062

31 ttg atg L M AB049089, AB285041, AB285042, AJ507158, AJ507163, AJ507182, AY808034, AY808035, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, DQ492194, DQ492195, EF407478, EU155219, EU155262, EU155315, EU155327, EU256061, EU256065, EU256101, EU482833, EU482885, FJ024279, FJ390397

31 ttg tcg L S DQ492001

31 ttg tgg L W DQ492078, DQ492088

32 ccg tcg P S AM401883, DQ492093

33 gga aga G R AM401968

34 gtc gcc V A AJ507170, AJ507182, AM401879, AM402058, DQ492009

34 gtc gac V D DQ491947, DQ492136, X61596

34 gtc atc V I AB285042, AB285043, AB285045, AB285047, AB285050, AB285061, AB285063, AB285065, AB285069, AB442222, AF165053, AF165054, AF165055, AF165056, AJ000009, AJ507189, AJ507194, AJ507197, AJ507199, AM494937, AY808027, AY808028, AY808033, D50482,

107

D50484, D63857, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, EF407472, EF407480, EF407488, EF407491, EU155227, EU155235, EU155253, EU255961, EU256101, GQ913869, HM042054, HM042055, HM042056, HM042058, HM042061

34 gtc att V I AB285078, EU155316, EU256065, HM042063

34 gtc ctc V L AB285044, AB285059, AF165051, AF165052, AJ507163, AJ507167, AJ507172, AM402056, D90208, EF407483, EF407500, EF407501, EU155220, EU155228, EU155300, EU155362, EU239714, EU256000, EU256066, EU256079, EU482880, GQ913870, HM042053, HM042059, HM042060

36 ttc ctc F L AB285040, DQ491997, DQ492017, EU256078

36 ttc ctt F L AM401947

36 ttc tac F Y DQ492128

37 ttc atc F I AB442222, AJ507163, AJ507182, AJ507188, AJ507197, AY808023, EF032892, EF032893, EF032894, EF407461, EF407468, EU155254, EU155256, EU155257, EU155279, EU155316, EU256064, EU482833, EU660388, EU862837, FJ390396

37 ttc att F I AY808036

37 ttc cta F L AJ507177, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AY808016, AY808030, EU155221, EU155307, EU155365, EU256066, EU256085, EU529682, EU862835, FJ390397

37 ttc ctc F L AB049090, AB049095, AB154177, AB154178, AB154179, AB154180, AB154183, AB154184, AB154185, AB154186, AB154187, AB154188, AB154189, AB154190, AB154191, AB154192, AB154193, AB154195, AB154196, AB154197, AB154198, AB154199, AB154200, AB154201, AB154202, AB154203, AB154204, AB154205, AB154206, AB285037, AB285038, AB285039, AB285040, AB285042, AB285051, AB285062, AB285069, AB285072, AB285077, AF033373, AF034151, AF165047, AF165048, AF165051, AF165052, AF165059, AF165060, AF165063, AF165064, AF176573, AF207752, AF207753, AF207760, AF207762, AF207766, AF207770, AF207772, AF265142, AF265143, AF265144, AF265145, AF265146, AF265147, AF265148, AF265149, AF265150, AF265158, AF265159, AF265160, AF265161, AF265162, AF265163, AF265164, AF265165, AF313916, AJ133096, AJ507156, AJ507160, AJ507162, AJ507166, AJ507167, AJ507168, AJ507175, AJ507178, AJ507179, AJ507191, AJ507192, AJ507194, AJ507195, AJ507196, AJ507199, AM401881, AM401883, AM401890, AM401895, AM402269, AY240969, AY460204, AY587844, AY808010, AY808012, AY808014, AY808015, AY808025, AY808026, AY808027, AY808029, AY808031, AY808033, AY808040, AY808041, AY808043, D50481, D50485, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ492019, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492074, DQ492075, DQ492076, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088,

108

DQ492089, DQ492090, DQ492091, DQ492092, DQ492093, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407459, EF407460, EF407462, EF407464, EF407465, EF407466, EF407471, EF407476, EF407478, EF407479, EF407480, EF407483, EF407485, EF407486, EF407488, EF407499, EF407500, EF407502, EF407504, EF638081, EU155218, EU155220, EU155222, EU155224, EU155225, EU155227, EU155228, EU155229, EU155231, EU155235, EU155255, EU155259, EU155263, EU155264, EU155281, EU155300, EU155305, EU155308, EU155315, EU155318, EU155325, EU155327, EU155334, EU155335, EU155336, EU155337, EU155356, EU155357, EU155358, EU155359, EU155360, EU155362, EU155368, EU155372, EU155374, EU155375, EU155376, EU234061, EU234062, EU255962, EU256000, EU256001, EU256045, EU256059, EU256061, EU256062, EU256075, EU256076, EU256079, EU256081, EU256082, EU256083, EU256088, EU256089, EU256091, EU256098, EU256099, EU256102, EU256103, EU482839, EU482849, EU482859, EU482879, EU482888, EU781825, EU781827, EU781830, EU781832, FJ024277, FJ024279, FJ390398, FJ478453, FJ958890, FJ958891, FJ958893, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958934, FJ958935, GQ913868, GQ913869, GQ913870, GQ913871, GQ913873, GU451218, GU451219, GU451221, GU451222, GU451223, GU451224, HM042051, HM042053, HM042054, HM042055, HM042056, HM042057, HM042058, HM042059, HM042060, HM042061, HM042063, L02836, U45476

37 ttc ctg F L AF207758, D90208, EU155302, EU256084

37 ttc ctt F L AB036519, AB036520, AB036521, AB109543, AB154194, AB191333, AB285035, AB285043, AB285047, AB285050, AB285056, AB285078, AB442220, AF165053, AF165054, AF165055, AF165056, AF207757, AF207771, AJ507172, AM494937, AY044867, D63857, EF407470, EU155364, EU239714, EU256065, EU256080, HM042062

37 ttc ttg F L AJ507174

37 ttc atg F M AJ507171

37 ttc cca F P AM402099

37 ttc ccc F P DQ491970

37 ttc tcc F S AM401869, DQ492094, DQ492109

37 ttc gta F V EF407489

37 ttc gtc F V EU155217, EU155304, EU155367, EU660386

37 ttc tac F Y AJ000009, AJ507159, AJ507190, AJ507198, EU256101

38 tca tta S L AM401851

38 tca ttg S L FJ958915

38 tca cca S P AM402086

39 tgt cgc C R AM402374, DQ492192

39 tgt cgt C R DQ491977

39 tgt tac C Y DQ492161, DQ492165

40 caa cac Q H AM493496

41 cgt cac R H AM401908

41 cgt cat R H AM401920, AM401949, AM401954, AM401977, DQ492027, DQ492028, DQ492034, DQ492036, DQ492037, DQ492039, EU155235

41 cgt agc R S EU256089

42 ggg gag G E AM401910, DQ492083

42 ggg agg G R AM402066, DQ492044, DQ492045, DQ492051, DQ492052

109

43 tac tgc Y C DQ492085

44 aag gag K E AM401900, AM401903, AM402380, DQ492152, DQ492168, DQ492174

44 aag aga K R AB285048, AB285052, AY808037, EU155362

44 aag agg K R AB049094, AB049101, AB154183, AB154184, AB154185, AB154186, AB154195, AB154196, AB154197, AB154198, AB154205, AB154206, AB285044, AB285059, AB285070, AB285078, AB442219, AB442222, AF165055, AF165056, AF207764, AF265142, AF265143, AF265144, AF265160, AF265161, AF356827, AJ133096, AJ507159, AJ507164, AJ507165, AJ507168, AJ507169, AJ507170, AJ507184, AJ507185, AJ507194, AM401896, AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401931, AM401932, AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401944, AM401945, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AY808010, AY808021, AY808031, AY808034, AY808035, AY808042, AY808043, D50480, D50483, D85516, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, DQ491984, DQ491992, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, DQ492111, DQ492112, DQ492115, DQ492116, EF407467, EF407471, EF407491, EF407498, EU155222, EU155228, EU155256, EU155261, EU155316, EU155330, EU155332, EU155337, EU155370, EU155371, EU255960, EU256062, EU256077, EU256080, EU256084, EU256090, EU256092, EU256101, EU256103, EU482877, EU660388, FJ390398, FJ958914, GQ913868, GQ913872, HM042062, U45476, X61596

45 gga gag G E AM401979

45 gga aga G R AM401904, AM402058, DQ492115

46 gtc gcc V A AB285039, AF165063, AF165064, DQ491982

46 gtc ttt V F AB285076

46 gtc ata V I AB154201, AB154202

46 gtc atc V I AB049098, AB154177, AB154178, AB154179, AB154180, AB154181, AB154182, AB154183, AB154187, AB154188, AB154191, AB154192, AB154193, AB154194, AB154197, AB154198, AB154199, AB154200, AB154203, AB154204, AB154205, AB154206, AF207760, AF207761, AF207769, AM401888, AM401973, AY008261, DQ492090, EF407461, EF407468, EF407469, EF407488, EU155306, EU256085, EU482888, GQ913873

46 gtc att V I AB154184, AB154195, AB154196, AB285040, AJ507168, AY808026, AY808038


47 tgg cgg W R DQ492087, DQ492106

48 cgg ctg R L AB049087, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488

48 cgg ccg R P AM402294, AM402302

110

48 cgg cag R Q AB154203, AB154204, AB285051, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AM494937, AY808027, DQ492056, DQ492157, EF407463, EU482875

49 ggc gcc G A HM042062

49 ggc gcg G A EU155306, HM042056, HM042058

49 ggc gaa G E DQ491971, DQ491993, EU155381

49 ggc gag G E AB049100, AF265142, AF265143, AF265144, AF265160, AF265161, AF265162

49 ggc tca G S AJ507197

50 gac gcc D A AM402115

50 gac gaa D E AB049099, AB285036, AB285038, D14484

50 gac gag D E EU256099

50 gac aac D N DQ492064

51 ggc gac G D AM402303

51 ggc agt G S AM402384

52 atc gcc I A DQ492151

52 atc atg I M DQ244140, EU155261

52 atc acc I T AB285038, DQ491996, DQ492024, DQ492103

52 atc gtc I V AB036519, AB036520, AB036521, AB049087, AB049092, AB049094, AB049095, AB049096, AB109543, AB191333, AB285040, AB285043, AB285045, AB285047, AB285048, AB285049, AB285050, AB285051, AB285052, AB285053, AB285057, AB285064, AB285068, AB285073, AB285075, AB285078, AB285079, AB442219, AB442220, AF165047, AF165048, AF165053, AF165054, AF165055, AF165056, AF207757, AF207768, AF207772, AF265154, AF265155, AJ507175, AJ507189, AM401864, AM401869, AM401871, AM401872, AM401874, AM401875, AM401877, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AM494937, AY808011, AY808016, AY808023, AY808036, D50482, D50484, DQ492124, DQ492125, DQ492126, DQ492127, DQ492128, DQ492129, DQ492130, DQ492131, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492139, DQ492140, DQ492141, DQ492142, DQ492144, DQ492145, DQ492147, DQ492148, DQ492149, DQ492150, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492162, EU155224, EU256088, EU482859, EU529682, GU451218, GU451219, HM042061, X61596

52 atc gtt I V AB049089, AB285054, AB285055, EU256102, FJ958914

53 atg ata M I AM402107, AM402108, DQ491967

53 atg acg M T DQ492062, DQ492076, DQ492193

53 atg gtg M V AF165047, AF165048, AM401944, DQ492064, EU155373, EU482875, HM042053

54 caa tgt Q C AY808012

54 caa cac Q H AB036519, AB036520, AB036521, AB049089, AB049098, AB109543,

111

AB154184, AB154193, AB191333, AB285036, AB285043, AB285047, AB285053, AB285057, AB285062, AB285073, AB285075, AB285081, AB429050, AF165047, AF165048, AF165055, AF165056, AF165061, AF165062, AF207761, AF207762, AF207764, AF265142, AF265143, AF265144, AF265160, AF265161, AF265162, AF483269, AJ133096, AJ507160, AJ507162, AJ507163, AJ507164, AJ507168, AJ507170, AJ507174, AJ507175, AJ507178, AJ507184, AJ507199, AM402266, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AM494937, AY808023, AY808026, AY808027, AY808039, AY808040, AY808043, D50482, D50484, D85516, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ492076, EF407460, EF407464, EF407468, EF407478, EF407483, EF407498, EU155218, EU155220, EU155222, EU155225, EU155232, EU155235, EU155256, EU155261, EU155281, EU155325, EU155330, EU155337, EU155356, EU155360, EU239714, EU256000, EU256062, EU256076, EU256077, EU256079, EU256083, EU256090, EU482839, EU482860, EU482875, EU482877, EU482886, EU482888, EU781825, EU781830, FJ024279, FJ390398, FJ958929, GQ913869, GQ913873, HM042051, HM042052, HM042054, HM042055, HM042056, HM042057, HM042058, HM042059, HM042063, M96362, U16362, U45476

54 caa cat Q H AB049091, AB049092, AB154203, AB154204, AB285037, AB285040, AB285041, AB285048, AB285060, AB285064, AF207766, AF207769, AF207770, AJ507166, AJ507167, AJ507169, AJ507177, AJ507179, AJ507182, AJ507192, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AY008261, AY808009, AY808015, AY808017, AY808021, AY808024, AY808036, AY808037, DQ244140, DQ244141, EF407462, EF407467, EF407481, EF407482, EF407484, EF407488, EF407497, EF407500, EF407501, EU155219, EU155228, EU155259, EU155279, EU155300, EU155303, EU155304, EU155306, EU155327, EU155332, EU155333, EU155357, EU155359, EU155369, EU155372, EU155373, EU155381, EU256089, EU256091, EU256102, EU482874, EU482881, EU482885, EU660386, EU781827, FJ024277, FJ390396, FJ958890, FJ958896, FJ958897, FJ958903, FJ958905, FJ958906, FJ958910, FJ958914, FJ958917, FJ958920, FJ958924, FJ958934, HM042061, HM042062

54 caa cta Q L AY587844, AY808032, D63857, EF407489

54 caa ctc Q L EU155358

54 caa aac Q N AF034151, EU255962, FJ958892, FJ958894

54 caa aat Q N EF407476, EU256066, FJ958891, FJ958893, FJ958895, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958904, FJ958907, FJ958909, FJ958911, FJ958912, FJ958913, FJ958915, FJ958916, FJ958918, FJ958919, FJ958921, FJ958922, FJ958923, FJ958925, FJ958926, FJ958927, FJ958928, FJ958930, FJ958931, FJ958932, FJ958933, FJ958935

54 caa ccg Q P AM493484

54 caa cgg Q R AM401932

54 caa cgt Q R AM402325

112

54 caa tac Q Y EU256064, EU256078

54 caa tat Q Y AB010250, AB285065, AF165063, AF165064, AJ507171, AY587016, D30613, D45172, D89815, EU155368, EU857431

55 acc gcc T A AB285038, AJ507169, AJ507179, AM402296, DQ492034, DQ492039, GQ913873, AJ507162, AM401848, AM401973, AM493484, DQ492024, EU482879

55 acc gct T A DQ492163

56 acc atc T I AB285038, AB285041, AB285046, AB285049, AB285066, AB285068, AB285075, AB442219, AF165045, AF165046, AF207755, AF207760, AF265152, AF265153, AJ507190, AM401958, AY808016, D50482, D50484, EF407499, EU155219

56 acc att T I EF407469

56 acc aac T N EU256064

56 acc gtc T V AY808035, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, EU256088

57 tgc cgc C R AM402334, DQ492017, DQ492080, DQ492193

57 tgc agc C S DQ492164

57 tgc tgg C W DQ491967

57 tgc tac C Y DQ492101

58 cca gca P A FJ390396

58 cca cta P L AB154197, AB154198, EU155225

58 cca ctg P L AJ507179

58 cca caa P Q AF165049, AF165050, AY808033

58 cca cag P Q AY808032

58 cca tca P S AB154195, AB154196, AB285037, AB285045, AB285048, AB285052, AB285057, AB285077, AF483269, AJ507180, AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401931, AM401932, AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401944, AM401945, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AM493491, AM493492, AY808012, DQ492050, EF407459, EF407488, EU155235, EU155261, EU155317, EU155330, EU155382, EU256090, EU482859

58 cca tct P S EU155253

58 cca aca P T EF407482, EF407497, EF407500, EU155279, EU155303, EU482881

58 cca acg P T FJ024277

59 tgt cgt C R AM401854, AM402351, AM402358, DQ491970, DQ492002

60 gga gca G A AF034151

60 gga gag G E DQ492008

60 gga aga G R AM402315

61 gca gga A G AF207765, AM401928, AM401932, AY808042, EF407482, EF407497, EF407500, EU155262, EU155279, EU155303, FJ024277, FJ390396

61 gca aca A T AM402332

61 gca gta A V AM401853, AM401858

62 cag gac Q D AB049089

62 cag gaa Q E AB285053, AJ133096, AM494937, EF407462, EU155256, EU155337, EU781827

62 cag gag Q E EF407481, EF407497

62 cag cac Q H EU256080, EU256084

113

62 cag cat Q H AF207760

62 cag aaa Q K EF407501, EU155235

62 cag cga Q R DQ492151

62 cag cgg Q R AB154195, AB154196, DQ859970, EU862835

62 cag tcg Q S AB442221

63 atc ctc I L AB285072, EU255961, M84754

63 atc ctg I L EU155261

63 atc ctt I L EF407467

63 atc ttg I L GU451219

63 atc atg I M AM401865, AM401866, AM401870, U45476

63 atc aac I N DQ492133

63 atc acc I T DQ492072, DQ492112, DQ492142

63 atc gtc I V EU155365, EU256001

64 acc gca T A AF265154, AF265155, AF265156, EU482879

64 acc gcc T A AB049090, AB049091, AB049095, AB049099, AB285036, AB285038, AB285046, AB285049, AB285050, AB285068, AB285077, AF176573, AF207758, AF207760, AF207762, AF207772, AF265145, AF265146, AF265147, AF265148, AF265149, AF265150, AF265152, AF265153, AF265158, AF265159, AF265163, AF265164, AF265165, AJ507156, AJ507166, AJ507195, AJ507196, AM401967, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AY808016, AY808025, AY808030, AY808035, AY808041, D14484, D50482, D50484, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, EF407465, EF407471, EF407479, EF407485, EF407486, EF407502, EF407504, EU155217, EU155218, EU155221, EU155231, EU155263, EU155302, EU155305, EU155307, EU155315, EU155318, EU155335, EU155336, EU155365, EU155367, EU155374, EU155376, EU256045, EU256059, EU256061, EU256075, EU256082, EU256085, EU256088, EU256098, EU256099, EU256103, EU482859, EU482883, EU529682, EU660388, EU862835, GQ913870, GU451218, HM042054, HM042060, HM042061, U45476

64 acc gct T A AB049094, AB285039, AB285043, AB285051, AB285052, AB285056, AB442220, AF165045, AF165046, AF165053, AF165054, AF207755, AF207757, AF207771, AJ507189, EF407459, EF407461, EF407464

64 acc agc T S AB049096, AB285042, AB285045, AB285054, AB285055, AB285079, AB442219

64 acc agt T S AB285035, AB429050

64 acc tca T S EF407466

64 acc tcc T S AB049087, AB049088, AB049092, AF165058, AF207769, AY240968, AY240969, AY808029, DQ492063, DQ492064, DQ492065, DQ492068, DQ492069, DQ492071, DQ492072, DQ492077, DQ492083, EF032892, EF032893, EF032894, EF407467, EF407499, EU155229, EU155254, EU155260, EU155363, EU234062, EU255961, EU256065, EU482849

64 acc tct T S AB285069, AF165057, AF356827, AY808027, EU155253

66 cat att H I GU451219

114

66 cat caa H Q DQ491972, DQ491973

66 cat cgt H R DQ492083, EF638081

67 gtg gcc V A AM401864

67 gtg gac V D DQ492098

67 gtg ttc V F D50482, D50484

68 aaa gaa K E AM402354, DQ491979

68 aaa agg K R DQ492114, DQ492120

68 aaa aca K T AY044867

69 aac gcc N A DQ491998

69 aac aag N K AB285064

69 aac agt N S AM402350, AM402352, AM402353

69 aac acc N T AJ507172, AM402271, DQ491995, DQ491996, DQ491997, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, EF407495

69 aac act N T HM042050

70 ggt agt G S AM402262, DQ491955

71 tcc ttc S F DQ492182

71 tcc ccc S P DQ859970

71 tcc acc S T AM401881

71 tcc act S T EU256103

72 atg aag M K DQ492040

72 atg acg M T DQ492109

72 atg gtg M V AM402356

73 agg ggg R G DQ492145

73 agg aag R K AB049087, AB049094, AB285044, AB285057, AF165045, AF165046, AF165053, AF165054, AF207755, AF207771, D50482, D50484, HM042059

74 atc ctc I L EU482860, HM042056

74 atc ctt I L AB285062

74 atc acc I T DQ491965, DQ492046, FJ958904

74 atc gtc I V AF265153, AM401896, AM401901, AY808035, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492047, DQ492048, DQ492049, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, EF407464, EF407498

75 gtg gcc V A EU660386

75 gtg gct V A AB049096, AB080299, AB285047, AB285048, AB285058, AB285064, AJ507155, AJ507163, AY240969, AY808009, AY808036, DQ244140, DQ244141, DQ492092, EU155235, EU155281, EU155356, EU155360, HM042055

75 gtg ttc V F AJ507169, GQ913873

75 gtg ttt V F AJ238800

75 gtg ggt V G AM402382


75 gtg atc V I EU155227

75 gtg att V I AB285044, AF208024, EU155263, EU155365, EU256103

75 gtg aca V T AF207758, EU155302

75 gtg acc V T AB285062, EU155367, EU482883

75 gtg act V T AB154197, AB154198, AF265154, AF265155, AF265156, AJ507170, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096,

115

AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AY808030, EU529682

75 gtg tgg V W M84754

75 gtg tat V Y AB285041, EF407468, HM042056

76 ggg gag G E DQ492068

76 ggg agg G R AM401892

77 cct ctc P L DQ492009

77 cct ctt P L FJ958892, FJ958894

77 cct tct P S AB154190, AM402312

78 agg ggg R G AM402120

78 agg aaa R K AB010249, AB036519, AB036520, AB036521, AB049088, AB049089, AB049092, AB049094, AB049095, AB049098, AB049099, AB049101, AB080299, AB109543, AB191333, AB249644, AB285035, AB285036, AB285037, AB285039, AB285040, AB285043, AB285044, AB285045, AB285046, AB285047, AB285048, AB285049, AB285050, AB285051, AB285052, AB285053, AB285054, AB285055, AB285056, AB285057, AB285058, AB285060, AB285061, AB285062, AB285063, AB285064, AB285069, AB285070, AB285071, AB285072, AB285073, AB285074, AB285075, AB285076, AB285077, AB285079, AB285081, AB426117, AB429050, AB442219, AB442220, AB442221, AF033373, AF034151, AF165045, AF165046, AF165047, AF165048, AF165053, AF165055, AF165056, AF165057, AF165058, AF165059, AF165060, AF165061, AF165062, AF207752, AF207753, AF207755, AF207756, AF207758, AF207760, AF207761, AF207762, AF207763, AF207765, AF207768, AF207770, AF207771, AF207774, AF265142, AF265143, AF265144, AF265160, AF265161, AF265162, AF483269, AJ000009, AJ507155, AJ507158, AJ507160, AJ507162, AJ507164, AJ507167, AJ507168, AJ507171, AJ507172, AJ507174, AJ507175, AJ507178, AJ507186, AJ507188, AJ507190, AJ507192, AJ507193, AJ507194, AJ507197, AJ507198, AJ507199, AM401867, AM401868, AM401876, AM401878, AM401880, AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401931, AM401932, AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401944, AM401945, AM401946, AM401947, AM401948, AM401954, AM401955, AM401957, AM401959, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AM402258, AM402259, AM402260, AM402261, AM402262, AM402264, AM402265, AM402266, AM402267, AM402268, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AY044867, AY045702, AY460204, AY808010, AY808011, AY808014, AY808015, AY808017, AY808020, AY808021, AY808022, AY808023, AY808024, AY808025, AY808027, AY808032, AY808035, AY808036, AY808037, AY808038, AY808039, AY808041, D10934, D11168, D16435, D50482, D50484, D63857, D85516, D89872, D90208, DQ071885, DQ244141, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964,

116

DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, DQ492076, DQ492093, DQ492103, DQ492105, DQ492106, DQ492124, DQ492125, DQ492126, DQ492128, DQ492129, DQ492130, DQ492131, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492139, DQ492140, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492148, DQ492149, DQ492150, DQ492151, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492162, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407459, EF407462, EF407468, EF407472, EF407474, EF407475, EF407477, EF407480, EF407483, EF407486, EF407487, EF407489, EF407491, EF407494, EF407495, EF407497, EF407498, EF407504, EU155218, EU155219, EU155220, EU155222, EU155223, EU155224, EU155225, EU155226, EU155227, EU155228, EU155231, EU155258, EU155260, EU155262, EU155263, EU155279, EU155300, EU155303, EU155315, EU155316, EU155317, EU155318, EU155327, EU155329, EU155332, EU155335, EU155336, EU155356, EU155358, EU155361, EU155362, EU155368, EU155372, EU155373, EU155374, EU155381, EU155382, EU239714, EU255960, EU255962, EU256000, EU256001, EU256045, EU256061, EU256062, EU256064, EU256066, EU256075, EU256076, EU256078, EU256079, EU256080, EU256083, EU256084, EU256085, EU256099, EU256101, EU256102, EU256103, EU482839, EU482875, EU482879, EU482881, EU482883, EU482885, EU482888, EU660388, FJ390396, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958904, FJ958906, FJ958907, FJ958909, FJ958911, FJ958912, FJ958913, FJ958915, FJ958916, FJ958918, FJ958919, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958935, GQ913869, GQ913873, GU451218, GU451219, GU451220, HM042050, HM042051, HM042052, HM042053, HM042055, HM042056, HM042057, HM042058, HM042059, HM042061, HM042063, M84754, M96362, U01214, U16362, U45476, X61596

78 agg aag R K AB049087, AB049091, AB049096, AB285066, AB285067, AB285068, AB285078, AF165051, AF165052, AF207757, AF207764, AF208024, AF265154, AF265155, AF265156, AF265158, AF265159, AF333324, AJ507157, AJ507165, AJ507166, AJ507169, AJ507173, AJ507177, AJ507181, AJ507182, AJ507183, AJ507184, AJ507189, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401877, AM401879, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AM493495, AY587844, AY808018, AY808019, AY808026, AY808028, AY808033, AY808042, D50480, D50481, D50483, D50485, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492082, DQ492094, DQ492095, DQ492096, DQ492097, DQ492098, DQ492099, DQ492100, DQ492101, DQ492102, DQ492104,

117

DQ492107, DQ492108, DQ492109, DQ492110, DQ492111, DQ492112, DQ492113, DQ492114, DQ492115, DQ492116, DQ492117, DQ492118, DQ492119, DQ492120, DQ492121, DQ492122, DQ492123, DQ859970, EF407460, EF407463, EF407466, EF407470, EF407476, EF407478, EF407479, EF407482, EF407490, EF407492, EF407496, EF407503, EU155232, EU155261, EU155301, EU155306, EU155308, EU155325, EU155326, EU155328, EU155330, EU155357, EU155359, EU155360, EU155363, EU155364, EU155366, EU155370, EU155371, EU255961, EU256059, EU256065, EU256098, EU482833, EU482877, EU482880, EU862837, FJ958890, FJ958896, FJ958897, FJ958903, FJ958905, FJ958910, FJ958914, FJ958917, FJ958920, FJ958926, FJ958934, FN435993, GQ913872, GU451221, GU451222, GU451223, GU451224, HM042054, HM042062, L02836, M58335

78 agg aac R N EU482886

78 agg agt R S DQ491944

78 agg aca R T DQ492127

79 acc gcc T A AB285041, AF207768, AF265153, AM401882, AM401911, AY008261, AY808020, AY808022, EF407481, EU155227, EU155333, EU155337, EU155362, EU256089, EU256090, EU781827, FJ390398

79 acc gct T A AB285072

79 acc att T I AM402290, AM402291, AM402293, AM402294, AM402296, AM402298, AM402299, AM402300, AM402303, AM402304, AM402305

79 acc atg T M AB285037

79 acc tcc T S AF356827

80 tgt ggc C G DQ492197

80 tgt cgc C R DQ491954

80 tgt tgg C W DQ491992

80 tgt tac C Y AM493498

81 agt gcc S A FN435993

81 agt tgc S C AB285074

81 agt ggc S G AM402359, DQ491952

82 aac gac N D DQ491971, DQ492082, DQ492168, DQ492174

82 aac agc N S AM402095, AY045702, DQ492131

83 acg gcg T A DQ491981, DQ491984, DQ491988, DQ491989, DQ491992

83 acg atg T M AB285041, AB285047, AF033373, AF165063, AF165064, AJ507155, AJ507179, AJ507186, AJ507188, AJ507198, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AM494937, AY240967, AY240968, AY240969, AY808009, AY808011, AY808014, AY808032, DQ492040, DQ492048, DQ492085, DQ492093, DQ492124, DQ492125, DQ492126, DQ492127, DQ492128, DQ492129, DQ492130, DQ492131, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492139, DQ492140, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492148, DQ492149, DQ492150, DQ492151, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492161, DQ492162, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182,

118

DQ492183, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407468, EF407496, EF407503, EU155220, EU155223, EU155226, EU155230, EU155300, EU155301, EU155358, EU255961, GU451221, GU451222, GU451223, GU451224, HM042051

83 acg gtg T V AB049099, DQ491980, DQ492184

84 tgg tgt W C AY044867

84 tgg cgg W R AM402121, AM402382, DQ492060, DQ492151

85 cat tgc H C AY460204, EF638081, GU451221, GU451222, GU451223, GU451224

85 cat tgt H C AB049092, AF265154, AF265155, AF265156, EU482859

85 cat gac H D D50482, D50484

85 cat ctc H L AY008261

85 cat aat H N AY808039, DQ492150

85 cat caa H Q AB049096, AJ507188

85 cat cag H Q AB285074

85 cat cgc H R AB285040, AM493477

85 cat cgt H R AB285052, AM401911, AM401918, AY808041, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ492128, DQ492186, EF407468, EU155300, HM042051, HM042062

85 cat agt H S AF265148, AF265149, AF265150, AF265163, AF265164, AF265165

85 cat tac H Y AB285047, AJ507174, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AM494937, AY808026, M96362, U16362

85 cat tat H Y AJ507164, AJ507170, AJ507199, DQ492046, DQ492100, EU155220, EU155228, EU239714, EU256077, FJ958890, FJ958892, FJ958894, FJ958896, FJ958897, FJ958910, FJ958914, FJ958920, GQ913869, HM042054, HM042061, HM042063, U45476

86 gga gaa G E DQ492061

87 aca gca T A AB154196, AJ507170, AM401937, DQ491962, EU155228, EU256062, AJ507164, AM401879

87 aca ata T I DQ492017

87 aca aga T R DQ492167

87 aca tca T S U45476

88 ttc ctc F L DQ492097

88 ttc ctt F L DQ491968

88 ttc tcc F S AM402350, DQ492057, DQ492171, FJ958915

89 ccc ctc P L AM402315

89 ccc tcc P S AY240968, AY240969

90 att acc I T AF034151, AM401896, DQ492111, FJ958931

90 att gtc I V AB049089, AB285037, AB285063, AF207766, AJ507171, AY808021, DQ492035, DQ492048, DQ492188, EF407494

91 aac gac N D AM401975, DQ492008

91 aac agc N S AM402315, DQ491981, DQ492087

92 gcg gga A G D50482, D50484

92 gcg aca A T AB429050, AM402258, AM402259, AM402260, AM402261, AM402262,

119

AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AY808039, DQ492196, EF407496

92 gcg acg A T AB285042, AJ507184, AM401857, AM493475, AY587844, AY808015, EU155357

92 gcg gta A V DQ244141, EF407489, AJ507188, AM401869, AM402059, EU155281, EU155358

93 tac tgc Y C AY240968, DQ491979

93 tac cac Y H AB049101, AB154189, AB154190, AB285065, AB285073, AB285075, AF207770, AJ507157, AJ507162, AJ507163, AY808032, AY808038, DQ491981, DQ491983, DQ491990, DQ491991, DQ491994, DQ492048, DQ492050, EF407460, EF407500, EF407501, EU155232, EU155256, EU155373, EU155374, EU256083, EU482874, EU781825, EU781827, HM042051, HM042055

94 acc atc T I AM402088, AM493437, DQ492015

95 acg gcg T A AB285066, DQ492116

95 acg atg T M AM493477

96 ggc gac G D AM493450, AY044867, DQ492164

96 ggc agc G S AM401950

97 ccc cac P H AF207769

97 ccc cgt P R AY808038

97 ccc tcc P S AB036519, AB036520, AB036521, AB109543, AB154179, AB191333, AB285068, AB285071, AJ507161, AJ507163, AJ507196, AM493487, AM493489, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493498, AM493499, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, D50482, D50484, EU155368, EU155372, EU256083

97 ccc tct P S AF207762

98 tgc ggc C G DQ492026, DQ492030, DQ492038, DQ492039

98 tgc aac C N DQ492015

98 tgc agc C S AF207762, AJ507191, AY808034, D50482, D50484, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492027, DQ492028, DQ492029, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, EF407477, EU155232, EU155262, FJ958890, FJ958896, FJ958897, FJ958903, FJ958905, FJ958910, FJ958920, FJ958926, FJ958934

98 tgc agt C S FJ958914

98 tgc tcc C S AB285049, AF207774, AJ507193, EU155369

98 tgc tgg C W AY460204

98 tgc tac C Y AM402113, AM402121, DQ492078, DQ492088, DQ492168, DQ492174

99 acg gca T A AY045702, EU155260

99 acg gcc T A AM402322

99 acg atg T M AJ507162

99 acg tca T S EU155224, EU256065, EU256103

99 acg tcg T S AJ507166, AY808041, D14484, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970

120

99 acg gta T V AB285071, AJ507161

99 acg gtg T V AF207765, AF265158, AF265159, EU862837

100 ccc gcc P A EU155222, HM042052


100 ccc ctt P L DQ492155

101 tcc gcc S A AB285069, AF356827, AY808013, EU155356, GQ913873, U01214

101 tcc cca S P FJ958928

101 tcc ccc S P AM402373, DQ492001

101 tcc acc S T AB049098, AB285036, AB285038, AF165062, AF207757, AF207762, AF265152, AF265153, AY587844, AY808009, AY808014, AY808025, AY808032, AY808039, D50482, D50484, DQ492093, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407473, EU155218, EU155332

101 tcc gtc S V EU155306

102 ccg ctg P L DQ491979

102 ccg acg P T DQ492048

103 gcg acg A T EU255961, U01214

104 cca tca P S AM402083, D50482, D50484

105 aat gac N D AF165057, AF165058, AM401853

105 aat agc N S AB285057, AM401923, AM401976, AM493444, AY240968, AY587844, AY808037, DQ491958, DQ491969, EU256045, EU256075, EU482888

105 aat agt N S AB285048, AB285059, EF407494, EU660388

106 tat tgt Y C AM401885, DQ492156

106 tat cat Y H DQ492192

107 tct gcc S A DQ244141, EU155363

107 tct ttt S F AJ132996, AJ507180

107 tct ccc S P DQ492069, FJ958891

107 tct acc S T AB285042, AB285053, AF207771, AF265145, AF265146, AF265147, AM401906, AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401931, AM401932, AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401944, AM401945, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AY808019, DQ492094, DQ492095, DQ492096, DQ492097, DQ492098, DQ492099, DQ492100, DQ492101, DQ492102, DQ492103, DQ492104, DQ492105, DQ492106, DQ492107, DQ492108, DQ492109, DQ492110, DQ492111, DQ492112, DQ492113, DQ492114, DQ492115, DQ492116, DQ492117, DQ492118, DQ492119, DQ492120, DQ492121, DQ492122, DQ492123, EF407485, EF407495, EU155370, EU155371, FN435993

107 tct act S T AF483269, AY808043

108 agg ggg R G AM401912, AM402363, AM402372

108 agg aaa R K AF207766, AJ507171

108 agg aag R K AB049089, AB049092, AB049095, AB049098, AB049099, AB285043, AB285045, AB285050, AB285053, AB285058, AB285070, AF033373, AF165058, AF207758, AF207762, AF207763, AF207764, AF207765, AF207772, AF265158, AF265159, AJ507166, AJ507168, AJ507173, AJ507180, AJ507190, AJ507197, AM402339, AY460204, AY808039, D50482, D50484, DQ071885, DQ244140, DQ492168, DQ492174, EF407480, EU155257, EU155302, EU155306, EU155369, EU256045, EU256103, EU482849, HM042054, HM042055, HM042056, M96362,

121

U16362

108 agg atg R M DQ492120

108 agg aat R N AY240968

108 agg aca R T EF407460, EU155362

108 agg acg R T AY808029, DQ492063, DQ492064, DQ492068, DQ492069, DQ492071, DQ492072, DQ492077, EU155365

109 gcg ggg A G AB049100

109 gcg ggc A G AY008261

109 gcg gtg A V AM402325

110 ctg ccg L P AM401858, AM402109, AM402311

110 ctg cgg L R DQ492159

111 tgg cgg W R AM402373, DQ492050, DQ492180

111 tgg tcg W S DQ492058

112 cgg cag R Q DQ492063, DQ492153

112 cgg tgg R W AM402301, AM402308, DQ492056

113 gtg gcg V A AM402128, DQ492082

113 gtg atg V M AJ507192

114 gct agt A S AJ507197

114 gct tcc A S DQ491946, DQ491956, EU155369

114 gct tct A S AB285041, AF207770, AY808040, DQ491943, DQ491944, DQ491945, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491957, EU256076

114 gct aca A T DQ492035

114 gct act A T AB154177, AB154178, AB154185, AB154186, AB154191, AB154192, AB285044, AB442219, AF313916

114 gct gtt A V D50482, D50484

115 gct ttt A F D50482, D50484

115 gct cct A P AB285075, L02836

115 gct tct A S AJ507185, AM401887, AM401894, AM402107

115 gct gtt A V DQ492015, DQ492042

116 gag gat E D AJ507162, DQ492120, EU155307

116 gag ggg E G AM402311, DQ492101

117 gag gac E D AB049096, AB285076, EU155281

117 gag ggg E G AM402112

117 gag aag E K DQ492089, DQ492096

117 gag gtg E V DQ492041, DQ492043

118 tac tgc Y C AM402384

119 gtg gcg V A AM401889, AM493438, DQ491979, DQ492176, DQ492183

119 gtg ttg V L AF313916

120 gag gat E D DQ492187

120 gag ggg E G AM401888, AM402333

121 gtt gcc V A AM402092

121 gtt atc V I AB285054, AB285055, AB442220, AM402346, AM402351, AY808027, EU256001, EU256078, EU482833

121 gtt att V I AB010249, AB049090, AF176573, AF207760, AF265145, AF265146, AF265147, D11168, D16435, D89872, EF407491, EF407498, EU155223, EU155332, EU862837

122 acg gcg T A AF165055, AF165056, AF165062, AM401942, AM401946, DQ491961, DQ491964, DQ491966, DQ492018

122 acg aag T K AB154195, AB154196, AB285076

122 acg ccg T P FJ958914

122 acg agg T R AJ507166, AY808029, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492074, DQ492075, DQ492076, DQ492077, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085,

122

DQ492086, DQ492087, DQ492089, DQ492090, DQ492091, DQ492092, EF407460, EU155224, EU234062, EU482849, EU862835, M84754

122 acg gtg T V AF207769, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, EF407492, EU155306, EU155376

122 acg tgg T W DQ492078, DQ492088

123 cgg caa R Q AM401924

123 cgg cag R Q AB154177, AB154194, AF313916, AJ507177, EU155221, EU155381, EU256045, EU482860, EU660388

123 cgg tgg R W DQ492011, DQ492012, DQ492013

124 gtg ttg V L AJ507162, AJ507195, EU256061

124 gtg atg V M AF165045, AF165046, AF207755, AY808034, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, FJ958902, FJ958935

125 ggg gcg G A AM493499


126 gat gaa D E AB049087

126 gat gag D E AB049094, AF165053

126 gat ggt D G DQ492034

127 ttc tgc F C AY808027

127 ttc ctc F L DQ491976, DQ492038, DQ492081, DQ492126, DQ492127, DQ492131

127 ttc tcc F S AB285036, AF207760, AF265148, AF265149, AF265150, AF265163, AF265164, AF265165, AM402055, AM402056, AM402057, AM402058, AM402060, AM402061, AM402062, AM402063, AM402064, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402080, AM402081, AM402082, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402112, AM402113, AM402114, AM402115, AM402116, AM402118, AM402119, AM402120, AM402121, AM402122, AM402125, AM402127, AM402128, AM402129, AY808022, DQ491974, DQ492015, EF407482, EF407488, EU482883

127 ttc tac F Y AB049091, AB285039

128 cac ctc H L DQ492047

128 cac cgc H R DQ492103, DQ492179

129 tac tgc Y C DQ492188

129 tac cac Y H AY044867, DQ492179

129 tac tcc Y S AM402275

130 gtg gcg V A DQ492091

130 gtg ata V I AF207774, AF265164, AJ507159, AY808038, D10934, EU155256

123

130 gtg atc V I AB049091, EU256061

130 gtg ctg V L DQ492170

131 acg gcg T A DQ492016

131 acg ata T I AF165062

131 acg atg T M AM401953

132 ggc gcc G A AM401914

132 ggc gac G D AY240968, AY240969

132 ggc agt G S AJ507164

133 atg ata M I AB285036, AB285046, AF265142, AF265143, AF265144, AF265160, AJ507155, EF407473, EF407492, EU256088

133 atg atc M I DQ492197, EU155336, EU256092

133 atg att M I EU155230

133 atg ttg M L AB049092

133 atg aca M T AF265162

133 atg acg M T AM401882, AM402373, AM402377, AM402382, AM402384, DQ491988, DQ492127, DQ492130, DQ492131, DQ492140, DQ492155, EU155259

133 atg gta M V AF265161, EU155222, EU256090

133 atg gtg M V AB154194, AB285041, AF165063, AF165064, AJ507179, AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401928, AM401929, AM401930, AM401931, AM401932, AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401944, AM401945, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AY808017, AY808041, D14484, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, EF407461, EF407471, EU155365, EU256061, EU256085, EU482875, EU660388, EU862835, HM042056, HM042058

133 atg gtt M V AY808010

134 acc gcc T A DQ492172, FJ958897

135 act gcc T A AF265154, AF265155, AF265156

135 act gct T A AB285035, AB285038, AB285046, AB285063, AB442220, AF207757, AF207772, AJ507194, AM401906, AY808025, AY808043, DQ491994, DQ492041, DQ492043, DQ492090, EU155224, EU155259, EU155382, EU256079

135 act att T I AJ507178

135 act aac T N AY808033

135 act aat T N AF165051, AF165052

135 act tcc T S EF407459

135 act gtc T V EU155279

135 act gtt T V AJ507168, EU155361

136 gac ggc D G FJ958920

136 gac aac D N DQ492184, EU155306

137 aac gac N D EU482839

137 aac tac N Y DQ491953

138 gta ata V I AB036519, AB036520, AB036521, AB049090, AB049091, AB049095, AB109543, AB154203, AB154204, AB191333, AB285037, AB285044, AB285051, AB285057, AB285060, AB285062, AB285077, AB285078, AF165051, AF165052, AF165056, AF165062, AF165063, AF165064, AF207757, AF207760, AF207765, AF265142, AF265143, AF265144, AF265148, AF265149, AF265150, AF265158, AF265159, AF265160, AF265161, AF265162, AF265163, AF265164, AF265165, AJ133096, AJ507159, AJ507164, AJ507166, AJ507167, AJ507168, AJ507170, AJ507174, AJ507178, AJ507179, AJ507186, AJ507189, AJ507196,

124

AJ507198, AJ507199, AM402057, AM402058, AM402063, AM402069, AM402071, AM402085, AM402086, AM402089, AM402098, AM402103, AM402120, AM402121, AM402129, AM494937, AY240967, AY808012, AY808026, AY808033, AY808037, AY808040, D10934, DQ071885, DQ244140, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, EF407460, EF407464, EF407467, EF407468, EF407470, EF407478, EF407483, EF407485, EF407486, EF407488, EF407491, EF407497, EF407500, EF407501, EU155219, EU155220, EU155221, EU155222, EU155224, EU155228, EU155231, EU155235, EU155279, EU155302, EU155303, EU155316, EU155318, EU155325, EU155327, EU155332, EU155333, EU155356, EU155359, EU155367, EU255960, EU256000, EU256045, EU256061, EU256062, EU256064, EU256066, EU256076, EU256077, EU256085, EU256091, EU256103, EU482839, EU482859, EU482875, EU482877, EU482885, EU660388, EU781825, EU781830, EU862837, FJ024277, FJ024279, FJ390396, FJ390398, GQ913869, HM042051, HM042052, HM042055, HM042056, HM042057, HM042058, HM042059, HM042061, HM042063, U45476

138 gta atc V I AF207771, AJ507169, AJ507184

138 gta att V I AY808041, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, EU155225, EU155281

138 gta cta V L AB285072, AY808032, D50481, EU155373, EU529682

138 gta ctg V L AB285041, EU155357, EU155375, EU482874

138 gta tta V L AB049088, AB285035, AB285040, AB285066, AB285076, AF165061, AF207769, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AY008261, AY240968, AY808028, D50485, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, EF407474, EF407487, EF407498, EU155217, EU155226, EU155304, EU256079, EU482880, GQ913873, HM042062

138 gta ttg V L AB285063, AB442221, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, EU155361

139 aag gaa K E AJ507183

139 aag gag K E DQ492190

139 aag aga K R AB049092, AB049095, AB285054, AB285055, AB429050

139 aag agg K R AB285045, AM401849, AY808039, DQ492181

140 tgc cgc C R DQ492021

142 tgt ggt C G DQ492103

142 tgt cgc C R AM401892

142 tgt tac C Y AM402309, DQ492144

142 tgt tat C Y DQ492120

143 cag cgg Q R AM401895, AM402112, AM402300, AM402346, DQ492127

125

144 gtt gct V A AM493451, DQ491978, DQ491989, DQ492056, DQ492067, DQ492158

145 ccg ctg P L AM401894, DQ492010

145 ccg tcg P S DQ492018

146 gcc ggc A G DQ492047, DQ492050, EU256059, GQ913872

146 gcc ccc A P D50480, D50482, D50483, D50484

146 gcc agc A S AB049087, EU155260

146 gcc tcc A S AB285044, DQ071885, EF407480

146 gcc acc A T AB049088, AB285042, AB285053, AB442221, AB442222, AJ507168, AM402268, AM493476, EU155254, EU155357

146 gcc act A T AB285039

146 gcc gtc A V AM401858

147 ccc ctc P L AM402103, DQ492097

147 ccc ctt P L AJ238800, FJ958934

148 gaa gat E D AB154193

148 gaa gta E V DQ492019

149 ttc atc F I DQ492039


149 ttc tta F L DQ492195

149 ttc tcc F S DQ491949

150 ttc ctc F L AM401874

150 ttc tcc F S DQ491951, DQ492010

150 ttc tac F Y EU256079

151 aca gcg T A AM402100, AM402369, DQ492103

151 aca aaa T K D50481, D50485

151 aca atg T M DQ491975

151 aca tcg T S AF333324, M58335

152 gaa gac E D AM402316

152 gaa gga E G DQ492019

152 gaa caa E Q EU256085

153 gtg gcg V A AM401869, AM493507

153 gtg ata V I EU255962, GQ913869

153 gtg cta V L AB049090

153 gtg ctc V L AF207768

153 gtg ctg V L AB049094, AB285073, AF207760, AF207769, AF207771, AJ133096, AJ507186, AJ507189, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, D50481, D50485, DQ244140, EF407467, EF407469, EF407485, EU155223, EU155224, EU155226, EU155255, EU155279, EU155281, EU155326, EU155332, EU256001, EU529682, EU862837, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958934, FJ958935

153 gtg ttg V L AB010249, AB010250, AB036519, AB036520, AB036521, AB049089, AB049092, AB049095, AB049096, AB109543, AB191333, AB285035, AB285038, AB285042, AB285043, AB285045, AB285047, AB285048, AB285049, AB285050, AB285052, AB285054, AB285055, AB285057, AB285060, AB285064, AB285068, AB285075, AB285078, AB285079, AB429050, AB442219, AB442220, AF165047, AF165048, AF165053, AF165054, AF165055, AF165056, AF165063, AF165064, AF207757, AF207762, AF207763, AF207764, AF207772, AJ507172, AJ507178, AY240968, AY587016, AY808013, AY808036, D11168, D14484,

126

D16435, D30613, D45172, D50482, D50484, D89815, D89872, DQ071885, DQ244141, EF407498, EU256080, EU256084, EU256085, EU482874, EU482880, EU857431, GU451218, GU451219, GU451220

153 gtg atg V M AM401875

154 gat ggc D G AM401937

154 gat ggt D G AM401872

155 ggg gag G E AM402330

155 ggg agg G R AM402277, DQ491974

156 gtg gcg V A AM401870, AM402322, DQ492189

156 gtg ata V I AF207761, EF407488, EU155222, EU256061, EU256062, EU256064, HM042053

157 cgg ggg R G DQ492144

157 cgg ctg R L AM401879

157 cgg cag R Q AB080299, AB154183, AB154184, AM493472, D85516, EU155300

157 cgg tgg R W AM401891

158 ttg ata L I AB285063

158 ttg atg L M DQ491991, EU155336

158 ttg ccg L P AM401871, AM402298, DQ491967, DQ492037, DQ492073

158 ttg gta L V EU155222

158 ttg gtg L V EU155304

159 cac cgc H R AM402300, AM402312, DQ492008, DQ492141

160 agg gga R G AM402323


160 agg tgg R W AM401886


161 tac tgt Y C DQ491981

161 tac ttc Y F AB154193, AB154194, EF407480

161 tac cac Y H DQ492158

161 tac cat Y H AM493480

161 tac aac Y N D50480, D50483, DQ492022

162 gct act A T AM401866, AM401977, AM402088

162 gct gtt A V AB285042, AB285075

163 cca ctg P L DQ492160

163 cca cag P Q AM402290

164 gcg gag A E EF407463

164 gcg cca A P AB154189, AB154190, AB154200, AB154203, AB154204

164 gcg ccg A P AB154177, AB154178, AB154179, AB154180, AB154181, AB154182, AB154183, AB154184, AB154185, AB154186, AB154187, AB154188, AB154191, AB154192, AB154193, AB154194, AB154195, AB154196, AB154197, AB154198, AB154199, AB154205, AB154206, AB285077, AF313916, EU155224, EU660388, EU862837

164 gcg aca A T AB429050

164 gcg acg A T DQ492038, EU781827

164 gcg gtg A V AB049095, AB049101, AB285043, AB285049, AB285051, AB285078, AB442221, AF033373, AF165053, AF165054, AF176573, AF207756, AF207763, AF207764, AJ507196, AJ507199, AM402087, AM402119, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, D50482, D50484, D90208, DQ244140, DQ492061, EF407480, EU155219, EU155308, EU256075, EU256084, HM042054, HM042059, HM042060

165 tgc cgc C R AM401879, AM402349, DQ492069, DQ492120

165 tgc agc C S AB285043

165 tgc tcc C S D50481, D50482, D50484, D50485

165 tgc tac C Y AM401884, AM402301, DQ492158, EF407482

127

166 aaa gaa K E DQ492111

166 aaa gag K E AM402085

166 aaa gga K G AM401964, D50480, D50483

166 aaa ata K I DQ492126

166 aaa atg K M AM493504

166 aaa aga K R AB010249, AB154180, AB154181, AB154182, AB154187, AB154188, AB154200, AB154205, AB249644, AB285061, AB285067, AB285074, AB426117, AF207768, AF207774, AJ132997, AJ507192, AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401931, AM401932, AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401945, AM401946, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AY460204, AY808013, AY808014, AY808038, D11168, D16435, D63857, D89872, DQ491950, DQ492093, DQ492125, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407469, EF407477, EF407480, EF638081, EU155220, EU155226, EU155227, EU155262, EU155373, EU155375, EU256100, EU482881, FJ390396, M96362, U16362

166 aaa agg K R AB154206, AF165063, AF165064, AF333324, AJ507155, AM402102, DQ492184, DQ492185, EF407465, EU155257, EU256092, EU482833, FJ390397, GU451221, GU451222, GU451223, GU451224, M58335

166 aaa cga K R AM401944, AM401947

167 ccc gcc P A AJ507168, EU155316


167 ccc ctt P L DQ492040

167 ccc tct P S EF407496

167 ccc act P T U01214

168 ctc ccc L P AM401934, DQ492080, FJ958911

168 ctc gtc L V D10934

169 cta ata L I AB154188, AF265155, EU482879

169 cta atg L M AF265154, AF265156, EU155327

170 cgg cac R H AF176573

170 cgg ccg R P AY240968

170 cgg tgg R W AM401898, AM401905, AM402330

171 gag gac E D AB285044, AB285047, AB285066, AB285073, AJ132997, AJ133096, AJ507164, AJ507167, AJ507169, AJ507170, AJ507178, AJ507184, AJ507199, AM402055, AM402059, AM402060, AM402061, AM402065, AM402066, AM402070, AM402073, AM402074, AM402075, AM402077, AM402079, AM402081, AM402083, AM402084, AM402085, AM402088, AM402090, AM402091, AM402093, AM402094, AM402099, AM402102, AM402104, AM402106, AM402108, AM402111, AM402112, AM402113, AM402115, AM402117, AM402123, AM402124, AM402125, AM402126, AM402127, AY008261, AY240968, AY460204, AY808017, AY808030, AY808041, AY808042, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, EF407472, EF407480, EF407482, EF407488, EF638081, EU155222, EU155227, EU155330, EU155365, EU155366, EU155367, EU155368, EU256000, EU256001, EU256062, EU256066, EU256091, EU256102, EU482881, EU482885, EU862835, GQ913869, GQ913873, GU451221, GU451222,

128

GU451223, GU451224, HM042052, HM042054, HM042056, HM042057, HM042058, HM042061, HM042063

171 gag gat E D AB010250, AB036519, AB036520, AB036521, AB049087, AB049089, AB049090, AB049092, AB049094, AB049095, AB049096, AB049098, AB049099, AB049100, AB049101, AB080299, AB109543, AB154181, AB154182, AB154205, AB154206, AB191333, AB285035, AB285036, AB285037, AB285038, AB285040, AB285041, AB285042, AB285043, AB285045, AB285048, AB285049, AB285050, AB285052, AB285053, AB285054, AB285055, AB285056, AB285057, AB285059, AB285060, AB285064, AB285068, AB285072, AB285075, AB285076, AB285078, AB285079, AB285081, AB429050, AB442219, AB442220, AF034151, AF165045, AF165046, AF165053, AF165054, AF165061, AF165062, AF265152, AF265153, AJ000009, AJ507159, AJ507160, AJ507163, AJ507171, AJ507172, AJ507174, AJ507185, AJ507186, AJ507189, AJ507190, AM402056, AM402057, AM402058, AM402062, AM402063, AM402064, AM402067, AM402068, AM402069, AM402071, AM402072, AM402076, AM402078, AM402080, AM402082, AM402086, AM402087, AM402089, AM402092, AM402095, AM402096, AM402097, AM402098, AM402100, AM402101, AM402103, AM402105, AM402107, AM402109, AM402110, AM402114, AM402116, AM402118, AM402119, AM402120, AM402121, AM402122, AM402128, AM402129, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AM494937, AY240967, AY240969, AY587016, AY808013, AY808023, AY808026, AY808027, AY808032, AY808033, AY808037, AY808039, D14484, D30613, D45172, D50481, D50482, D50484, D50485, D85516, D89815, DQ071885, DQ244141, DQ859970, EF407459, EF407461, EF407463, EF407466, EF407467, EF407468, EF407469, EF407471, EF407477, EF407478, EF407479, EF407485, EF407487, EF407491, EF407498, EF407504, EU155217, EU155218, EU155219, EU155223, EU155228, EU155229, EU155230, EU155256, EU155307, EU155326, EU155329, EU155333, EU155335, EU155336, EU155356, EU155359, EU155360, EU155364, EU155370, EU155371, EU155372, EU155374, EU239714, EU255962, EU256075, EU256076, EU256077, EU256080, EU256081, EU256084, EU256085, EU256088, EU256092, EU256099, EU482833, EU482859, EU482877, EU482880, EU482883, EU529682, EU660386, EU857431, FJ390396, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958934, FJ958935, GU451218, GU451219, GU451220, HM042051, HM042055, HM042059, HM042062, U45476, X61596

171 gag aag E K AY808043, DQ492152, EU256059

171 gag agt E S EU155226

171 gag acg E T AY808035, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062

171 gag gtg E V AB010249, D16435, D89872

172 gag gac E D AF207765, AM402055, AM402056, AM402057, AM402058, AM402060, AM402061, AM402062, AM402063, AM402064, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402080, AM402081, AM402082, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112,

129

AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402125, AM402126, AM402127, AM402128, AM402129, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, EU155376

172 gag gat E D AB010249, AF176573, AJ507196, AM402059, AM402065, AM402079, AM402083, AM402091, AM402123, AM402124, D11168, D16435, D89872, EU256085, EU660388

172 gag ggg E G AY044867, DQ492071

173 gtc gcc V A AB049091, EF407503, EU256103, EU482874

173 gtc atc V I FJ958895

173 gtc acc V T AY808043

174 aca gca T A AJ507196, EU155318, EU155359, EU155368, GU451221, GU451222, GU451223, GU451224

174 aca gac T D EU155306

174 aca gat T D AY240967, D10934

174 aca gga T G EU256090

174 aca aaa T K D63857

174 aca tta T L DQ244140, EU862835

174 aca atg T M AB285062, AJ507172, AM401867, AM401868, AM401876, AM401878, AM401880, EU155264, EU155307

174 aca aac T N EU155377

174 aca agt T S AB285069

174 aca tca T S AB049099, AB285036, AB285038, AB285068, AB285081, AF165049, AF165050, AJ507155, AJ507182, AM402056, AM402057, AM402058, AM402060, AM402062, AM402063, AM402064, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402080, AM402081, AM402082, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402125, AM402126, AM402127, AM402128, AM402129, AY808016, AY808023, AY808030, D14484, EF407467, EU155221, EU155336, EU155365, EU155367, EU155381, EU256064, EU256066, EU256089, EU256091, EU256099, EU862837, FJ390397, HM042056, M84754

174 aca tcg T S AM402055, AM402059, AM402061, AM402065, AM402079, AM402083, AM402091, AM402113, AM402123, AM402124, EU482875, U45476

174 aca tct T S HM042053

174 aca gta T V AB049088, AB049091, AB154181, AB154182, AB285041, AB285058, AB285070, AB442221, AF034151, AF207752, AF207758, AY460204, D90208, EF638081, M96362, U16362

174 aca gtc T V AY808013

174 aca gtg T V AB285061, AB285077, AB442222, AF207772, AJ507158, AY808038, EF407465, EF407472, EF407498, EU155280, EU155302, EU781832, L02836

174 aca gtt T V AF207774

175 ttc ctc F L DQ491952, DQ492064

175 ttc tcc F S DQ492050

175 ttc tac F Y AF207762, AF207764

176 ctg tgt L C EU256085

176 ctg gag L E EU155279

176 ctg cat L H HM042059

176 ctg atg L M AB154189, AB154190, AF033373, AJ507188, AM402059, AM402065, AM402073, AM402079, AM402083, AM402091, AM402093, AM402111, AM402117, AM402123, AM402124, AM402126, AY808015, AY808036,

130

EU234062, EU482849, EU862837

176 ctg caa L Q AB285063, AB285079, AF207769, AJ507185, AY240968, EU155224, EU155318, GQ913872

176 ctg cag L Q AB010249, AB010250, AB036519, AB036520, AB036521, AB049087, AB049088, AB049089, AB049090, AB049091, AB049092, AB049094, AB049095, AB049096, AB049098, AB049099, AB049100, AB049101, AB080299, AB109543, AB154177, AB154178, AB154179, AB154180, AB154181, AB154182, AB154183, AB154184, AB154185, AB154186, AB154187, AB154188, AB154191, AB154192, AB154193, AB154194, AB154195, AB154196, AB154197, AB154198, AB154199, AB154200, AB154201, AB154202, AB154203, AB154204, AB154205, AB154206, AB191333, AB249644, AB285035, AB285036, AB285037, AB285038, AB285039, AB285040, AB285041, AB285042, AB285043, AB285044, AB285045, AB285046, AB285047, AB285048, AB285049, AB285050, AB285051, AB285052, AB285053, AB285054, AB285055, AB285056, AB285057, AB285058, AB285059, AB285060, AB285061, AB285062, AB285064, AB285065, AB285066, AB285067, AB285068, AB285069, AB285070, AB285071, AB285072, AB285073, AB285074, AB285075, AB285076, AB285077, AB285078, AB285081, AB426117, AB429050, AB442219, AB442220, AB442221, AB442222, AF034151, AF165045, AF165046, AF165047, AF165048, AF165049, AF165050, AF165051, AF165052, AF165053, AF165054, AF165055, AF165056, AF165057, AF165058, AF165059, AF165060, AF165061, AF165062, AF165063, AF165064, AF176573, AF207752, AF207753, AF207754, AF207755, AF207756, AF207757, AF207758, AF207760, AF207761, AF207762, AF207763, AF207764, AF207765, AF207766, AF207768, AF207770, AF207771, AF207772, AF207774, AF208024, AF265142, AF265143, AF265144, AF265145, AF265146, AF265147, AF265152, AF265153, AF265154, AF265155, AF265156, AF265158, AF265159, AF265160, AF265161, AF265162, AF313916, AF333324, AF356827, AF483269, AJ000009, AJ132996, AJ132997, AJ507155, AJ507157, AJ507158, AJ507159, AJ507160, AJ507161, AJ507162, AJ507163, AJ507164, AJ507165, AJ507166, AJ507168, AJ507169, AJ507170, AJ507171, AJ507172, AJ507173, AJ507174, AJ507175, AJ507177, AJ507178, AJ507179, AJ507180, AJ507181, AJ507182, AJ507183, AJ507184, AJ507186, AJ507187, AJ507189, AJ507190, AJ507191, AJ507192, AJ507193, AJ507194, AJ507195, AJ507196, AJ507197, AJ507198, AJ507199, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401931, AM401932, AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401944, AM401945, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303,

131

AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AM494937, AY045702, AY240967, AY240969, AY460204, AY587016, AY587844, AY808009, AY808011, AY808012, AY808013, AY808014, AY808016, AY808017, AY808018, AY808020, AY808021, AY808022, AY808024, AY808025, AY808026, AY808027, AY808028, AY808029, AY808030, AY808032, AY808033, AY808034, AY808035, AY808037, AY808038, AY808039, AY808040, AY808041, AY808042, D10934, D11168, D14484, D16435, D30613, D45172, D50480, D50481, D50482, D50483, D50484, D50485, D63857, D85516, D89815, D89872, D90208, DQ071885, DQ244140, DQ244141, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492074, DQ492075, DQ492076, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, DQ492093, DQ492124, DQ492125, DQ492126, DQ492127, DQ492128, DQ492129, DQ492130, DQ492131, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492139, DQ492140, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492148, DQ492149, DQ492150, DQ492151, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492161, DQ492162, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, DQ859970, EF032892, EF032893, EF032894, EF407459, EF407460, EF407461, EF407462, EF407463, EF407465, EF407466, EF407467, EF407468, EF407469, EF407470, EF407471, EF407472, EF407473, EF407475, EF407476, EF407477, EF407478, EF407479, EF407480, EF407481, EF407482, EF407483, EF407484, EF407485, EF407486, EF407487, EF407488, EF407489, EF407490, EF407491, EF407492, EF407494, EF407495, EF407496, EF407497, EF407498, EF407499, EF407500, EF407501, EF407502, EF407503, EF407504, EF638081,

132

EU155217, EU155218, EU155219, EU155220, EU155221, EU155222, EU155223, EU155225, EU155226, EU155227, EU155228, EU155229, EU155230, EU155231, EU155232, EU155235, EU155253, EU155254, EU155255, EU155256, EU155257, EU155259, EU155260, EU155261, EU155262, EU155263, EU155280, EU155281, EU155300, EU155301, EU155302, EU155303, EU155305, EU155306, EU155307, EU155308, EU155315, EU155316, EU155317, EU155324, EU155325, EU155326, EU155327, EU155328, EU155329, EU155330, EU155331, EU155332, EU155334, EU155335, EU155336, EU155337, EU155356, EU155357, EU155358, EU155359, EU155360, EU155361, EU155362, EU155363, EU155364, EU155365, EU155366, EU155367, EU155368, EU155369, EU155370, EU155371, EU155372, EU155373, EU155374, EU155375, EU155376, EU155377, EU155381, EU155382, EU234061, EU239714, EU255960, EU255961, EU255962, EU256000, EU256001, EU256045, EU256059, EU256061, EU256062, EU256064, EU256065, EU256066, EU256075, EU256076, EU256078, EU256079, EU256080, EU256081, EU256082, EU256083, EU256084, EU256088, EU256089, EU256090, EU256092, EU256098, EU256099, EU256100, EU256101, EU256102, EU256103, EU482833, EU482859, EU482860, EU482875, EU482877, EU482879, EU482880, EU482881, EU482883, EU482886, EU482888, EU529682, EU660386, EU660388, EU781825, EU781827, EU781830, EU781832, EU857431, EU862835, FJ024277, FJ024279, FJ390396, FJ390397, FJ390398, FJ478453, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958934, FJ958935, FN435993, GQ913868, GQ913869, GQ913870, GQ913871, GQ913873, GU451218, GU451219, GU451220, GU451221, GU451222, GU451223, GU451224, HM042050, HM042051, HM042052, HM042054, HM042055, HM042056, HM042057, HM042058, HM042060, HM042061, HM042062, HM042063, L02836, M58335, M84754, M96362, U01214, U16362, U45476, X61596

176 ctg cgg L R EU482839

176 ctg acg L T AY808023

176 ctg tgg L W EU155264

177 gtc gcc V A AM402110, DQ492137

177 gtc atc V I DQ492040, EF407487, EU155303

178 ggg gaa G E AM402320

178 ggg cgg G R AY240968

179 ctc tgc L C DQ244140, EU155280

179 ctc tgt L C EU482875

179 ctc ttc L F AB285068, AM402283, FJ958923

179 ctc cac L H AM493442, DQ492027

179 ctc ccc L P AM401957, DQ491994, DQ492046, DQ492067, DQ492097, DQ492151

180 aat gac N D AM401859

180 aat cac N H AB285041, AB285059, AJ133096, AJ507158, AJ507177, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AY808016, AY808030, EF407491, EU155221,

133

EU155307, EU155331, EU155363, EU155374, EU155381, EU234062, EU256066, EU256085, EU256100, EU529682, EU862835, FJ390397, GU451221, GU451222, GU451223, GU451224, HM042051, M96362, U16362

180 aat cat N H EU155328, EU155330, EU155365, EU155367

180 aat agc N S AM402283, AM402352, DQ492166, FJ958920

181 caa gaa Q E AM402056, AM402057, AM402058, AM402062, AM402063, AM402064, AM402067, AM402068, AM402069, AM402071, AM402072, AM402076, AM402078, AM402080, AM402082, AM402086, AM402087, AM402089, AM402092, AM402095, AM402096, AM402097, AM402098, AM402099, AM402101, AM402103, AM402105, AM402107, AM402110, AM402114, AM402116, AM402118, AM402119, AM402120, AM402121, AM402122, AM402128, AM402129, AY808026

181 caa gag Q E AM402100

181 caa cac Q H AB442219, U45476

181 caa cat Q H AB285079

181 caa aag Q K AM402109

181 caa cta Q L DQ492131, DQ492146

181 caa cga Q R AM402370, DQ492011, DQ492012, DQ492013, DQ492059, DQ492137, EU155257

181 caa cgc Q R D50482, D50484

181 caa cgg Q R AM494937, AY808023

182 tac tgc Y C AM402278, DQ492084

182 tac ttc Y F AB049095, AB049096, AB285035, AB285045, AB285048, AB285054, AB285055, AB285068, AB285071, AB285079, AB442219, AF034151, AF207763, AJ507189, EF407480, EU155219, X61596

182 tac ttt Y F AB285049, AB285066, AB285072

182 tac cac Y H DQ492003

182 tac cat Y H DQ492138

182 tac aac Y N AM494937

183 ctg gca L A AB285047, EU155365, EU155366, EU256076, EU529682

183 ctg gcg L A AF207760, AJ507160, AJ507164, AM402055, AM402060, AM402061, AM402066, AM402070, AM402074, AM402075, AM402077, AM402081, AM402084, AM402085, AM402088, AM402090, AM402094, AM402102, AM402106, AM402108, AM402111, AM402112, AM402113, AM402127, AY808030, D50484, EF407469, EU155222, EU155367, EU256062, EU256085, EU482849, EU482885, FJ958934, HM042059, HM042063

183 ctg ata L I AF165063, AF165064

183 ctg cca L P AB285073, AF165047, AF165048, AJ507171

183 ctg ccg L P AB049087, AB049089, AB049092, AB049094, AB049095, AB049096, AB154181, AB154182, AB154205, AB154206, AB285035, AB285038, AB285040, AB285042, AB285043, AB285044, AB285045, AB285048, AB285049, AB285050, AB285052, AB285053, AB285054, AB285055, AB285056, AB285057, AB285059, AB285060, AB285068, AB285075, AB285078, AB285079, AB429050, AB442219, AB442220, AF034151, AF165045, AF165046, AF165053, AF165055, AF165056, AF207755, AF207757, AF207763, AF207768, AF207769, AF207771, AF265149, AJ507169, AJ507172, AJ507185, AJ507189, AY240967, AY240969, AY808013, AY808037, D10934, D50482, DQ071885, DQ244141, DQ492134, EF407467, EF407480, EF407491, EF407498, EU155227, EU155306, EU155307, EU155326, EU155330, EU155336, EU155359, EU155360, EU155368, EU256075, EU256077, EU256084, EU256088, EU256102, EU482859, EU482881, EU660386, EU862835, GU451219, GU451220, GU451221, GU451222, GU451223, GU451224, HM042062, U45476, X61596

183 ctg cct L P AJ133096, EU256080, GU451218

183 ctg acg L T AB010250, AF165054, AM402115, AM402125, AY587016, D30613, D45172, D89815, EU857431

183 ctg gta L V AB285066, GQ913873

183 ctg gtg L V AB036519, AB036520, AB036521, AB049090, AB109543, AB154195, AB154196, AB191333, AB285036, AB285041, AB285072, AB285076,

134

AB285081, AJ000009, AJ507166, AJ507167, AJ507170, AJ507178, AJ507184, AJ507186, AJ507188, AJ507199, AM402056, AM402057, AM402058, AM402059, AM402062, AM402063, AM402064, AM402065, AM402067, AM402068, AM402069, AM402071, AM402072, AM402073, AM402076, AM402078, AM402079, AM402080, AM402082, AM402083, AM402086, AM402087, AM402089, AM402091, AM402092, AM402093, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402103, AM402104, AM402105, AM402107, AM402109, AM402110, AM402114, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402126, AM402128, AM402129, AY008261, AY808016, AY808023, AY808026, AY808027, AY808033, AY808041, D50481, D50485, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, EF407459, EF407461, EF407468, EF407477, EF407482, EF407487, EF407488, EU155217, EU155219, EU155223, EU155224, EU155228, EU155255, EU155318, EU155333, EU155356, EU155364, EU155370, EU155371, EU155376, EU155377, EU234062, EU255962, EU256001, EU256066, EU256090, EU256091, EU256099, EU482874, EU482883, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958935, GQ913869, HM042051, HM042052, HM042054, HM042055, HM042056, HM042057, HM042058, HM042061, M84754

184 gtt gct V A DQ491954

184 gtt atc V I AY808024

184 gtt att V I AB285036, AJ507194

185 ggg gaa G E AM402129

185 ggg gag G E AM401894, AM401940, DQ491947

186 tca tta S L DQ492011, DQ492012, DQ492013

186 tca cca S P FJ958904

186 tca cct S P AM402330

187 cag cgg Q R AM401971, AM402119, AM402326, DQ492109, DQ492133, DQ492168, DQ492174

188 ctc ttc L F AM401907

188 ctc cac L H DQ492115

188 ctc ccc L P FJ958935

189 cca cta P L DQ491967

189 cca tca P S DQ492173

190 tgc cgt C R DQ492154

191 gag ggg E G AM401860, AM402124, DQ492075, DQ492102, DQ492131

191 gag aag E K DQ492162

191 gag gtg E V AM402339

192 ccc gcc P A DQ492000

192 ccc tcc P S DQ492076

193 gaa gca E A DQ492187


193 gaa gat E D AM401933

193 gaa gta E V DQ492093

194 ccg ctg P L AJ507174, AJ507193, AM402323, EU155301, EU155316

195 gac ggc D G AJ132996, AM401886, AM493441

195 gac aac D N AJ133096

195 gac aat D N AM402061

196 gta gca V A DQ492133

135

196 gta gcg V A AM401907, AY808041, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970

196 gta aca V T AY808025

197 gca gaa A E EU256079

197 gca gag A E EF407491

197 gca ata A I AB285035

197 gca atg A M AB285048, AB285073, EF407467

197 gca caa A Q EF407488

197 gca aga A R EU256085

197 gca tca A S AB285067, AB285074, AB285081, AJ507174, EU155220, EU155226, EU155259, EU155279, EU155358

197 gca tcg A S EU155256

197 gca aca A T AB010250, AB036519, AB036520, AB036521, AB049089, AB049090, AB049092, AB049094, AB049095, AB049096, AB049100, AB049101, AB109543, AB191333, AB285040, AB285042, AB285043, AB285045, AB285047, AB285049, AB285050, AB285053, AB285054, AB285055, AB285056, AB285057, AB285058, AB285060, AB285064, AB285068, AB285075, AB285078, AB429050, AB442219, AB442220, AF033373, AF165045, AF165046, AF165047, AF165048, AF165049, AF165050, AF165053, AF165055, AF165056, AF207755, AF207757, AF207760, AF207768, AF265145, AF265146, AF265147, AF265148, AF265149, AF265150, AF265154, AF265155, AF265156, AF265158, AF265159, AF265163, AF265164, AF265165, AF483269, AJ132996, AJ132997, AJ133096, AJ507155, AJ507157, AJ507161, AJ507165, AJ507168, AJ507169, AJ507171, AJ507175, AJ507177, AJ507181, AJ507182, AJ507186, AJ507187, AJ507189, AJ507190, AJ507191, AJ507193, AJ507194, AJ507196, AJ507198, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM402329, AM402330, AM402331, AM402332, AM402333, AM402334, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AY240969, AY587016, AY808010, AY808011, AY808013, AY808014, AY808015, AY808020, AY808022, AY808023, AY808024, AY808025, AY808026, AY808027, AY808028, AY808029, AY808036, AY808037, AY808039, AY808043, D30613, D45172, D50484, D89815, DQ244141, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492074, DQ492075, DQ492076, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088,

136

DQ492089, DQ492090, DQ492091, DQ492092, DQ492093, DQ492124, DQ492125, DQ492127, DQ492128, DQ492129, DQ492130, DQ492131, DQ492132, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492139, DQ492140, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492148, DQ492149, DQ492150, DQ492151, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492161, DQ492162, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407460, EF407462, EF407466, EF407469, EF407470, EF407474, EF407475, EF407476, EF407477, EF407479, EF407485, EF407486, EF407489, EF407490, EF407499, EF407500, EF407502, EF407503, EF407504, EU155218, EU155223, EU155224, EU155225, EU155227, EU155230, EU155231, EU155255, EU155258, EU155280, EU155300, EU155301, EU155302, EU155307, EU155316, EU155318, EU155324, EU155325, EU155326, EU155327, EU155330, EU155335, EU155360, EU155361, EU155363, EU155368, EU155371, EU155373, EU155374, EU155375, EU255962, EU256001, EU256045, EU256059, EU256075, EU256077, EU256078, EU256082, EU256088, EU256089, EU256100, EU256103, EU482833, EU482849, EU482874, EU482875, EU482877, EU482879, EU482883, EU482888, EU857431, FJ024279, FJ390397, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958934, FJ958935, GQ913868, GU451218, GU451219, GU451220, HM042060, L02836, X61596

197 gca acc A T AY044867, EU256083

197 gca acg A T AB154177, AB154178, AB154192, AB285037, AB285052, AB285066, AB285079, AF165054, AJ507167, AJ507188, AY808034, D50480, EU155232, EU234062, EU482880, EU862837

197 gca act A T AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507

197 gca gta A V AM402066, AM402119, EU155260, EU256092, EU256101

197 gca gtg A V AF207771, AJ000009, DQ492025, DQ492033, EF407495

197 gca gtt A V AY808041, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970

198 gtg gcg V A AM402374, DQ491970, DQ492099, EU256059


198 gtg atg V M AM401914

198 gtg aca V T EU256092

198 gtg acg V T AF483269, EU482833

199 ctc gcc L A AM402326

199 ctc ttc L F AF207754, AM401941, DQ492179

199 ctc atc L I AB049089, AB049096, AB285040, AB285042, AB285043, AB285056, AB285068, AB429050, AJ507171, D50484, EU256077, EU256085, EU256101

199 ctc att L I AJ507167

199 ctc ccc L P AF165050

199 ctc gtc L V AB010250, AB049094, AB285035, AB285048, AB285050, AB285052, AB285064, AB285073, AB285078, AB285079, AB442219, AB442220, AF165045, AF165046, AF165053, AF165054, AF207755, AF207757, AF207771, AF265148, AF265149, AF265150, AF265154, AF265163,

137

AF265164, AF265165, AJ000009, AJ507169, AJ507189, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AY587016, D30613, D45172, D89815, DQ244141, EF407467, EF407502, EF407504, EU155260, EU155318, EU155335, EU155368, EU256088, EU482877, EU857431

199 ctc gtt L V AB285049, EF407495

200 act gct T A AB154177, AB154178, AF207774, AM402363, AY808041, DQ491957, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ491977, DQ492079, DQ492173, EF407500

200 act tct T S DQ492175

201 tcc ccc S P DQ492039, DQ492095, DQ492144

202 atg ata M I DQ491989, DQ492081

202 atg ctg M L AB049101, AY808037

202 atg ttg M L EU155225


202 atg gtg M V DQ492023, DQ492028, DQ492035, DQ492158

203 ctc ttc L F AM402059, AM402062, AM402065, AM402079, AM402083, AM402091, AM402123, AM402124, AY808016, AY808030, AY808032, DQ492074, DQ492181, EU155365, EU155367, EU529682

203 ctc ttt L F EU482833

203 ctc ccc L P AM402086

204 acc gcc T A AM402117, AY240969, DQ244140, DQ492068, FJ958905

204 acc atc T I AF483269, EU155263

204 acc att T I DQ492087

204 acc tcc T S AF207769, AF265158, AF265159

205 gac ggc D G AM493486, DQ492073

205 gac aac D N DQ492068

207 tcc gcc S A AY808041, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, EU155369, EU155374, EU256065, EU256066

207 tcc ccc S P AB049092, AF207768, AF265154, AF265155, AF265156, AM493490, AY044867, EU155223

207 tcc cct S P AM401871

208 cac cgc H R DQ491945, DQ492137

209 att acc I T AM401858, AM402129

209 att act I T DQ492042

210 acg gca T A AM401855, AM402372

210 acg gcc T A AM402301, AM402308

210 acg ata T I DQ491945, DQ492187

211 gcg gga A G D50480, D50483

211 gcg gta A V DQ491948

212 gag gac E D EU255961

212 gag ggg E G AM401882

212 gag cac E H AM401868, AM401879

138

213 acg gca T A FJ958907

213 acg gcg T A AB010250, AB049089, AB049092, AB049098, AB154177, AB154178, AB154179, AB154181, AB154182, AB154185, AB154186, AB154187, AB154188, AB154191, AB154192, AB154193, AB154194, AB154197, AB154198, AB154199, AB154200, AB154201, AB154202, AB154205, AB154206, AB285037, AB285040, AB285042, AB285046, AB285047, AB285051, AB285054, AB285055, AB285060, AB285065, AB285066, AB285067, AB285071, AB285072, AB285073, AB285076, AB285081, AB429050, AB442222, AF033373, AF165055, AF207760, AF207768, AF208024, AF265142, AF265143, AF265144, AF265160, AF265162, AF313916, AJ132997, AJ507159, AJ507162, AJ507169, AJ507171, AJ507172, AJ507195, AM401916, AM401919, AM401920, AM401921, AM401923, AM401925, AM401926, AM401929, AM401933, AM401934, AM401935, AM401937, AM401938, AM401942, AM401946, AM401948, AM401955, AM401957, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AM402339, AM494937, AY587016, AY587844, AY808013, AY808014, AY808016, AY808023, AY808027, AY808030, D10934, D30613, D45172, D50480, D50485, D85516, D89815, DQ244141, DQ492093, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407459, EF407492, EF407497, EF407503, EU155219, EU155221, EU155232, EU155254, EU155255, EU155300, EU155301, EU155307, EU155316, EU155317, EU155365, EU155367, EU155368, EU155369, EU155377, EU234062, EU256045, EU256075, EU256085, EU482849, EU482875, EU482886, EU529682, EU857431, EU862835, FJ390397, FJ390398, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958934, FJ958935, FN435993, GQ913870, HM042060

213 acg atg T M AM493481, DQ492028

213 acg tcg T S EU155217

213 acg gtg T V AY044867

214 gct gtt A V AM402115, DQ492050

215 aag gcg K A AJ507156

215 aag gaa K E DQ492132, DQ492135

215 aag gga K G DQ492197

215 aag ggc K G AY808015

215 aag ggg K G AB285045, AY808014, AY808019, AY808021, AY808041, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ492093, DQ492094, DQ492095, DQ492096, DQ492097, DQ492098, DQ492099, DQ492100, DQ492101, DQ492102, DQ492103, DQ492104, DQ492105, DQ492106, DQ492107, DQ492108, DQ492109, DQ492110, DQ492111, DQ492112, DQ492113, DQ492114, DQ492115, DQ492116,

139

DQ492117, DQ492118, DQ492119, DQ492120, DQ492121, DQ492122, DQ492123, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, EF407473, EF407489, EU155224, EU155377, EU256075

215 aag ata K I DQ492133

215 aag aga K R AB285049, AB442222

215 aag agg K R AB010250, AB049087, AB049092, AB049094, AB049099, AB049101, AB154185, AB154186, AB285040, AB285042, AB285046, AB285052, AB285058, AB285064, AB285073, AB285076, AB285078, AB285079, AB442219, AF165051, AF165052, AF165053, AF165054, AF165061, AF165062, AF207762, AF207764, AF207768, AF207769, AF207772, AF265154, AF265155, AF265156, AJ507171, AJ507189, AM401876, AM402330, AM402332, AM402333, AM402335, AM402337, AM402338, AM402345, AM402346, AM402347, AM402348, AM402350, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402381, AM402382, AM402383, AM402384, AY587016, AY808025, D30613, D45172, D89815, DQ244141, EF407467, EF407485, EF407498, EF407502, EU155219, EU155227, EU155231, EU155255, EU155382, EU256079, EU482879, EU857431, HM042058, HM042059

216 cgt cac R H DQ491954, DQ492161

216 cgt ccc R P DQ492157

217 agg aaa R K AB049094, GQ913869

217 agg aag R K AF034151

217 agg atg R M AJ507165

217 agg agc R S AM401863

218 ctg ccg L P AB010249, AM402117, AM402332, D16435, D89872

218 ctg cgg L R AM401915

218 ctg tcg L S DQ492182

219 gcc gac A D AJ000009, AJ132996, AM493493, AY044867, EF407499, EU155374, EU660388

219 gcc gat A D AF356827

219 gcc gaa A E AJ507172

219 gcc ggc A G AY587844, EU155264, EU482879

219 gcc ccc A P AY240968

219 gcc acc A T AM401907

219 gcc gtc A V AM401902, FN435993


222 tct tgt S C AB049089, AM401905

222 tct cca S P AM402295

222 tct ccc S P DQ492060, FJ958931

222 tct cct S P D63857, DQ491976, DQ491990, DQ492064, DQ492095

222 tct acc S T EU155219, EU155333, EU482885

223 ccc gct P A EU155219, EU155333, EU482885

223 ccc ggg P G AY808017

223 ccc tcc P S AF176573, EF407465

224 ccc ggg P G AY808017

224 ccc tcc P S DQ492154

225 tcc ttc S F AB285075

226 ttg gca L A EU155305

226 ttg atg L M AB049091, AJ133096

140

226 ttg ccg L P AM402269

226 ttg cgg L R DQ492171

226 ttg tcg L S AM402086, AM402329, DQ492100, DQ492149

226 ttg gtg L V FJ390396

226 ttg tgg L W AM401918

227 gcc ccc A P DQ492142

227 gcc gtc A V DQ492080

228 agc ggc S G AM401850, AM402291, AM402308, DQ492021, HM042059

228 agc ggt S G D89815, FJ958903

228 agc aac S N DQ492162

228 agc agg S R DQ492100

228 agc cgc S R AB109543, AB191333, AB285050


230 tca gca S A FJ958905

230 tca tta S L AM401864

230 tca cca S P DQ491991, DQ492103

231 gct acc A T AM401962

231 gct act A T AM402266, AM402299, AM402361

231 gct gtt A V AM402337

232 agc tgt S C DQ492186

232 agc acc S T AB285063

232 agc act S T AB049092

233 cag cgg Q R DQ492042, DQ492161

234 ctg ttc L F DQ492042

234 ctg ccg L P DQ492172

234 ctg tcg L S AM402320, DQ492019, DQ492032

235 tct ttt S F AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401877, AM401879, AM402303

236 gcg acg A T DQ492034

236 gcg gtg A V AM493485

237 cct gct P A EU482849, EU529682, EU862835

237 cct ttt P F AJ507169

237 cct att P I GQ913873

237 cct ctt P L AB010250, AB285041, AY008261, AY587016, D30613, D45172, D89815, EF407461, EF407468, EU857431, M84754

237 cct tct P S AM402315

237 cct act P T DQ492048

237 cct gtc P V AB285040

237 cct gtt P V AY240968, EF407467, EU155306, HM042056

238 tcc gcc S A DQ492070

238 tcc ccc S P AM402087

238 tcc cct S P AM401900, AM401903

239 ttg atg L M EU482874, HM042054

239 ttg ccg L P DQ492025, DQ492033

239 ttg tcg L S AM401862, AM402312, AY808031, D50483

240 aag gag K E AM402274, AM402299, DQ492181, DQ492191

240 aag agg K R AB285054, AB285055, AJ507183, AM402103, AY808013, AY808025, EU256081, EU482879, FJ024279, FJ958890, FJ958896, GQ913868, GQ913871, HM042050, HM042062

240 aag tgg K W AY240967

141

241 gca ccg A P EF407461

241 gca gtg A V AY240969, FJ958895

242 aca gca T A AB049091, AF165047, AF165048, AJ507169, AM401884, AM402327, DQ492034, DQ492136, EF407467, EF407468, EU155306, EU255961, EU529682, GQ913871, GQ913873, HM042056

242 aca gcg T A AB285040, AY240968, DQ492165, M84754

242 aca cac T H HM042062

242 aca aag T K AY240969

243 tgc cgc C R AM401939, AM402346, DQ491972, DQ491973

243 tgc tac C Y AM401936, D50483, DQ492046, DQ492142

244 act gcc T A AB285060

244 act gct T A DQ244141, DQ492016, DQ492047, HM042062

244 act ggt T G HM042058

244 act att T I AY808031, D50483, DQ492158

244 act aat T N DQ244140

244 act ccc T P AB285076

244 act cct T P AB154195, AB154196, AF165062, DQ492160

245 acc gcc T A AB285040, AB429050, AF265154, AF265155, AF265156, AJ507175, AM402308, AY008261, AY808025, AY808029, AY808043, DQ492063, DQ492064, DQ492065, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492074, DQ492075, DQ492076, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, EU155260, EU155306, EU155360, EU155361, EU482879, EU482880, EU482888, EU529682, HM042059

245 acc atc T I AB285070, AY240969, D14484, DQ492131

245 acc aac T N EU155332, EU862835

245 acc tac T Y AB154195, AB154196

246 cgt tgc R C AB049087, AB049092, AF207768, D50484, EU155360

246 cgt tgt R C AB285054, AB285055, AB285076, AJ507189, AM401908, AY240969, AY808013, AY808043, DQ492113, DQ492114, DQ492117, DQ492118, DQ492119, EF407494, EU482875, HM042059

246 cgt cac R H AB010249, AB049099, AB080299, AB285035, AB285039, AB285048, AB285057, AB285060, AB285061, AB285072, AF207753, AF207754, AF207758, AF207769, AF207770, AF333324, AF483269, AJ507159, AJ507163, AJ507179, AJ507182, AJ507183, AM493484, AY808025, AY808032, AY808039, AY808040, D11168, D16435, D50482, D85516, D89872, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ492080, EF407480, EF407498, EF407500, EF407504, EU155254, EU155302, EU155316, EU155336, EU155361, EU155375, EU155382, EU255961, EU256099, EU482874, EU482885, EU482886, GU451218, GU451219, GU451220, M58335

246 cgt cat R H AB010250, AB036519, AB036520, AB036521, AB049088, AB049091, AB049094, AB049096, AB049098, AB049100, AB049101, AB109543, AB191333, AB249644, AB285036, AB285037, AB285038, AB285043, AB285044, AB285053, AB285058, AB285059, AB285062, AB285063, AB285064, AB285069, AB285070, AB285073, AB285077, AB285078, AB426117, AB442219, AB442220, AB442221, AB442222, AF165047, AF165048, AF165049, AF165050, AF165055, AF165056, AF165057, AF165058, AF165059, AF165060, AF165061, AF207752, AF207756, AF207762, AF207763, AF207764, AF207765, AF207772, AF207774, AF208024, AF265148, AF265158, AF265159, AJ133096, AJ507155, AJ507156, AJ507162, AJ507165, AJ507172, AJ507175, AJ507177, AJ507180, AJ507181, AJ507184, AJ507192, AJ507197, AJ507198, AM402329, AM402331, AM402334, AM402336, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM493497, AM493501, AM493504, AY045702, AY240967, AY240968, AY460204, AY587016, AY808010, AY808012, AY808017, AY808023, AY808024, AY808027,

142

AY808034, AY808042, D10934, D14484, D30613, D45172, D50480, D50481, D63857, D89815, D90208, DQ244140, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492049, DQ492051, DQ492052, DQ492053, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, DQ492065, DQ492066, DQ492067, DQ492068, DQ492070, DQ492073, DQ492074, DQ492078, DQ492079, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, DQ492127, DQ492129, DQ492130, DQ492131, DQ492140, DQ492157, EF032892, EF032893, EF032894, EF407462, EF407466, EF407477, EF407479, EF407485, EF407487, EF407491, EF407497, EF407502, EF638081, EU155219, EU155226, EU155227, EU155253, EU155257, EU155259, EU155262, EU155279, EU155328, EU155335, EU155357, EU155358, EU155359, EU155362, EU155367, EU155368, EU155369, EU155370, EU155371, EU155372, EU155377, EU155381, EU239714, EU255960, EU255962, EU256059, EU256077, EU256079, EU256082, EU256083, EU256088, EU256090, EU256101, EU256102, EU482833, EU482839, EU482859, EU482860, EU482877, EU482883, EU781832, EU857431, FJ024277, FJ390396, FJ478453, GU451221, GU451222, GU451223, GU451224, L02836, M96362, U01214, U16362

246 cgt aaa R K EU155317, EU256080, EU256091

246 cgt ctt R L AB154195, AB154196, AB285051, AY808026

246 cgt aac R N EU155325

246 cgt aat R N AB285047, AF165051, AF165052, AF165062, AY808009, DQ859970, EU155306, EU256084

246 cgt caa R Q AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401931, AM401932, AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401944, AM401945, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AM402330, AM402332, AM402333, AM402335, AM402337, AM402338, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, AY808031, D50483, EU155376

246 cgt cag R Q AB154205, AB154206, AM401930, EU482880

246 cgt tac R Y AB049089

246 cgt tat R Y EF407481

247 cat ttt H F HM042058

247 cat aaa H K AB285059

247 cat ctt H L AJ507189

247 cat aac H N AY240969, HM042062

247 cat cct H P AY808031

247 cat caa H Q AB049090, AM402077, EU155377

247 cat cgt H R AB285038, AB285044, AB285060, AF207762, EU155302

247 cat tat H Y D50483

143

248 gac gaa D E AF265156, EU256078

248 gac ggc D G AB010250, AB049089, AF207769, AJ507189, AM401938, AY587016, D30613, D45172, D89815, EF407481, EU857431, HM042062

248 gac cac D H AB049091

248 gac aac D N AB049092, DQ244141, DQ492054, DQ492055, DQ492085

248 gac aat D N EU256084

248 gac cgc D R AB285043, DQ244140

248 gac acc D T M84754

248 gac gtc D V AF333324, AY808032, EU482875, M58335

249 tcc gcc S A AB010250, AB049092, AY587016, D30613, D45172, D89815, EU155326, EU857431

249 tcc tgc S C AJ507189

249 tcc ttc S F AY808031, DQ492096

249 tcc ccc S P AB285076, EF407467, EF407481, M84754

249 tcc cct S P AJ507169, HM042062

250 ccg gca P A AB285078

250 ccg ctg P L DQ492191

250 ccg caa P Q EU255962

250 ccg acg P T AY460204, EF638081

251 gac gcc D A AB285067

251 gac gag D E AB049100

251 gac ggc D G AF165062, AM402382, DQ859970

251 gac aac D N AJ507189, DQ491979

252 gct ttt A F D50483

252 gct att A I AB285056

252 gct ctc A L DQ491983

252 gct ctt A L AB049091, AB285043, DQ244140, DQ244141, DQ491980, DQ491981, DQ491984, DQ491989, DQ491994, DQ492047, DQ492048, DQ492050, DQ492054, DQ492055, EF407480

252 gct cct A P AB049100

252 gct aca A T DQ492090

252 gct acc A T AF207769, DQ492021, DQ492129, DQ492130, DQ492140, DQ492150

252 gct act A T AB010250, AY587016, D30613, D45172, D89815, EU482849, EU857431

252 gct gta A V EU529682

252 gct gtc A V AB285044, AB429050, AM493443, AY008261, EF407467, EU155332, EU155366, HM042059, HM042062

252 gct gtt A V AB285040, AB285076, AB285078, AF165061, AF165062, AF207757, AF207762, AJ507191, AY045702, AY240967, AY240969, AY808039, DQ491967, EF407461, EF407473, EF407488, EU155220, EU155221, EU155222, EU155264, EU155280, EU155306, EU155324, EU234061, EU255962, EU482839, FJ390397, FJ478453, GQ913872, GQ913873, HM042051

253 gac gcc D A DQ491980, DQ491981, DQ491983, DQ491984, DQ491989, DQ491994

253 gac gaa D E AB442220, EF407468

253 gac gag D E AJ507169, AY240968

253 gac ggc D G AF356827

254 ctc ttc L F AM401890, AM402370, DQ492127

254 ctc ccc L P DQ492106

255 atc ctc I L AY808039, EU155306

255 atc agc I S DQ492017

255 atc acc I T EF407480, HM042062

255 atc act I T AM401882

255 atc gtc I V AB285040, AJ507169, AJ507196, AM402335, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484,

144

AM493485, AM493486, AM493487, AM493488, AY240969, DQ492098, EF407459, EF407478, EF407481

255 atc gtt I V AF207769, AY008261, HM042058, HM042059

256 gag gac E D AY808016

256 gag gat E D AM401849, AY008261

257 gcc acc A T AM402076, DQ492129, DQ492130, DQ492140

257 gcc gtc A V AM493477, DQ491955

258 aac gac N D AM401856, DQ491955

258 aac gat N D AM402296, AM402315

258 aac cac N H U45476

258 aac atc N I DQ492172

258 aac agc N S EF407473, EU155367

258 aac agt N S AB154203, AB154204

259 ctc ttc L F DQ492182

259 ctc ccc L P AM402072, DQ492054, DQ492055

260 ctg atg L M AF207769, AY808028, DQ491980, DQ491981, DQ491983, DQ491984, DQ491989, DQ491994, DQ492040, DQ492047, DQ492048, DQ492050, DQ492054, DQ492055, EF407481, HM042059

260 ctg ccg L P DQ491967, DQ492087, DQ492099

261 tgg cgg W R AM402313, DQ492175

262 cgg cac R H AF165061, AF165062

262 cgg aag R K AJ507155

262 cgg cag R Q AB285040, DQ491965, DQ492114, DQ492169, EU155306, EU529682

262 cgg tgg R W AB049088, AB285043, AB285044, AB285064, AY808026, AY808028, DQ244140, DQ244141, DQ491980, DQ491981, DQ491983, DQ491984, DQ491989, DQ491994, DQ492015, DQ492040, DQ492047, DQ492048, DQ492050, DQ492054, DQ492055, DQ492125, DQ492172, EF407480, EF407481, HM042059, HM042062

263 cag cat Q H AB285076, AY808026

263 cag cgg Q R AM401962, AM402287, AM402384, HM042059

264 gag gca E A AJ507173

264 gag gcg E A EU155232, EU155281, EU155359

264 gag aag E K AM493487, AM493488

264 gag cag E Q AB285044

264 gag acg E T EF407494

264 gag gtg E V AF207769, EU529682

265 atg ata M I AM402102, DQ492072, DQ492151

265 atg aaa M K DQ491980, DQ491981, DQ491983, DQ491984, DQ491989, DQ491994

265 atg aag M K AB285043, AB285064, DQ244140, DQ244141, DQ492040, DQ492047, DQ492048, DQ492050, DQ492054, DQ492055

265 atg ctg M L AY808014, AY808026, DQ492093, DQ492163, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492179, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492187, DQ492190, DQ492191, DQ492192, DQ492193, DQ492196


265 atg gtg M V DQ492075, DQ492104, EF407480, EU482880

266 ggc gcc G A EF407480

266 ggc tgc G C AY587844

266 ggc gac G D DQ492019

266 ggc gat G D EU256088

266 ggc aac G N EU155281

266 ggc aat G N EF407489

267 ggg gcg G A EF407503

267 ggg gat G D AB154203, AB154204

145

267 ggg gag G E DQ491978, DQ492151

267 ggg aat G N EU256075

267 ggg tca G S AY808014, DQ492093, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197

268 aac gac N D AB285076, AM401964, AM402124, AM402275, AY808031, EF407481

268 aac gat N D EF407466

268 aac gaa N E AY008261

268 aac atc N I AJ507163

268 aac aaa N K AY808026

268 aac agc N S AF207769, AJ507169, AY808037, DQ492167, EF407467, HM042062

268 aac acc N T AB285040, GQ913873

269 atc cac I H AY240969

269 atc acc I T AM401853, AM401890, AM401976, AM402379

269 atc gtc I V AM402083, AM402259, AY808026, DQ492020, DQ492026, DQ492144

270 acc gcc T A AM401867, AM401878, AM402353, DQ492180

270 acc atc T I DQ491997

270 acc ttg T L EU155325

270 acc tcc T S AB049088, AB285044, AY808037

270 acc gtc T V EU256084

270 acc tac T Y EF407480

271 cgc cac R H AM401966, AM402321

271 cgc ccg R P DQ492011

272 gtg gcg V A DQ492113, DQ492117, DQ492118, DQ492119, DQ492191

272 gtg gag V E AM401858

273 gag gat E D AB285073

273 gag ggg E G AM402356, DQ492002, DQ492087, FJ958895


273 gag gtg E V DQ492132, DQ492135


275 gaa gac E D AY240967

275 gaa ggg E G AM401973, DQ491957, DQ492034, DQ492188

276 aat gcc N A EU256091

276 aat gct N A AY587844

276 aat gac N D AM401974

276 aat aaa N K HM042056

276 aat aag N K EU155306

276 aat agc N S AM401857, DQ492114, DQ492167, EF407471, EU155325, EU862837

276 aat agt N S DQ492053, U01214

277 aag gaa K E DQ492176, DQ492183

277 aag gag K E DQ492085

278 gta gca V A AM402315, AM402328, AM402383

278 gta ata V I AB010250, AF165053, AF165054, AY587016, D30613, D45172, D89815, DQ492028, EU155260, EU155362, EU857431

278 gta aca V T AY808017

279 gta gcg V A AM402113

279 gta ata V I AB049088, AB049101, AB285043, AB285064, AB285071, AB442222, AJ507169, AM401877, AY808014, AY808028, DQ244140, DQ244141, DQ491980, DQ491981, DQ491983, DQ491984, DQ491989, DQ492040, DQ492047, DQ492048, DQ492050, DQ492054, DQ492055, DQ492093,

146

DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407503, EU155333, EU482880, GU451219, GU451220

279 gta att V I D50480

280 att acc I T D50483

280 att gtc I V AF207757, AF333324, AJ507199, AY808030, AY808042, AY808043, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, EF407459, EF407465, EF407471, EF407494, EU155217, EU155221, EU155264, EU155329, EU155367, EU234062, EU256085, EU256103, EU781832, FJ478453, M58335

280 att gtt I V AB285046, AB285056, AB285081, AF265145, AF265146, AF265147, AJ507173, AY808011, DQ492124, DQ492125, DQ492126, DQ492128, DQ492129, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492139, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492148, DQ492149, DQ492150, DQ492151, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492161, DQ492162, EF407473, EF407484, EF407486, EU155223, EU155225, EU155226, EU155229, EU155230, EU155263, EU155324, EU155330, EU155332, EU155334, EU155375, EU155376, EU256061, EU256084, EU256090, EU256091, EU482839, EU482877, EU660386, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958934, FJ958935

281 ttg atg L M AB285043, DQ244140, HM042051

281 ttg cca L P HM042059

281 ttg ccg L P AM401947, DQ492017, EF407481

282 gac ggc D G AM402299, DQ492094, EF638081

283 tct gcc S A DQ491977

283 tct ccc S P DQ491963

283 tct cct S P AM401953, DQ492054, DQ492055

284 ttc tta F L DQ492188

284 ttc ttg F L AM402106

284 ttc tcc F S DQ492004, DQ492116, DQ492145

284 ttc gtc F V DQ492044, DQ492045, DQ492051, DQ492052

285 gag tgc E C AY808034, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039

285 gag gac E D AB010249, AB036519, AB036520, AB036521, AB049087, AB049088, AB049089, AB049094, AB049096, AB049099, AB049100, AB080299, AB109543, AB191333, AB285035, AB285036, AB285037, AB285038, AB285039, AB285044, AB285048, AB285050, AB285051, AB285052, AB285053, AB285054, AB285055, AB285056, AB285057, AB285061, AB285064, AB285069, AB285070, AB285073, AB285075, AB285077, AB285078, AB285079, AB442220, AF034151, AF165045, AF165046, AF165047, AF165048, AF165049, AF165050, AF165051, AF165052, AF165053, AF165054, AF165055, AF165056, AF165057, AF165058, AF165059, AF165060, AF165062, AF165063, AF165064, AF176573, AF207755, AF207756, AF207758, AF207763, AF207764, AF207765, AF207768, AF207770, AF207771, AF207772, AF207774, AF208024, AF265142, AF265143, AF265144, AF265154, AF265155, AF265156,

147

AF265158, AF265159, AF265160, AF265161, AF265162, AF333324, AF483269, AJ000009, AJ133096, AJ507156, AJ507159, AJ507162, AJ507163, AJ507164, AJ507165, AJ507166, AJ507167, AJ507168, AJ507169, AJ507170, AJ507174, AJ507175, AJ507177, AJ507178, AJ507179, AJ507180, AJ507181, AJ507182, AJ507183, AJ507184, AJ507188, AJ507189, AJ507190, AJ507191, AJ507192, AJ507194, AJ507197, AJ507198, AJ507199, AM401853, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AM401943, AM401959, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AM402329, AM402330, AM402331, AM402332, AM402333, AM402334, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AM494937, AY240967, AY240968, AY240969, AY460204, AY808009, AY808010, AY808012, AY808013, AY808014, AY808015, AY808016, AY808017, AY808018, AY808019, AY808021, AY808023, AY808024, AY808027, AY808029, AY808030, AY808031, AY808035, AY808037, AY808039, AY808040, AY808041, AY808043, D11168, D16435, D50480, D50482, D50483, D50484, D63857, D85516, D89872, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492074, DQ492075, DQ492076, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, DQ492093, DQ492094, DQ492095, DQ492096, DQ492097, DQ492098, DQ492099, DQ492100, DQ492101, DQ492102, DQ492103, DQ492104, DQ492105, DQ492106, DQ492107, DQ492108, DQ492109, DQ492110, DQ492111, DQ492112, DQ492113, DQ492114, DQ492115, DQ492116, DQ492117, DQ492118, DQ492119, DQ492120, DQ492121, DQ492122, DQ492123, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177,

148

DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, DQ859970, EF032892, EF032893, EF032894, EF407460, EF407462, EF407464, EF407466, EF407474, EF407475, EF407476, EF407477, EF407478, EF407479, EF407482, EF407483, EF407488, EF407497, EF407498, EF407501, EF407502, EF407504, EF638081, EU155218, EU155219, EU155220, EU155222, EU155227, EU155228, EU155231, EU155235, EU155253, EU155254, EU155257, EU155259, EU155279, EU155302, EU155303, EU155307, EU155315, EU155316, EU155317, EU155325, EU155327, EU155331, EU155333, EU155335, EU155336, EU155337, EU155356, EU155357, EU155358, EU155360, EU155362, EU155363, EU155364, EU155365, EU155366, EU155367, EU155368, EU155369, EU155370, EU155371, EU155372, EU155373, EU155375, EU155377, EU155381, EU155382, EU255961, EU256000, EU256045, EU256059, EU256062, EU256065, EU256066, EU256075, EU256076, EU256077, EU256078, EU256079, EU256081, EU256082, EU256085, EU256089, EU256090, EU256092, EU256101, EU256103, EU482833, EU482859, EU482860, EU482874, EU482875, EU482879, EU482883, EU482885, EU482886, EU482888, EU781825, EU862837, FJ024277, FJ390396, FJ390397, FJ390398, FN435993, GQ913869, GU451218, GU451219, GU451220, GU451221, GU451222, GU451223, GU451224, HM042050, HM042051, HM042052, HM042054, HM042055, HM042056, HM042057, HM042061, HM042063, L02836, M58335, M84754, M96362, U01214, U16362, U45476

285 gag gat E D AB049091, AB049095, AB049098, AB249644, AB285047, AB285060, AB285062, AB285066, AB285068, AB285072, AB426117, AB442219, AB442221, AB442222, AF033373, AF165061, AF207752, AF207753, AF207754, AJ132996, AJ132997, AJ507155, AJ507157, AJ507158, AJ507160, AJ507161, AJ507171, AJ507172, AJ507186, AJ507193, AM401848, AM401849, AM401850, AM401851, AM401852, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401931, AM401932, AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401944, AM401945, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401957, AM401958, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AY045702, AY808025, AY808032, AY808033, AY808036, AY808042, D90208, DQ244140, DQ244141, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, EF407468, EF407485, EF407487, EF407489, EF407491, EU155232, EU155256, EU155262, EU155318, EU155359, EU155361, EU255960, EU256064, EU256080, EU256083, EU256084, EU256088, EU256099, EU256102, EU482880, EU781827, EU781830,

149

GQ913873

285 gag aac E N DQ492171

285 gag acc E T AB285059

285 gag gta E V HM042059

285 gag gtc E V AF207769, AM402378, EF407461, EU529682, EU862835, HM042058

285 gag gtg E V EF407481

286 ccg aag P K AB285054, AB285055, AB285073

286 ccg ctg P L AM402076, DQ491989, DQ492053

286 ccg cag P Q AB049096, AM494937

286 ccg tcg P S AF165049, AF165050, EU256065, EU482886

287 ctc tgt L C AB049101

287 ctc ttt L F AM402277, HM042062

287 ctc att L I AB285037, AF165053, AF165054, AF207772, D14484, D90208

287 ctc cct L P AM401933

288 caa cac Q H AF207756

288 caa cat Q H AB049098, AF034151, AY808026, D85516, EF407490, EU155302, EU482874, HM042053

288 caa aat Q N EU155258

288 caa aga Q R AB154189, AB154190, AJ507166, AJ507169, AM402064, EU155331

288 caa cga Q R AB010249, AB010250, AB036519, AB036520, AB036521, AB049089, AB049092, AB049094, AB049095, AB049099, AB049101, AB080299, AB109543, AB154177, AB154178, AB154179, AB154180, AB154181, AB154182, AB154183, AB154184, AB154185, AB154186, AB154187, AB154188, AB154191, AB154194, AB154195, AB154196, AB154197, AB154198, AB154199, AB154200, AB154201, AB154202, AB154203, AB154204, AB154205, AB154206, AB191333, AB249644, AB285036, AB285037, AB285038, AB285039, AB285040, AB285047, AB285048, AB285050, AB285051, AB285052, AB285053, AB285056, AB285057, AB285058, AB285059, AB285061, AB285063, AB285064, AB285065, AB285067, AB285068, AB285069, AB285070, AB285071, AB285072, AB285073, AB285074, AB285075, AB285078, AB285079, AB285081, AB426117, AB442219, AB442220, AB442221, AB442222, AF033373, AF165045, AF165046, AF165047, AF165048, AF165049, AF165050, AF165051, AF165052, AF165053, AF165054, AF165055, AF165056, AF165057, AF165058, AF165059, AF165060, AF165061, AF165062, AF165063, AF165064, AF176573, AF207752, AF207754, AF207755, AF207757, AF207758, AF207762, AF207763, AF207764, AF207765, AF207766, AF207768, AF207769, AF207770, AF207771, AF207772, AF207774, AF208024, AF265142, AF265143, AF265144, AF265145, AF265146, AF265147, AF265152, AF265153, AF265154, AF265155, AF265156, AF265158, AF265159, AF265160, AF265161, AF265162, AF265163, AF313916, AF333324, AF356827, AF483269, AJ000009, AJ132996, AJ132997, AJ238800, AJ507156, AJ507157, AJ507158, AJ507159, AJ507162, AJ507163, AJ507164, AJ507165, AJ507167, AJ507168, AJ507170, AJ507171, AJ507173, AJ507175, AJ507178, AJ507179, AJ507180, AJ507181, AJ507182, AJ507185, AJ507186, AJ507189, AJ507190, AJ507191, AJ507192, AJ507193, AJ507194, AJ507195, AJ507196, AJ507197, AJ507198, AJ507199, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401931, AM401932,

150

AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401944, AM401945, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401956, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AM402329, AM402330, AM402331, AM402332, AM402333, AM402334, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AM494937, AY008261, AY044867, AY045702, AY240967, AY240968, AY240969, AY460204, AY587016, AY587844, AY808009, AY808010, AY808011, AY808012, AY808014, AY808017, AY808018, AY808019, AY808021, AY808023, AY808024, AY808025, AY808027, AY808028, AY808029, AY808030, AY808032, AY808033, AY808035, AY808036, AY808037, AY808038, AY808039, AY808040, AY808041, AY808042, AY808043, D10934, D11168, D14484, D16435, D30613, D45172, D50480, D50481, D50482, D50483, D50484, D50485, D63857, D89815, D89872, DQ244140, DQ244141, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492074, DQ492075, DQ492076, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, DQ492093, DQ492094, DQ492095, DQ492096, DQ492097, DQ492098, DQ492099, DQ492100, DQ492101, DQ492102, DQ492103, DQ492104, DQ492105, DQ492106, DQ492107, DQ492108, DQ492109, DQ492110, DQ492111, DQ492112, DQ492113, DQ492114,

151

DQ492115, DQ492116, DQ492117, DQ492118, DQ492119, DQ492120, DQ492121, DQ492122, DQ492123, DQ492124, DQ492125, DQ492126, DQ492127, DQ492128, DQ492129, DQ492130, DQ492131, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492139, DQ492140, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492148, DQ492149, DQ492150, DQ492151, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492161, DQ492162, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, DQ859970, EF032892, EF032893, EF032894, EF407459, EF407460, EF407461, EF407462, EF407463, EF407464, EF407465, EF407466, EF407468, EF407469, EF407470, EF407471, EF407472, EF407473, EF407475, EF407476, EF407477, EF407478, EF407480, EF407481, EF407482, EF407484, EF407485, EF407488, EF407489, EF407491, EF407492, EF407495, EF407497, EF407498, EF407499, EF407500, EF407501, EF407502, EF638081, EU155217, EU155218, EU155219, EU155221, EU155222, EU155223, EU155224, EU155226, EU155227, EU155228, EU155229, EU155230, EU155231, EU155232, EU155235, EU155253, EU155254, EU155256, EU155257, EU155259, EU155261, EU155262, EU155263, EU155264, EU155279, EU155280, EU155281, EU155303, EU155304, EU155306, EU155307, EU155315, EU155317, EU155318, EU155325, EU155326, EU155327, EU155328, EU155329, EU155330, EU155332, EU155334, EU155335, EU155336, EU155337, EU155356, EU155357, EU155358, EU155360, EU155361, EU155362, EU155364, EU155365, EU155366, EU155367, EU155368, EU155369, EU155370, EU155371, EU155372, EU155373, EU155375, EU155376, EU155377, EU155381, EU155382, EU234061, EU234062, EU255960, EU255961, EU255962, EU256000, EU256001, EU256045, EU256059, EU256061, EU256062, EU256064, EU256065, EU256075, EU256076, EU256079, EU256080, EU256081, EU256082, EU256083, EU256084, EU256085, EU256088, EU256089, EU256090, EU256101, EU256102, EU256103, EU482833, EU482839, EU482849, EU482859, EU482860, EU482875, EU482877, EU482883, EU482885, EU482886, EU482888, EU529682, EU660386, EU781825, EU781827, EU781830, EU781832, EU857431, EU862835, FJ024277, FJ390396, FJ390397, FJ390398, FJ478453, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958934, FJ958935, GQ913868, GQ913869, GQ913870, GQ913871, GQ913873, GU451218, GU451219, GU451220, GU451221, GU451222, GU451223, GU451224, HM042050, HM042051, HM042052, HM042054, HM042055, HM042056, HM042057, HM042058, HM042059, HM042061, HM042062, HM042063, M58335, M84754, M96362, U01214, U16362, U45476

288 caa cgc Q R AB285066, AB285077, AJ507174, AJ507177, AJ507183, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449,

152

AM493450, AM493451, EF407483, EU155220, EU155260

288 caa cgg Q R AB049087, AB049088, AB049090, AB049091, AB049096, AB285043, AB285046, AB285060, AB285062, AB285076, AB429050, AF207753, AF207760, AF265148, AF265149, AF265150, AF265164, AF265165, AJ133096, AJ507160, AJ507161, AJ507172, AJ507188, AY808016, AY808031, D90208, DQ491992, DQ492172, EF407467, EF407474, EF407486, EF407496, EU155324, EU155359, EU155363, EU256091, EU256092, EU256099, EU482880, EU660388, EU862837, FJ958907, L02836, X61596

288 caa cgt Q R AB049100, AM401955, DQ492178, EF407479, EU155225, EU155316, EU155333, EU256077, GQ913872

288 caa gta Q V AB285044

288 caa gtg Q V AJ507155

288 caa tgg Q W EU155305

288 caa tat Q Y AJ507184

289 gcg ccg A P AB285043, AM402086

289 gcg aca A T AF207769

289 gcg acg A T HM042062

289 gcg gta A V HM042059

289 gcg gtg A V AF165062, AY008261, DQ492017, EU155281, HM042056

290 gag gca E A AJ507187

290 gag gac E D EU256064

290 gag ggg E G AM402303, AY808023, DQ492002, DQ492105

290 gag aag E K AM401951

290 gag gtg E V D90208

291 gag gca E A EU155376

291 gag gcg E A EU155218

291 gag gat E D EU482879, M96362, U16362

291 gag ggg E G AM401861, AM401870, AM401969, DQ492039, DQ492105

291 gag aaa E K AJ507158

291 gag aag E K AY808040, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957

291 gag gtg E V EF407494

292 gat tgt D C EU155302

292 gat gaa D E EF407496

292 gat gag D E AF207757, EU256061

292 gat ggt D G AF265158, AF265159, DQ492120, EF638081, HM042062, U01214

292 gat aat D N AB285040, AF207755, EU155304, EU256092

292 gat agt D S AB285079, AJ507180, HM042056

293 gag gac E D AB285051, DQ244141, EF407498, EU155218, EU155258

293 gag gat E D AB285072, AY460204, AY808038, AY808043, DQ492148, EF407482, EF407483, EF407486, EF638081, EU155220, EU781830, EU862835, GU451221, GU451222, GU451223, GU451224

293 gag gga E G AY808041, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, EU155228

293 gag ggg E G AB049092, AJ507159, AM402327, AM402337, AM402352, AY808031

293 gag cag E Q AY808042, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977

293 gag cgg E R DQ491978, EU256079

294 agg gga R G EU256065

294 agg ggc R G AJ507185

294 agg ggg R G AB285076, AB442221, AF207760, AF265148, AF265150, AF265164, AF265165, AJ507168, AY240969, AY808041, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ491988,

153

DQ492072, DQ492114, EF407460, EF407467, EU155262, EU155336, EU155375, EU155381, EU256082, EU256084, EU256101, EU660386, HM042062, M96362, U01214, U16362

294 agg aag R K AB285043, AF034151, DQ492085, DQ859970, EU155372, EU482877

294 agg atg R M HM042056

294 agg cag R Q AM494937, EU256092

294 agg acg R T HM042051


295 gaa gga E G AM493496, DQ492136, DQ492149

295 gaa ggg E G FJ958916

296 gta gag V E AY808028

296 gta ggg V G AM401848

296 gta ata V I AB049096, AB154203, AB154204, AB285038, AB285040, AB285054, AB285055, AB285059, AB285073, AB285075, AB442221, AF207752, AF207753, AF207760, AF207761, AF265148, AF265149, AF265150, AF265154, AF265155, AF265156, AF265158, AF265159, AF265163, AF265164, AF265165, AJ132996, AJ132997, AJ507166, AJ507168, AJ507169, AJ507179, AJ507193, AJ507196, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AY240968, AY587844, AY808009, AY808014, AY808020, AY808021, AY808029, AY808035, AY808036, AY808040, D90208, DQ244140, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492074, DQ492075, DQ492076, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492083, DQ492084, DQ492085, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, DQ492093, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407460, EF407462, EF407464, EF407473, EF407494, EF407500, EU155218, EU155254, EU155256, EU155280, EU155326, EU155327, EU155329, EU155330, EU155331, EU155364, EU155365, EU155373, EU155375, EU155376, EU256075, EU256078, EU256079, EU256082, EU256084, EU256103, EU482833, EU482877, EU482879, EU482883, EU529682, EU660386, GU451220, HM042050, M96362, U16362

296 gta atc V I AJ507155, HM042055, HM042058

296 gta ttg V L DQ859970, EU155279

296 gta atg V M AB285065, AJ507170, AJ507178, AJ507186, AJ507199, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449,

154

AM493450, AM493451, AY240967, AY808033, DQ491989, EU155228, EU155229, EU155306, GQ913869, GQ913873, HM042051, HM042052, HM042054, HM042056, HM042061, HM042062, HM042063

296 gta cca V P AF165049, AF165050, AF165051, AF165052, AF165057, AF165058, AF165061, AF165062, AF207762, AF207765, AF207770, AY045702

296 gta ccg V P EU256059

296 gta aca V T DQ492086

297 tcc ttc S F DQ492025, DQ492033

298 gtt gcc V A DQ491948

298 gtt gct V A D89815, DQ491965

298 gtt ata V I EU155318

298 gtt atc V I AM402068, AY808016, AY808028, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, EF407482, EU155224, EU155335, EU256081, EU256088, EU256101

298 gtt att V I AB285061, AF165063, AF165064, AJ507158, AJ507191, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AY808030, AY808033, AY808043, D50480, D50483, EF407474, EF407475, EF407478, EF407488, EU155219, EU155221, EU155303, EU155307, EU155315, EU155361, EU155367, EU256045, EU256085, EU482859, EU482880, EU862835, FJ390396

298 gtt ctc V L D50485

298 gtt ctt V L D50481

298 gtt atg V M EF407464

298 gtt act V T EU155365, EU529682

299 ccg gca P A AB010249, AB285043, AB285053, AF207761, AM401911, AY008261, AY240967, AY460204, D11168, D16435, D89872, EF638081, EU155279, EU256090, EU256102, GU451221, GU451222, GU451223, GU451224

299 ccg gcg P A AB010250, AB049087, AB049090, AB049094, AB049095, AB049098, AB049100, AB049101, AB080299, AB249644, AB285035, AB285037, AB285040, AB285042, AB285044, AB285046, AB285048, AB285054, AB285055, AB285056, AB285057, AB285058, AB285060, AB285061, AB285063, AB285068, AB285069, AB285075, AB285077, AB285078, AB285079, AB426117, AB429050, AB442219, AB442220, AB442221, AF165045, AF165046, AF165047, AF165048, AF165053, AF165054, AF165055, AF165056, AF165059, AF165060, AF165061, AF165062, AF165063, AF165064, AF207752, AF207755, AF207756, AF207757, AF207758, AF207765, AF207766, AF207768, AF207771, AF207774, AJ000009, AJ507169, AJ507171, AJ507174, AJ507182, AJ507196, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907,

155

AM401908, AM401909, AM401910, AM401912, AM401913, AM401914, AM401915, AY045702, AY587016, AY808009, AY808031, AY808039, D10934, D30613, D45172, D50482, D50484, D63857, D85516, D89815, DQ244140, DQ244141, EU155258, EU155302, EU155332, EU155374, EU239714, EU482833, EU482874, EU857431, GU451218, GU451219, GU451220, L02836, U01214, X61596

299 ccg gct P A AB049089, AB285047

299 ccg gag P E AB049096, AB285052, AB285059, AB285064, AF207763, AF207764, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AY240968, DQ859970

299 ccg ctg P L DQ491967, DQ492127

299 ccg tcg P S AM494937, DQ492159, DQ492167, EU155330

300 gcg gag A E D63857

300 gcg tcg A S EU862835

300 gcg gtg A V AM493488, FJ958905

301 gag ggg E G AM402304, DQ492090, DQ492105, DQ492184, FJ958907


301 gag gtg E V DQ491976

302 atc ttc I F AB285073

302 atc acc I T AM402071, DQ244141, DQ492040, DQ492047, DQ492120

302 atc gtc I V AF207766, AY808031, DQ491982, DQ492074, EU482833, EU660388, EU862837, FJ958934

303 ctg ccg L P AM401923, DQ492170

303 ctg cag L Q DQ491971

304 cgg cta R L DQ492054, DQ492055, GQ913869

304 cgg ctg R L AJ507164, AJ507170, AJ507178, AJ507187, AJ507199, AY808035, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, EF407481, EU155222, EU155228, EU155256, EU155333, EU155356, EU256076, FJ390397, GQ913873, HM042051, HM042052, HM042054, HM042056, HM042061, HM042063

304 cgg cag R Q AM402089

304 cgg tcg R S EU155373

304 cgg tgg R W AB154205, AB154206, AM402354

305 agg gaa R E DQ492169

305 agg aaa R K AB010249, AB036519, AB036520, AB036521, AB049087, AB049088, AB049089, AB049090, AB049091, AB049094, AB049095, AB049096, AB049098, AB049099, AB049100, AB080299, AB109543, AB191333, AB249644, AB285035, AB285036, AB285037, AB285038, AB285039, AB285040, AB285042, AB285043, AB285044, AB285046, AB285047, AB285048, AB285050, AB285051, AB285052, AB285053, AB285054, AB285055, AB285056, AB285057, AB285058, AB285059, AB285060, AB285062, AB285063, AB285065, AB285066, AB285067, AB285068, AB285069, AB285070, AB285071, AB285072, AB285074, AB285075, AB285076, AB285077, AB285078, AB285081, AB426117, AB429050, AB442219, AB442221, AF165045, AF165046, AF165049, AF165050, AF165051, AF165052, AF165053, AF165054, AF165055, AF165056, AF165057, AF165058, AF165061, AF165062, AF165063, AF165064, AF176573, AF207752, AF207753, AF207755, AF207756, AF207757, AF207758, AF207760, AF207761, AF207762, AF207763, AF207764, AF207765, AF207766, AF207768, AF207770, AF207771, AF207772, AF207774, AF208024, AF265142, AF265143, AF265144, AF265145, AF265146, AF265147, AF265148, AF265149, AF265150, AF265152, AF265153, AF265154, AF265155, AF265156, AF265160, AF265161, AF265162, AF265163, AF265164, AF265165, AF333324, AF356827, AF483269, AJ000009, AJ132997, AJ133096, AJ507156, AJ507158, AJ507159, AJ507160, AJ507161, AJ507162, AJ507163, AJ507164, AJ507166, AJ507167, AJ507168, AJ507169, AJ507170, AJ507171,

156

AJ507172, AJ507173, AJ507174, AJ507175, AJ507179, AJ507180, AJ507181, AJ507182, AJ507183, AJ507184, AJ507185, AJ507186, AJ507187, AJ507188, AJ507189, AJ507190, AJ507191, AJ507192, AJ507193, AJ507194, AJ507195, AJ507197, AJ507198, AJ507199, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AM401916, AM401919, AM401920, AM401921, AM401925, AM401929, AM401931, AM401934, AM401935, AM401937, AM401938, AM401942, AM401946, AM401948, AM401957, AM402058, AM402070, AM402098, AM402103, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM402329, AM402330, AM402331, AM402332, AM402333, AM402334, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AM494937, AY008261, AY044867, AY045702, AY240969, AY460204, AY587844, AY808009, AY808010, AY808011, AY808012, AY808013, AY808014, AY808015, AY808017, AY808018, AY808019, AY808020, AY808023, AY808024, AY808025, AY808026, AY808027, AY808028, AY808030, AY808031, AY808032, AY808034, AY808035, AY808036, AY808038, AY808040, AY808041, AY808042, D10934, D11168, D16435, D50480, D50481, D50482, D50483, D50484, D50485, D85516, D89872, D90208, DQ071885, DQ244141, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012,

157

DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, DQ492063, DQ492065, DQ492066, DQ492067, DQ492068, DQ492070, DQ492073, DQ492074, DQ492078, DQ492079, DQ492080, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, DQ492093, DQ492094, DQ492095, DQ492096, DQ492097, DQ492098, DQ492099, DQ492100, DQ492101, DQ492102, DQ492103, DQ492104, DQ492105, DQ492106, DQ492107, DQ492108, DQ492109, DQ492110, DQ492111, DQ492112, DQ492113, DQ492114, DQ492115, DQ492116, DQ492117, DQ492118, DQ492119, DQ492120, DQ492121, DQ492122, DQ492123, DQ492124, DQ492125, DQ492126, DQ492127, DQ492128, DQ492129, DQ492130, DQ492131, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492139, DQ492140, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492148, DQ492149, DQ492150, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492161, DQ492162, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, DQ859970, EF032892, EF032893, EF032894, EF407459, EF407460, EF407462, EF407463, EF407464, EF407465, EF407466, EF407467, EF407468, EF407469, EF407471, EF407472, EF407474, EF407475, EF407476, EF407477, EF407478, EF407480, EF407481, EF407483, EF407484, EF407485, EF407486, EF407487, EF407488, EF407489, EF407490, EF407492, EF407494, EF407495, EF407496, EF407498, EF407499, EF407502, EF407503, EF407504, EF638081, EU155217, EU155218, EU155219, EU155220, EU155221, EU155223, EU155224, EU155225, EU155226, EU155227, EU155228, EU155230, EU155231, EU155232, EU155235, EU155253, EU155254, EU155255, EU155256, EU155257, EU155258, EU155259, EU155260, EU155261, EU155263, EU155264, EU155280, EU155281, EU155300, EU155301, EU155302, EU155305, EU155306, EU155307, EU155308, EU155315, EU155316, EU155317, EU155318, EU155324, EU155326, EU155327, EU155328, EU155331, EU155332, EU155333, EU155335, EU155336, EU155337, EU155356, EU155357, EU155358, EU155359, EU155361, EU155362, EU155363, EU155364, EU155366, EU155368, EU155369, EU155370, EU155372, EU155373, EU155374, EU155375, EU155377, EU155381, EU155382, EU234061, EU234062, EU239714, EU255960, EU255961, EU255962, EU256000, EU256001, EU256045, EU256059, EU256061, EU256062, EU256065, EU256066, EU256075, EU256076, EU256077, EU256078, EU256079, EU256080, EU256081, EU256082, EU256083, EU256084, EU256085, EU256090, EU256091, EU256092, EU256098, EU256099, EU256100, EU256101, EU482833, EU482839, EU482849, EU482860, EU482874, EU482877, EU482879, EU482880, EU482883, EU482886, EU482888, EU660386, EU660388, EU781825, EU781827, EU781830, EU781832, EU862835, EU862837, FJ024279, FJ390397, FJ390398, FJ478453, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958931, FJ958933, FJ958934, FJ958935, FN435993, GQ913869, GQ913871, GQ913872, GQ913873, GU451218, GU451219, GU451220, GU451221, GU451222, GU451223, GU451224, HM042051, HM042052, HM042054, HM042055, HM042057, HM042058, HM042059, HM042061,

158

HM042062, HM042063, L02836, M58335, M84754, M96362, U01214, U16362, U45476, X61596

305 agg aag R K AB010250, AB285061, AB285079, AB442220, AB442222, AF033373, AF034151, AF165047, AF165048, AF207754, AF207769, AF265158, AF265159, AJ507155, AM401917, AM401918, AM401922, AM401923, AM401924, AM401926, AM401927, AM401928, AM401930, AM401932, AM401933, AM401936, AM401939, AM401940, AM401941, AM401943, AM401944, AM401945, AM401947, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AM402351, AY240967, AY240968, AY587016, AY808043, D30613, D45172, D89815, EF407461, EF407479, EF407482, EF407497, EF407500, EU155279, EU155303, EU155329, EU256064, EU256088, EU256089, EU256102, EU256103, EU482875, EU857431, FJ024277, FJ390396, FJ958930, FJ958932, HM042050, HM042056

305 agg cca R P EF407491, EU155222

305 agg caa R Q DQ244140

305 agg aca R T AB285064, AY808037, EU155262, EU482885

306 tcc gct S A EU256088

306 tcc ttc S F AM402107

306 tcc cac S H EU155318

306 tcc aag S K EU155228

306 tcc aat S N AF207764

306 tcc ccc S P AF207753, AJ507157, AJ507192, D90208, DQ491978, DQ492016, DQ492020, EU155376, EU862837, GQ913870, HM042053

306 tcc acc S T AB010250, AB036519, AB036520, AB036521, AB049087, AB049089, AB049092, AB049094, AB049095, AB049096, AB109543, AB191333, AB285035, AB285040, AB285043, AB285047, AB285048, AB285050, AB285052, AB285053, AB285054, AB285055, AB285056, AB285057, AB285060, AB285061, AB285064, AB285068, AB285073, AB285075, AB285077, AB285078, AB285079, AB429050, AB442219, AB442220, AF165045, AF165046, AF165047, AF165048, AF165053, AF165054, AF165055, AF165056, AF165057, AF165058, AF165063, AF165064, AF176573, AF207755, AF207757, AF207769, AF207771, AF265158, AF265159, AJ507158, AJ507161, AJ507164, AJ507184, AJ507189, AM401864, AM401865, AM401866, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401877, AM401879, AM401883, AM401884, AM401885, AM401887, AM401888, AM401889, AM401890, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AY045702, AY460204, AY587016, AY808025, AY808041, D10934, D30613, D45172, D50482, D50484, D89815, DQ071885, DQ244141, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, EF407478, EF407479, EF407490, EF407498, EF638081, EU155222, EU155231, EU155253, EU155324, EU155361, EU155369, EU155382, EU256064, EU256065, EU256076, EU256089, EU256100, EU256101, EU482874, EU482875, EU482880, EU857431, FJ958911, FJ958924, GU451218, GU451219, GU451220, GU451221, GU451222, GU451223, GU451224, HM042056

306 tcc acg S T EF407487

306 tcc act S T AB285042, AM493500, AY808012, EF032892, EF032893, EF032894

307 agg ggg R G EU155368

307 agg aaa R K AB010249, AB285061, AB442221, EF407468, EF407487, EU155303, EU155361, EU155372, EU256066, EU482839, EU482881, EU529682,

159

EU862835

307 agg aag R K AB285044, AB285053, AB285055, AB285056, AB285064, AB285070, AF034151, AF165049, AF165050, AF165057, AF165058, AF207762, AF207765, AF333324, AJ507166, AJ507171, AJ507172, AJ507174, AJ507180, AJ507183, AJ507186, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AM494937, AY044867, AY045702, AY240969, AY808016, AY808030, AY808033, AY808035, AY808036, D11168, D16435, D50480, D50483, D50484, D89872, DQ244141, DQ491971, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, EF407460, EF407462, EF407464, EF407466, EF407475, EF407479, EF407488, EF407503, EF638081, EU155221, EU155228, EU155232, EU155255, EU155257, EU155307, EU155315, EU155357, EU155365, EU155367, EU155375, EU255962, EU256064, EU256078, EU256081, EU256085, EU256090, EU256091, EU256103, EU482859, EU482875, EU482880, EU781825, FJ390397, FJ390398, GU451221, GU451222, GU451223, GU451224, HM042050, HM042056, M58335, U01214

308 aaa gca K A EU862835

308 aaa gcc K A AM402109

308 aaa gct K A AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402099, AM402101, AM402102, AM402106, AM402107, AM402108, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AY808030, EU155221, EU155365, EU155367, EU529682

308 aaa gaa K E AY808034, DQ491946, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492017, DQ492018, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492040, DQ492046, DQ492047, DQ492048, DQ492050, DQ492054, DQ492055, EU256079

308 aaa gag K E AB049088

308 aaa gga K G DQ492019

308 aaa ggc K G EU256085

308 aaa ggg K G AB049099

308 aaa ata K I EU256066

308 aaa atg K M EU256075

308 aaa aac K N AB285039, EF407485, EF407500, EU482885

308 aaa aat K N AB010249, AB049091, AF207752, DQ491980, DQ491981, DQ491983, DQ491984, DQ491989, DQ492065, DQ492067, DQ492068, DQ492070, DQ492074, DQ492080, DQ492082, DQ492085, DQ492086, DQ492089, DQ492090, DQ492091, DQ492092, EU155257

160

308 aaa aga K R AB049089, AF207760, AF207768, AF483269, AJ000009, AJ507155, AJ507171, AJ507175, AM402266, AM402329, AM402330, AM402331, AM402332, AM402333, AM402334, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493498, AM493499, AM493500, AM493502, AM493503, AM493505, AM493506, AM493507, AY044867, D10934, EF407460, EF407488, EU155220, EU155256, EU155318, EU155336, EU155372, EU256064, EU256081, EU482880, EU862837, HM042050

308 aaa agg K R AB049101, AB285056, AB285076, AB442221, AF034151, AF207762, AF207765, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401867, AM401868, AM401876, AM401878, AM401880, AM401881, AM401882, AM401886, AM401891, AM401892, AM494937, AY045702, EU155306, EU155315, EU155376

308 aaa agc K S AJ132997, EU256091

308 aaa agt K S AF165063, AF165064, HM042056

308 aaa gta K V FJ390397

308 aaa gtc K V AM402100

308 aaa gtt K V AM402090, AM402098, AM402103, AM402104, AM402105, AY808016


309 ttc tcc F S AM401977, DQ491961, DQ491964, DQ491966

309 ttc tac F Y AB285048, AY460204, EF407462, EF638081, GU451221, GU451222, GU451223, GU451224

310 cct ctc P L AM402092, DQ492180

310 cct tcc P S EU155219

310 cct acc P T AJ507155, AM402329, AM402330, AM402331, AM402332, AM402333, AM402334, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384

311 cga gca R A AB010250, AB249644, AB285037, AB285040, AB285044, AB285062, AB285063, AB426117, AF207762, AF207772, AF207774, AF333324, AJ507179, AY587016, AY808022, D14484, D30613, D45172, D89815, EU155318, EU155335, EU155376, EU256081, EU857431, M58335

311 cga gcg R A AB049098, AB285070, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401877, AM401879, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, DQ244141, EF407459, EU155358, EU256099

311 cga gct R A AB285061, EU155382

311 cga cac R H EU256061

311 cga cat R H AB285076

161

311 cga cta R L AB049092, AB049096, AB285069, AF165050, AJ507170, EF407502, HM042062

311 cga ctc R L EU155316

311 cga ctg R L AB154188, AF165049, EF407462, HM042058

311 cga atg R M AF165051, AF165052

311 cga cca R P AB049087, AB049088, AB049089, AB049091, AB049094, AB049095, AB049099, AB154189, AB154190, AB285035, AB285042, AB285047, AB285051, AB285053, AB285054, AB285055, AB285056, AB285057, AB285058, AB285064, AB285068, AB285071, AB285073, AB285077, AB285078, AB285079, AB442219, AB442222, AF034151, AF165045, AF165046, AF165055, AF165056, AF165058, AF165059, AF165060, AF165061, AF165062, AF165064, AF207753, AF207755, AF207758, AF207764, AF207766, AF207768, AF207769, AF207770, AF265142, AF265143, AF265144, AF265158, AF265159, AF265160, AF265162, AJ507156, AJ507158, AJ507159, AJ507160, AJ507163, AJ507164, AJ507167, AJ507168, AJ507174, AJ507178, AJ507181, AJ507182, AJ507190, AJ507193, AJ507194, AM402329, AM402330, AM402331, AM402332, AM402333, AM402334, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, AM494937, AY008261, AY240967, AY240968, AY240969, AY460204, AY587844, AY808009, AY808017, AY808020, AY808021, AY808025, AY808026, AY808031, AY808034, AY808035, AY808038, AY808039, AY808040, D10934, D50480, D50482, D50484, D63857, D85516, D90208, DQ244140, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, DQ492064, DQ492069, DQ492071, DQ492072, DQ492075, DQ492076, DQ492077, DQ492081, EF407460, EF407461, EF407464, EF407468, EF407477, EF407478, EF407483, EF407488, EF407491, EF407501, EF407503, EF407504, EF638081, EU155219, EU155220, EU155222, EU155227, EU155228, EU155229, EU155231, EU155232, EU155235, EU155256, EU155257, EU155281, EU155302, EU155306, EU155325, EU155330, EU155334, EU155337, EU155356, EU155359, EU155360, EU155361, EU155363, EU155364, EU155366, EU155369, EU155373, EU155375, EU155377, EU234061, EU234062, EU255960, EU256045, EU256064, EU256075, EU256076, EU256083, EU256084, EU256088, EU256089, EU256090, EU256092, EU482833, EU482849, EU482859, EU482886, FJ024277, FJ390397, GQ913869, GQ913873, GU451218, GU451219, GU451220, GU451221, GU451222, GU451223, GU451224, HM042050, HM042051, HM042052, HM042054, HM042055, HM042056, HM042057, HM042061, HM042063, M84754, M96362, U16362, U45476, X61596

311 cga ccc R P AB285043, AB285066, AF265161, AJ507157, AJ507183, AY808019, AY808042, D11168, D16435, D89872, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, DQ492094, DQ492095, DQ492096, DQ492097, DQ492098, DQ492099, DQ492100, DQ492101, DQ492102, DQ492103, DQ492104, DQ492105, DQ492106, DQ492107, DQ492108, DQ492109, DQ492110, DQ492111, DQ492112, DQ492113, DQ492114, DQ492115, DQ492116, DQ492117, DQ492118, DQ492119, DQ492120, DQ492121, DQ492122, DQ492123, EU155263, EU155327, EU155336, EU482874, FJ390398, FJ958934

162

311 cga ccg R P AB010249, AB049090, AB049100, AB285048, AB285052, AB285065, AB429050, AB442221, AF165047, AF165048, AF165053, AF165054, AF165063, AF207757, AF207771, AF208024, AF265145, AF265146, AF265147, AJ507155, AJ507161, AJ507165, AJ507166, AJ507171, AJ507180, AJ507184, AJ507186, AJ507187, AJ507189, AJ507192, AJ507199, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AY045702, AY808016, AY808018, AY808029, AY808030, AY808033, D50485, DQ492018, DQ492063, DQ492065, DQ492066, DQ492067, DQ492068, DQ492070, DQ492073, DQ492074, DQ492078, DQ492079, DQ492080, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, EF407469, EF407475, EF407476, EF407485, EF407486, EF407490, EU155221, EU155223, EU155307, EU155315, EU155317, EU155332, EU155365, EU155367, EU155368, EU155370, EU155371, EU155372, EU155374, EU256080, EU256085, EU256091, EU256100, EU256103, EU482880, EU482885, EU482888, EU529682, EU781827, EU862835, EU862837, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958935, FN435993, U01214

311 cga cct R P AB080299, AB442220, AF165057, AY808012, DQ492040

311 cga caa R Q AJ133096, AJ507191, AY808028, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, EF407496, EU155254, EU155305, EU155324, EU155328, HM042059

311 cga cag R Q AJ507177

311 cga tca R S AB036519, AB036520, AB036521, AB109543, AB191333, AB285036, AB285038, AB285050, AB285059, AB285060, AB285067, AB285072, AB285074, AB285075, AB285081, AF033373, AF176573, AF207752, AF207754, AF207765, AF483269, AJ507162, AJ507172, AJ507175, AJ507197, AJ507198, AY044867, AY808036, AY808043, D50483, DQ859970, EF407487, EU155357, EU155362, EU256062, EU256066, EU256078, EU781825, EU781830, FJ478453, L02836

311 cga tcg R S AB049101, AB285046, AJ507188, AM401867, AM401868, AM401876, AM401878, AM401880, AY808013, AY808024, EU256000

311 cga aca R T AF207756, EF407479, EF407498, EU482839

312 gcg acg A T DQ492070

313 atg ata M I AB049087, AB049094, AB049095, AB049099, AB285052, AB285053, AB285056, AB285060, AB285068, AB429050, AF165045, AF165046, AF165047, AF165048, AF165057, AF165058, AF207755, AF207764, AM401926, DQ492128, EF407467, EU155365, EU256059, GQ913873, HM042051, HM042056, HM042058

313 atg att M I AF165053, AF165054, AF165063, AF165064

163

313 atg cta M L AF207757, AF207771, AY808032

313 atg ctc M L D50482, D50484

313 atg ctg M L AB049088, AB049089, AB049090, AB049092, AB049100, AB080299, AB285043, AB285047, AB285048, AB285051, AB285065, AB285075, AB285077, AB442221, AF165059, AF165060, AF207754, AF207756, AF207762, AF207763, AF207765, AF265150, AF265164, AJ507155, AJ507156, AJ507163, AJ507164, AJ507180, AJ507182, AJ507187, AJ507191, AJ507192, AJ507194, AY240967, AY240968, AY240969, AY460204, AY587844, AY808028, AY808033, AY808043, DQ071885, DQ491979, DQ491982, DQ491985, DQ491986, DQ491987, DQ491988, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492057, DQ492189, DQ859970, EF407462, EF407465, EF407470, EF407476, EF407490, EF407494, EU155219, EU155255, EU155256, EU155327, EU155328, EU155364, EU155374, EU155377, EU239714, EU256078, EU256079, EU256080, EU256085, EU482833, EU482859, EU482880, EU529682, EU862837, GQ913868, L02836, M96362, U16362

313 atg tta M L AB285035, D50480, D50483, DQ492184, DQ492185

313 atg ttg M L AB010249, AB036519, AB036520, AB036521, AB049101, AB109543, AB154190, AB191333, AB249644, AB285042, AB285050, AB285058, AB285066, AB285069, AB285073, AB285076, AB285079, AB426117, AB442222, AF034151, AF165055, AF165056, AF207753, AF207758, AF207766, AF207768, AF207770, AJ507157, AJ507173, AJ507179, AJ507190, AJ507196, AM402060, AM402066, AM402070, AM402074, AM402075, AM402081, AM402084, AM402088, AM402090, AM402096, AM402101, AM402102, AM402106, AM402108, AM402111, AM402112, AM402115, AM402127, AM402329, AM402330, AM402331, AM402332, AM402333, AM402334, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AM494937, AY044867, AY808014, D11168, D16435, D85516, D89872, D90208, DQ244140, DQ491980, DQ491981, DQ491983, DQ491984, DQ491989, DQ492093, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492186, DQ492187, DQ492188, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407471, EF407477, EF407486, EF407491, EF407503, EF407504, EU155227, EU155281, EU155302, EU155316, EU155335, EU155360, EU155369, EU256083, EU256089, EU256091, FN435993, HM042062, M84754, U01214, X61596

313 atg tcg M S DQ492163


313 atg gtg M V AM401853, AM402276, AM402282, AY808017, HM042059

313 atg tgg M W AM402125

314 ccc ctc P L DQ492109

314 ccc tcc P S AM493438, AM493445, DQ492192

315 ata gaa I E U01214

315 ata ttt I F AB285067

315 ata aca I T DQ492188

315 ata gta I V AB010250, AB049090, AB049098, AB049100, AB080299, AB154179, AB154180, AB154188, AB285037, AB285065, AB285066, AB285069, AB285074, AF165051, AF165052, AF165055, AF165056, AF165059,

164

AF165060, AF165063, AF165064, AF207752, AF207760, AF207762, AF207763, AF207769, AF207774, AJ507158, AJ507160, AJ507167, AJ507179, AJ507181, AJ507188, AJ507191, AJ507196, AJ507198, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AM401932, AM401933, AM401939, AM401940, AM401945, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM402330, AM402331, AM402332, AM402333, AM402335, AM402337, AM402338, AM402340, AM402341, AM402346, AM402348, AM402349, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402361, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402381, AM402383, AM402384, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AM493491, AM493492, AM493493, AM493496, AM493498, AM493499, AM493501, AM493502, AM493503, AM493504, AM493507, AY044867, AY460204, AY587016, AY808009, AY808011, AY808018, AY808028, AY808031, AY808032, AY808033, D10934, D30613, D45172, D89815, DQ244140, DQ244141, DQ491946, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492124, DQ492125, DQ492126, DQ492127, DQ492128, DQ492129, DQ492130, DQ492131, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492139, DQ492140, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492148, DQ492149, DQ492150, DQ492151, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492161, DQ492162, EF407469, EF407479, EF407483, EF407487, EF407491, EF407497, EF407499, EF407502, EF407504, EU155227, EU155258, EU155261, EU155317, EU155331, EU155335, EU155337,

165

EU155356, EU155357, EU155359, EU155361, EU155366, EU155369, EU155371, EU155374, EU155375, EU155376, EU155377, EU155382, EU255961, EU256062, EU256077, EU256079, EU256083, EU256088, EU256089, EU256099, EU256101, EU482859, EU482888, EU529682, EU660388, EU857431, FJ024279, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958934, FJ958935, GU451221, GU451222, GU451223, GU451224, HM042057, M84754

315 ata gtc I V AJ507178

315 ata gtg I V AB285040, AF207761, AF207765, AF483269, AJ507168, AJ507184, AY808025, EU155232, EU155336, EU255960, EU862837, FJ390398

315 ata gtt I V AJ507197, EF407498, EU155358, FN435993

316 tgg agg W R DQ492038

316 tgg cgg W R AM402314, DQ492024, DQ492029

317 gca aca A T AM401913

317 gca gta A V DQ492077

318 cgc tgc R C DQ491947, DQ492050

318 cgc tgt R C DQ492082

318 cgc cac R H AB285050, AB285078, AY808033

318 cgc ctc R L DQ491950

318 cgc ccg R P M96362, U16362

318 cgc cag R Q EU529682

318 cgc agc R S EF407500

319 ccg gcg P A AF207757, EU155328

319 ccg cta P L AM493488

319 ccg ctg P L AM401889, DQ491976

319 ccg tcg P S AM401977, DQ492022

319 ccg aca P T DQ491965

320 gat gag D E AY808017, EF407500, HM042059

320 gat ggc D G AM493473, DQ492132, DQ492135


320 gat aat D N AB285078, AY808033

320 gat agc D S X61596

321 tac tgc Y C AM401892, AM401896, AM401974

321 tac gac Y D DQ491959, DQ491960, DQ491965

321 tac aac Y N DQ491996

322 aac gac N D AM401885, DQ492027, DQ492130, DQ492139, DQ492140

322 aac cac N H AM493497

322 aac agc N S AM402118, AM402349

323 cct ctt P L AM402306


324 cca cgg P R DQ492028

325 ctg cca L P AM402305

325 ctg gtg L V AF207754

326 tta ata L I AB049090, AB154183, AB154184, AB154195, AB154196, AB285048, AB285062, AB285075, AB285081, AB442220, AF165045, AF165046, AF165053, AF165054, AF207755, AF207758, AF207760, AF207771, AF265142, AF265143, AF265144, AF265145, AF265146, AF265147, AF265158, AF265159, AF265161, AJ507164, AJ507166, AJ507167, AJ507170, AJ507178, AJ507181, AJ507182, AJ507186, AJ507189, AJ507199, AM402055, AM402056, AM402057, AM402058, AM402059,

166

AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AM493489, AM493490, AM493493, AM493494, AM493495, AM493496, AM493497, AM493499, AM493500, AM493502, AM493505, AM493506, AY044867, AY808016, AY808018, AY808027, AY808029, AY808035, D50481, D50485, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492074, DQ492075, DQ492076, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, DQ859970, EF407460, EF407461, EF407475, EF407479, EF407482, EF407483, EF407485, EF407488, EF407492, EF407497, EF407500, EF407501, EF407502, EF407503, EU155219, EU155220, EU155221, EU155222, EU155223, EU155224, EU155226, EU155228, EU155229, EU155232, EU155255, EU155256, EU155279, EU155280, EU155300, EU155301, EU155303, EU155317, EU155326, EU155327, EU155337, EU155356, EU155357, EU155364, EU255962, EU256001, EU256045, EU256062, EU256076, EU256078, EU256081, EU256083, EU256091, EU256100, EU482833, EU781825, EU781827, EU862835, FJ024279, FJ390396, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958935, GQ913869, GQ913870, GQ913873, HM042050, HM042051, HM042052, HM042054, HM042055, HM042056, HM042057, HM042058, HM042061

326 tta att L I EU155218, EU256000, FJ024277

326 tta cca L P AM401855, DQ492170

326 tta gta L V AB049096, AB154178, AB154179, AB154180, AB154181, AB154182, AB154187, AB154188, AB154191, AB154193, AB154194, AB154198, AB154199, AB154200, AB154201, AB154202, AB154203, AB154204, AB154205, AB154206, AB285038, AB285052, AB285074, AF265160, AF265162, AF313916, AJ507155, AJ507174, AJ507185, AJ507195, AY808012, DQ491999, DQ492016, EF407464, EF407469, EF407478, EU155235, EU155315, EU155325, EU256064, EU781830, FJ958934, HM042063

326 tta gtc L V AY808034, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492017, DQ492018, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039

326 tta gtg L V AB154185, AB154186, AB154189, AB154190, AB154192, AB154197, AB285078, DQ492019, EU482881

327 gag ggg E G AM401849, FJ958917

327 gag caa E Q AJ507163

327 gag cag E Q AB285073, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493500, AM493501,

167

AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, DQ071885, EF407504, EU660386

327 gag cgg E R AM493489

328 tcc gca S A DQ492165

328 tcc gcc S A AM494937, AY808009, AY808014, AY808032, DQ492093, DQ492163, DQ492164, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197

328 tcc gct S A DQ492166, DQ492191

328 tcc ctc S L EF407471

328 tcc cca S P AY587844

328 tcc ccc S P AB080299, AB154189, AB154190, AB285038, AB285067, AB285073, AB285079, AF034151, AF165061, AF165062, AF207769, AF265162, AJ507157, AJ507169, AM401875, AM402094, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AY808033, AY808034, AY808042, D50482, D50483, DQ071885, DQ244140, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492040, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492054, DQ492055, DQ492120, DQ492121, DQ492122, DQ492123, EF407461, EF407477, EF407491, EF407500, EF407501, EU155235, EU155256, EU155315, EU155357, EU155364, EU256079, EU256083, EU256091, EU862835, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958934, FJ958935, FN435993, GQ913873, HM042050, HM042054, HM042055, HM042056, HM042058, M84754

328 tcc cct S P AB049096, AB049100, AB285043, AB285048, AB285075, AB285078, AF165055, AF165056, AF207771, AJ507163, AY808023, DQ244141, EU155306, GU451218

328 tcc acc S T AB285056, AF265160, AY808028, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007

328 tcc acg S T AJ507155

328 tcc act S T AB285047


329 tgg cgg W R AM401888, AM401913

330 aag gag K E AF483269

330 aag aga K R AB036519, AB036520, AB036521, AB109543, AB191333, AB285050, AJ507157, DQ491971, DQ491972, DQ491973, DQ491974, DQ491976

330 aag agg K R AB049089, AB049094, AB049095, AB285040, AB285043, AB285052, AB285057, AB285068, AB442220, AF165045, AF165046, AF165053, AF165054, AF165055, AF165056, AF207755, AF207757, AF207771, AM402121, D50482, D50484, DQ244141, DQ491977, DQ492149, DQ492158, EF407467, EU155217, EU155281, FJ958891, FJ958892, FJ958894, FJ958907, FJ958912, FJ958913, FJ958928, GU451218,

168

GU451219, GU451220

330 aag acg K T DQ492084

331 gac gcc D A AF165057, AJ507162, AY240967, AY240968, AY808040, D50484, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, EU256075, HM042062

331 gac gga D G AB285074

331 gac ggc D G AF207768, AM402121, AM402129, DQ492067

331 gac cac D H AF265158, AF265159

331 gac aaa D K AB049089, AF207769, EU155263

331 gac aag D K AJ507155

331 gac aac D N AB010250, AB049101, AB285040, AB285044, AB285046, AB285053, AB285060, AB285061, AB429050, AB442219, AF207770, AF265148, AF265149, AF265150, AF265163, AF265164, AF356827, AF483269, AJ133096, AY587016, AY808025, AY808030, AY808036, AY808042, D30613, D45172, D89815, DQ244141, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, DQ491983, EF407473, EU155261, EU155306, EU155315, EU155375, EU155376, EU857431, FJ958933

331 gac aat D N AY008261

331 gac aga D R AY808031

331 gac agc D S AB049100, AB285051, AB285064

331 gac agt D S AB010249, D11168, D16435, D89872

331 gac gtc D V AB285058

333 gac gcc D A AJ507178, AJ507199, AY808034, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492191, EU155223, GQ913869, HM042051, HM042052, HM042054, HM042055, HM042056, HM042057, HM042058, HM042061, HM042063

333 gac gcg D A DQ492019

333 gac gct D A AJ507170, GQ913873

333 gac gaa D E AB285074, AB285079, AF207756

333 gac gag D E AF033373, AM401978

333 gac ggc D G AF165062, AY808035, DQ491955, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492051, DQ492052, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, EF407481, EU155264, EU256079, HM042059

333 gac aac D N AB049095, AB285042, AF207764, AJ507186, AM401882, AM402115, AM402125, DQ492176, DQ492183, EU155308, EU155328, EU256059, GQ913868, GU451218

333 gac aat D N AB285056

334 tac tag Y * DQ492113


334 tac cat Y H AM493506

335 gtc gcc V A AF265142, AF265143, AF265144, AF265161, AJ507156, AM401928, D50482, D50484, DQ491948, DQ492143, DQ859970, EU155222, HM042062

335 gtc gct V A EU155255

335 gtc gaa V E AJ507155

335 gtc atc V I AF207766, AM401911, DQ492094, DQ492177, EU155217

335 gtc att V I AJ507160, EF407469, FN435993

335 gtc aaa V K EF407480

335 gtc ctc V L DQ492113

335 gtc aac V N AJ507186

335 gtc ccc V P AF207768

169

335 gtc tcc V S AJ507165

335 gtc acc V T AB285065, AY008261, AY808018, EU155223, EU256091

335 gtc act V T EF407501, EU155361, EU155370

336 cct ctt P L AM402306, DQ492069, DQ492164

337 cca gcg P A EU234061

337 cca cgg P R DQ492114

337 cca acg P T DQ492094

338 gtg gcg V A AB010249, AJ507179, AM402073, AM402271, AM402306, AM402346, D11168, D16435, D89872

338 gtg aag V K DQ492194

338 gtg ctg V L AF207765, EU155327

338 gtg atg V M DQ492104, EU256084, FJ478453

338 gtg aca V T EU256080

339 gta gca V A AM493500

339 gta ata V I DQ492007

340 cac cgc H R AM402084, DQ491995

340 cac tcc H S AB285051

341 ggg gag G E AM402092, DQ492011, DQ492012, DQ492013


342 tgt ggc C G DQ492188

342 tgt cgc C R AM401856

342 tgt agc C S DQ491961, DQ491964, DQ491966

342 tgt tac C Y DQ492024, DQ492050

343 cca gca P A AY808038, HM042059


343 cca tca P S DQ492030, DQ492091

343 cca aca P T AM401921

344 ttg tgc L C DQ492105

345 ccg cac P H DQ492105

345 ccg tca P S AM493481

346 cct gcc P A AF265158, AF265159

346 cct ctt P L DQ492105

346 cct tca P S DQ492079

346 cct tcc P S AB285042, AB285048, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493498, AM493499, AM493502, AM493503, AM493505, AM493506, AY240969, GQ913872

346 cct tct P S AB049089, AB249644, AB426117, AJ507164, AJ507190, AY808029, D90208, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492070, DQ492071, DQ492073, DQ492074, DQ492075, DQ492076, DQ492078, DQ492080, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, EU482833

346 cct act P T FJ958921

347 gcc gac A D EU155256

347 gcc atc A I AB285055, AF265154, AF333324, AF356827, AJ507180, AJ507181, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493498, AM493499, AM493501, AM493502, AM493503, AM493505, AM493506, AM493507, EU155361, EU155372, EU155375, EU781825, M58335

347 gcc att A I AB154181, AB154182, AF165061, AF165062, AM402374, EU155227, EU256103

347 gcc atg A M AB285079

347 gcc aac A N AF034151, EU155337, EU155381, EU781827

347 gcc aat A N AB249644, AB426117

347 gcc cca A P AJ507155

170

347 gcc ccc A P AB285065, AB285075, AY808035, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062

347 gcc agc A S AF265155, AJ507161

347 gcc aca A T AB285056, AJ507197, DQ492149, DQ492151, EF407464, EU155258, EU155363, GQ913870, HM042053, HM042059, HM042060

347 gcc acc A T AB010249, AB010250, AB036519, AB036520, AB036521, AB049087, AB049088, AB049089, AB049091, AB049092, AB049096, AB049098, AB049100, AB049101, AB080299, AB109543, AB154191, AB191333, AB285035, AB285036, AB285037, AB285038, AB285040, AB285042, AB285043, AB285044, AB285046, AB285048, AB285050, AB285051, AB285054, AB285057, AB285060, AB285061, AB285062, AB285063, AB285064, AB285069, AB285070, AB285071, AB285072, AB285074, AB285076, AB285077, AB285078, AB285081, AB429050, AB442219, AB442222, AF165045, AF165046, AF165047, AF165048, AF165049, AF165050, AF165051, AF165052, AF165055, AF165056, AF165057, AF165058, AF165059, AF165060, AF165063, AF165064, AF176573, AF207752, AF207753, AF207754, AF207755, AF207756, AF207757, AF207758, AF207760, AF207761, AF207762, AF207763, AF207764, AF207766, AF207768, AF207769, AF207770, AF207771, AF207772, AF207774, AF208024, AF265142, AF265143, AF265144, AF265145, AF265146, AF265147, AF265156, AF265158, AF265159, AF265160, AF265161, AF265162, AF265165, AJ000009, AJ132997, AJ507156, AJ507158, AJ507160, AJ507162, AJ507164, AJ507167, AJ507169, AJ507170, AJ507171, AJ507172, AJ507173, AJ507179, AJ507183, AJ507184, AJ507185, AJ507186, AJ507187, AJ507188, AJ507190, AJ507191, AJ507195, AJ507196, AJ507198, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401931, AM401932, AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401944, AM401945, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AM402059, AM402062, AM402063, AM402065, AM402079, AM402083, AM402091, AM402123, AM402124, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493437, AM493438, AM493439, AM493441, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449,

171

AM493450, AM493451, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AM493497, AM493500, AM494937, AY044867, AY045702, AY240967, AY240968, AY240969, AY460204, AY587016, AY587844, AY808014, AY808017, AY808020, AY808021, AY808022, AY808025, AY808026, AY808027, AY808029, AY808031, AY808032, AY808034, AY808038, AY808039, AY808041, AY808042, AY808043, D11168, D14484, D16435, D30613, D45172, D50481, D50485, D85516, D89815, D89872, D90208, DQ244141, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492074, DQ492075, DQ492076, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, DQ492093, DQ492127, DQ492130, DQ492131, DQ492139, DQ492140, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, DQ859970, EF032892, EF032893, EF032894, EF407459, EF407460, EF407462, EF407463, EF407466, EF407468, EF407469, EF407470, EF407471, EF407473, EF407474, EF407475, EF407476, EF407478, EF407479, EF407480, EF407482, EF407483, EF407484, EF407485, EF407486, EF407487, EF407488, EF407490, EF407491, EF407492, EF407495, EF407497, EF407498, EF407500, EF407502, EF407503, EF407504, EF638081, EU155217, EU155218, EU155220, EU155222, EU155223, EU155224, EU155226, EU155229, EU155230, EU155232, EU155255, EU155257, EU155259, EU155260, EU155263, EU155279, EU155281, EU155300, EU155301, EU155302, EU155304, EU155308, EU155316, EU155318, EU155324, EU155325, EU155326, EU155327, EU155328, EU155329, EU155330, EU155331, EU155332, EU155333, EU155335, EU155336, EU155356, EU155358, EU155364, EU155366, EU155368, EU155369, EU155373, EU155374, EU155377, EU234061, EU234062, EU239714, EU255961, EU255962, EU256000, EU256001, EU256059, EU256061, EU256062, EU256064, EU256066, EU256075, EU256077, EU256079, EU256080, EU256081, EU256082, EU256083, EU256084, EU256085, EU256089, EU256092, EU256098, EU256099, EU256100, EU256101, EU482849, EU482859, EU482860, EU482877, EU482883, EU482885, EU482888, EU781830, EU857431, EU862837, FJ024277, FJ024279, FJ390398, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958934, GQ913868, GQ913871, GU451218, GU451219, GU451220, GU451221, GU451222, GU451223, GU451224, HM042050, HM042058, HM042062, HM042063, L02836, M96362, U16362, U45476, X61596

347 gcc acg A T AB285066

347 gcc act A T AB049090, AB049099, AB154177, AB154178, AB154179, AB154180, AB154183, AB154184, AB154185, AB154186, AB154187, AB154188, AB154189, AB154190, AB154192, AB154193, AB154194, AB154195, AB154196, AB154197, AB154198, AB154199, AB154200, AB154201,

172

AB154202, AB154203, AB154204, AB154205, AB154206, AB285047, AB285059, AB285067, AB442221, AF265148, AF265149, AF265150, AF265152, AF265153, AF265163, AF265164, AF313916, AF483269, AJ507157, AJ507159, AJ507163, AJ507166, AJ507178, AJ507182, AJ507192, AJ507199, AM402056, AM402057, AM402058, AM402060, AM402061, AM402064, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402080, AM402081, AM402082, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402125, AM402126, AM402127, AM402128, AM402129, AM402329, AM402330, AM402331, AM402332, AM402333, AM402334, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, AY808011, AY808012, AY808016, AY808024, AY808028, AY808030, AY808033, AY808036, AY808040, D10934, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492124, DQ492125, DQ492126, DQ492128, DQ492129, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492150, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492161, DQ492162, DQ492172, EF407481, EF407489, EF407496, EU155219, EU155221, EU155225, EU155228, EU155231, EU155235, EU155253, EU155254, EU155261, EU155262, EU155303, EU155305, EU155307, EU155317, EU155359, EU155360, EU155362, EU155367, EU255960, EU256045, EU256076, EU256078, EU256090, EU482833, EU482839, EU482875, EU482879, EU482880, EU529682, EU660386, EU660388, EU862835, FJ390396, FJ390397, GQ913869, GQ913872, HM042051, HM042052, HM042054, HM042055, HM042056, HM042057, HM042061

347 gcc gtc A V AB285058, AF207765, AJ507168, AJ507194, EF407501, EU256065, EU482874, M84754

347 gcc gtt A V AJ507175, EF407467

348 aag gga K G FJ958904

348 aag ggg K G AJ507189, AY808040, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, EU660388

348 aag atg K M DQ492189, X61596

348 aag ccg K P DQ492040, DQ492073

348 aag cag K Q AB285063

348 aag aga K R AM402328, FJ958890, FJ958893, FJ958896, FJ958900, FJ958901, FJ958902, FJ958903, FJ958906, FJ958914, FJ958915, FJ958916, FJ958917, FJ958919, FJ958920, FJ958921, FJ958927, FJ958928, FJ958931, FJ958935

348 aag agg K R AB049099, AB429050, AF165052, AJ132997, AJ507157, AJ507172, AM401904, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280,

173

AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402329, AM402330, AM402331, AM402332, AM402333, AM402334, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AM493504, AY808034, AY808043, DQ491967, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492017, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492124, DQ492125, DQ492129, DQ492138, EF407466, EF407470, EF407495, EF407497, EU155361, EU155370, HM042051

348 aag cgg K R AJ507155, AY808035, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062

348 aag acg K T AB010249, AB154188, AY808029, D11168, D16435, D89872, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492074, DQ492075, DQ492076, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, EU256079, FJ390398, GQ913871, L02836

349 gcc gac A D D50482, D50484

349 gcc gag A E AY808022

349 gcc ggc A G AB010249, AB049088, AB049089, AB285036, AB285038, AF207752, AF356827, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, D11168, D16435, D89872, EU155260, EU256001, EU256066, FN435993

349 gcc ggg A G AF207753

349 gcc atc A I AY808018, FJ958891, FJ958923

349 gcc aat A N DQ492041, DQ492043, DQ492044, DQ492045, DQ492051, DQ492052

349 gcc ccc A P AF165048, EF407485, EU155259

349 gcc agc A S AY808010

349 gcc acc A T AB285052, AB285054, AB285056, AB285059, AB285068, AB442220, AF165045, AF165046, AF165053, AF165054, AF207771, AJ507163, AJ507189, AM401931, AM402090, AM402299, AY808027, AY808043, EF407463, EF407494, EF407499, EU155226, EU155300, EU155332, EU155359, EU155377, EU255960, FJ958892, FJ958894, FJ958895, FJ958897, FJ958898, FJ958899, FJ958905, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958918, FJ958922, FJ958924, FJ958925, FJ958926, FJ958929, FJ958930, FJ958932, FJ958934

349 gcc acg A T AY808028, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001,

174

DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007

349 gcc act A T AB285063, AB285078, AF207757, AY808035, DQ244140, DQ492040, DQ492042, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062

349 gcc gta A V AB285042, FJ390396

349 gcc gtc A V AB285075, AF176573, AY008261, AY808030, DQ492156, EU155370, EU155371, FJ958917, GU451218, GU451219, GU451220

349 gcc gtg A V AJ507161

350 cct gcc P A AY808010, EU256102

350 cct gct P A D14484

350 cct cat P H AJ507192

350 cct ctt P L AF207762

350 cct tcc P S DQ492002

351 ccg tca P S AM402297, DQ244141

352 ata tta I L AF207753, AF207772

352 ata atg I M AM402066, AM402319

352 ata aca I T AY808025, DQ492019, DQ492153

352 ata acg I T EU482883

352 ata gta I V AB285038, AB285048, AB285063, AB285071, AB285077, AF165053, AF165054, AF265154, AF265155, AF265156, AJ132996, AJ507162, AJ507166, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AY008261, AY808023, AY808024, AY808043, DQ492090, DQ492172, EF407487, EU155305, EU155360, EU256080, EU256102, EU482881, L02836

352 ata gtg I V AB049101, AF165061, AF165062, AF207760, AF207762, AF207769, AJ507155, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, DQ244141, EF407489, EU155219, EU155315, EU155317, EU155376, EU482859, GQ913871

353 cca tca P S AM402071

354 cct ctt P L AM402125


355 cca caa P Q AF207772

355 cca tca P S AM402374, DQ492164

356 cgg ctg R L DQ492172, DQ492197

356 cgg cag R Q DQ492114

356 cgg tgg R W DQ492133


357 agg aaa R K AB010249, AB049094, AB285039, AB285078, AF207774, AJ000009, AJ132997, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AY587844, AY808020, AY808022, D11168, D16435, D89872, DQ492182, EF407471, EF407489, EF407496, EF407497, EU155331, EU155373, EU256079, EU256083, FJ024279

175

357 agg aag R K AB049095, AM493501, AY808011, AY808025, DQ492124, DQ492125, DQ492126, DQ492127, DQ492128, DQ492129, DQ492130, DQ492131, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492139, DQ492140, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492148, DQ492149, DQ492150, DQ492151, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492161, DQ492162, EU155364, EU256061, EU256077

357 agg tgg R W DQ492053

358 aag gaa K E DQ492090

358 aag gag K E AM493501

358 aag aga K R EF407459

358 aag agg K R DQ492083, EF407498, EU862835, FJ958921, FJ958933

359 agg gga R G DQ492072


359 agg aag R K AB049094, AB249644, AB285052, AB285069, AB426117, AF165045, AF165046, AF207755, AF207757, AF207771, AF265148, AF265149, AF265150, AF265163, AF265164, AF265165, AJ507161, AJ507179, AJ507189, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AY808028, AY808030, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492190, EF407459, EF407468, EF407480, EF407484, EU155221, EU155231, EU155257, EU155258, EU155304, EU155307, EU155308, EU155316, EU155318, EU155365, EU155367, EU155377, EU256001, EU256045, EU256066, EU256085, EU256088, EU256098, EU482886, EU529682, EU862835, FJ390397, FJ390398, GU451218, GU451219, GU451220

360 acg gca T A AM401855

360 acg gcc T A AJ507191

360 acg gcg T A EF407495, EU155304, EU256085, GQ913873

360 acg atg T M DQ491998, DQ492003, DQ492004, DQ492005

361 gtt gct V A AM401957, AM402059, AM402280, DQ492179

361 gtt ttc V F AF207760

361 gtt ata V I AJ507156, EU256061

361 gtt atc V I AB049099, AM493491, AM493492, DQ492131, DQ492155, EU155262, EU256059, FJ958909, FJ958927

361 gtt att V I AB285036, AB285060, AB442221, AF207764, AM401901, AM493489, AM493490, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AY044867, AY808011, D14484, DQ492124, DQ492125, DQ492126, DQ492127, DQ492128, DQ492129, DQ492130, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492139, DQ492140, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492148, DQ492149, DQ492150, DQ492151, DQ492152, DQ492153, DQ492154, DQ492156, DQ492157, DQ492159, DQ492160, DQ492161, DQ492162, EF407476, EU155261, EU155304, EU155305, EU155336, EU155376, EU256084, EU256090, EU256091, EU482880, FJ958890,

176

FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958934, FJ958935, HM042050

362 gtc gcc V A AB080299, AM402337, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493498, AM493499, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, DQ491947, EU155376

362 gtc gac V D AM402096

362 gtc atc V I AB036519, AB036520, AB036521, AB049087, AB049089, AB049096, AB109543, AB191333, AB285035, AB285042, AB285048, AB285050, AB285053, AB285060, AB285068, AB285073, AB429050, AF165055, AF165056, AF165063, AF165064, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, DQ492094, DQ492102, DQ859970, EU256000, EU256059, GU451218

362 gtc att V I AB285043, AB285054, AB285055, AB285079, AY240967

362 gtc act V T AM493497, AM493500

363 ctg ccg L P AM401979, AM402316, DQ492112

363 ctg cag L Q AM401951, DQ492115

364 tca gca S A AM401965, AM402069, AM402094, DQ491979, DQ492180

364 tca aca S T AB010249, AB010250, AB036519, AB036520, AB036521, AB049087, AB049089, AB049090, AB049091, AB049092, AB049095, AB049096, AB049098, AB049099, AB049101, AB080299, AB109543, AB154177, AB154178, AB154179, AB154180, AB154181, AB154182, AB154183, AB154184, AB154185, AB154186, AB154187, AB154188, AB154189, AB154190, AB154191, AB154192, AB154193, AB154194, AB154195, AB154196, AB154197, AB154198, AB154199, AB154200, AB154201, AB154202, AB154203, AB154204, AB154205, AB154206, AB191333, AB285035, AB285036, AB285037, AB285038, AB285039, AB285040, AB285042, AB285043, AB285044, AB285046, AB285047, AB285048, AB285050, AB285052, AB285053, AB285054, AB285055, AB285056, AB285058, AB285059, AB285060, AB285061, AB285062, AB285063, AB285064, AB285065, AB285066, AB285067, AB285068, AB285069, AB285070, AB285071, AB285072, AB285073, AB285075, AB285076, AB285077, AB285078, AB285079, AB285081, AB429050, AB442219, AB442220, AB442221, AB442222, AF033373, AF034151, AF165045, AF165046, AF165047, AF165048, AF165049, AF165050, AF165051, AF165052, AF165053, AF165054, AF165055, AF165056, AF165057, AF165058, AF165059, AF165060, AF165061, AF165062, AF165063, AF165064, AF207752, AF207753, AF207754, AF207755, AF207756, AF207758, AF207760, AF207762, AF207763, AF207764, AF207765, AF207766, AF207768, AF207769, AF207770, AF207771, AF207774, AF208024, AF265145, AF265146, AF265147, AF265152, AF265153, AF265158, AF265159, AF313916, AF333324, AF356827, AF483269, AJ000009, AJ132996, AJ132997, AJ507155, AJ507156, AJ507157, AJ507158, AJ507159, AJ507160, AJ507161, AJ507162, AJ507165, AJ507166, AJ507168, AJ507169, AJ507172, AJ507173, AJ507177, AJ507179, AJ507180, AJ507181, AJ507182, AJ507183, AJ507184,

177

AJ507185, AJ507186, AJ507187, AJ507188, AJ507189, AJ507191, AJ507192, AJ507193, AJ507195, AJ507196, AJ507198, AJ507199, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401932, AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401944, AM401945, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402092, AM402093, AM402095, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AM402329, AM402330, AM402331, AM402332, AM402333, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AM494937, AY008261, AY045702, AY240967, AY240968, AY240969, AY460204, AY587016, AY587844, AY808009, AY808010, AY808011, AY808012, AY808013, AY808014, AY808015, AY808016, AY808017, AY808018, AY808019, AY808020, AY808021, AY808022, AY808023, AY808024, AY808025, AY808026, AY808028, AY808030, AY808031, AY808032, AY808033, AY808034, AY808036, AY808037, AY808038, AY808039, AY808040, AY808041, AY808042, AY808043, D10934, D11168, D14484, D16435, D30613, D45172, D50480, D50481, D50482, D50483, D50484, D50485, D63857, D85516, D89815, D89872, D90208, DQ244140, DQ244141, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491988, DQ491989, DQ491991, DQ491992, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492008,

178

DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492093, DQ492094, DQ492095, DQ492096, DQ492097, DQ492098, DQ492099, DQ492100, DQ492101, DQ492102, DQ492103, DQ492104, DQ492105, DQ492106, DQ492107, DQ492108, DQ492109, DQ492110, DQ492111, DQ492112, DQ492113, DQ492114, DQ492115, DQ492116, DQ492117, DQ492118, DQ492119, DQ492120, DQ492121, DQ492122, DQ492123, DQ492124, DQ492125, DQ492126, DQ492127, DQ492128, DQ492129, DQ492130, DQ492131, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492139, DQ492140, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492148, DQ492150, DQ492151, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492161, DQ492162, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492197, DQ859970, EF032892, EF032893, EF032894, EF407459, EF407461, EF407463, EF407464, EF407465, EF407466, EF407467, EF407468, EF407469, EF407470, EF407471, EF407472, EF407474, EF407475, EF407476, EF407477, EF407478, EF407480, EF407482, EF407484, EF407485, EF407486, EF407487, EF407489, EF407490, EF407491, EF407494, EF407495, EF407496, EF407497, EF407498, EF407499, EF407500, EF407502, EF407503, EF407504, EU155218, EU155219, EU155221, EU155223, EU155224, EU155225, EU155226, EU155227, EU155229, EU155230, EU155231, EU155232, EU155253, EU155255, EU155256, EU155257, EU155260, EU155261, EU155262, EU155263, EU155264, EU155279, EU155280, EU155281, EU155300, EU155301, EU155302, EU155303, EU155304, EU155305, EU155306, EU155308, EU155316, EU155318, EU155324, EU155326, EU155327, EU155329, EU155330, EU155331, EU155332, EU155333, EU155334, EU155335, EU155336, EU155337, EU155358, EU155359, EU155360, EU155361, EU155362, EU155363, EU155364, EU155365, EU155366, EU155367, EU155368, EU155369, EU155370, EU155371, EU155372, EU155373, EU155374, EU155375, EU155376, EU155377, EU155382, EU234061, EU234062, EU255961, EU255962, EU256000, EU256001, EU256045, EU256059, EU256061, EU256064, EU256066, EU256075, EU256077, EU256078, EU256079, EU256080, EU256081, EU256083, EU256084, EU256085, EU256088, EU256089, EU256090, EU256092, EU256098, EU256099, EU256100, EU256101, EU256102, EU256103, EU482833, EU482839, EU482849, EU482859, EU482860, EU482875, EU482880, EU482881, EU482883, EU482885, EU482886, EU482888, EU529682, EU660386, EU781825, EU781832, EU857431, EU862835, EU862837, FJ024277, FJ024279, FJ390397, FJ390398, FJ478453, FJ958890, FJ958891, FJ958893, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958934, FJ958935, FN435993, GQ913870, GU451218, GU451219, GU451220, GU451221, GU451223, GU451224, HM042050, HM042053, HM042057, HM042059, HM042060, L02836, M58335, M84754, M96362, U01214, U16362, U45476, X61596

364 tca acc S T AF265156, AJ507167, AJ507175, EU255960

364 tca acg S T AB049088, AB049100, AB285051, AB285057, AB285074, AF176573, AF207757, AF207761, AF265154, AF265155, AJ133096, AJ507171, AJ507197, AM401931, AM402091, AM402096, AM402334, AY808027, AY808035, DQ491986, DQ491987, DQ491993, DQ492026, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046,

179

DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, DQ492149, DQ492196, EF407492, EF638081, EU155217, EU155258, EU155357, EU256065, EU482877, EU482879, FJ390396, FJ958892, FJ958894

364 tca act S T AB249644, AB426117, AJ507163, AJ507190, AJ507194, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AY808029, DQ491990, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492074, DQ492075, DQ492076, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, EU155307, EU155328, EU256082, GU451222

365 gaa gac E D AB049100, AB285079, AB429050, AF165047, AF165048, AJ507169, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, EU155260, EU155363, HM042059

365 gaa gat E D AB285055, AB285060, DQ859970, EU155372, HM042056

365 gaa gga E G AJ507174, EU256059

365 gaa ggg E G AB285076, DQ492164

366 tct ttc S F DQ492181

366 tct ttt S F DQ492150


367 acc gct T A DQ492182

367 acc atc T I EU155223, EU256084

367 acc att T I EU256089

367 acc aac T N AB285060, AJ507186, AY808038, EU155363

367 acc aat T N EU482833

367 acc agt T S AB285036, AF165060, AF207760

367 acc tcc T S AF333324, EF407474, EF407494, EU256076, L02836, M58335

367 acc tct T S AJ507177, AJ507184, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315,

180

AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, EU155217, EU155336, EU155360, EU482874

368 gtg gcg V A DQ492082, EU256084

368 gtg ata V I GQ913873, HM042056

368 gtg ctg V L AB285081

368 gtg ttg V L AB285078

368 gtg atg V M AM494937, AY240967, DQ491968, DQ492030, EU155232

369 tct gct S A AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AY808028, DQ491979, DQ491980, DQ491981, DQ491982, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, EF638081, EU239714

369 tct ttt S F AM402352, DQ492143

369 tct cct S P AY240967

369 tct acc S T EU155257, EU155337

369 tct gtt S V DQ491983

370 tct gct S A AF207754, EU239714

370 tct ctt S L EU155281

370 tct cct S P DQ492024

370 tct act S T AB285060, AB285067, AB285076, EU155333, HM042056

371 gcc ggc A G AM402263, AM402269

371 gcc acc A T AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, DQ492092

371 gcc gtc A V AB049094, AB285044, AY240967, DQ492165, EU155381, EU256001, EU482880

371 gcc gtt A V AB285076, AJ507191, DQ244140

372 ttg cag L Q AM402326

372 ttg tgg L W DQ492149

373 gcg ggg A G EU482880

373 gcg tcg A S DQ491995

373 gcg gta A V EU155263

373 gcg gtg A V DQ491967

374 gag gac E D FN435993

374 gag gat E D AY808010, EU155328

374 gag ggg E G AJ507175, AM402282, AM402315, EU155372, EU256075

374 gag aag E K AM401884, AM401889, AM401893, DQ491999

375 ctc ttc L F AF207762

375 ctc ttt L F AF207756, AM493439

375 ctc cct L P DQ492058, DQ492125

376 gcc acc A T AM402066, DQ492182, EU239714

376 gcc gtt A V AM401924, DQ491951

377 aca gca T A AJ507192, AM494937, AY808024, AY808026, AY808033, EF407469, EF407472, EU155225, EU155227, EU155328, EU155376, EU234062, EU529682

377 aca gcg T A AB285048, AB285055, AJ507160, AY240969, EU256085

377 aca gct T A AF207769

181

377 aca gag T E EU256075

377 aca ata T I AF265148, AF265149, AF265150, AF265163, AF265164, AF265165, AJ507161, AJ507175, AM493491, AM493492, AM493500, AM493501, AM493503, AM493504, AM493505, GQ913872, GU451222

377 aca aaa T K AY808042, DQ491975, DQ491978, DQ492108, EU155219

377 aca aag T K HM042062

377 aca agg T R EU256084

377 aca tcg T S EU256081

377 aca gta T V AF165045, AF165046, AF207755, AJ507179, EF407461, EU155372, EU482886

377 aca gtt T V AF165058, U01214

378 aag gag K E AJ507157, DQ492020, DQ492057, FJ958927

378 aag cag K Q AY808040, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957

378 aag agg K R AJ507189, AM493499, DQ491982, DQ491991, DQ492125, DQ492188, EU155280

378 aag tcg K S AJ507184

378 aag aca K T AJ507161

378 aag acc K T AB285048

379 acc gcc T A AB049092, AB285040, AM402055, AM402060, AM402066, AM402070, AM402071, AM402074, AM402075, AM402081, AM402088, AM402090, AM402096, AM402098, AM402101, AM402102, AM402106, AM402108, AM402111, AM402112, AM402115, AM402125, AM402127, AY460204, EF407470, EF407477, EU155218, EU155381, EU781832, EU862837, HM042053

379 acc gct T A AF165045, AF165046, AF207755

379 acc agc T S AF165057, AF165058

379 acc agt T S GQ913869

379 acc tcc T S AB442220, EF407481, EU256083

379 acc tct T S EU155375

380 ttc ctc F L AM402296, AM493443, DQ491949, DQ492017

380 ttc ctt F L DQ492049

380 ttc tcc F S DQ492027, DQ492151, DQ492165

381 ggc gcc G A AB285073

381 ggc gct G A EF407489, EU155373

381 ggc gac G D AF208024, AM402347, AY808026

381 ggc aat G N DQ492080

381 ggc agc G S AB049094, AB049098, AB049099, AB285037, AJ507160, AJ507186, AJ507192, AY240969, AY808024, AY808033, EF407461, EF407470, EF407476, EF407482, EF638081, EU155219, EU155227, EU155261, EU155326, EU155333, EU155334, EU155363, EU256084, EU482885, EU862837, FJ958898

381 ggc agt G S DQ492063, DQ492065, DQ492067, DQ492068, DQ492070, DQ492073, DQ492074, DQ492075, DQ492078, DQ492079, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, EF407490, EU256001

381 ggc act G T EU155281

382 agc gac S D AB285044, AB285055, EU155228, EU155370, EU155371, GU451222

382 agc gga S G AF207755

382 agc ggc S G AB049098, AB285062, AB442221, AF207774, AJ507160, AJ507182, AJ507188, AJ507189, AM402115, AY240969, AY808017, AY808031, AY808032, AY808033, D14484, EF407470, EU155279, EU155281, EU155305, EU155306, EU155307, EU155333, EU256001, EU256075, EU256084, EU256091, FJ958897, GQ913872

382 agc ggt S G AF207769, D89815, EU155369

382 agc aac S N AB285054, AF207771, AJ507192, AM402061, AM493493, GU451218, GU451219, GU451220

182

382 agc aga S R DQ491976

383 tcc ttc S F HM042058

383 tcc ttt S F AM402367

383 tcc ccc S P AF165061, AF207769, AM401885, AM493449, DQ244140, EF407461, EF407470, FJ958916

383 tcc cct S P EF407482, EU862837

384 gaa gca E A EU155260, EU155364, EU256080, EU256084

384 gaa gcg E A EU155232

384 gaa gac E D AM402329, AM402330, AM402331, AM402332, AM402333, AM402334, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402364, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, AY808027, HM042059

384 gaa gat E D AB285081

384 gaa gga E G AB010249, AB010250, AB036519, AB036520, AB036521, AB049087, AB049088, AB049089, AB049090, AB049092, AB049095, AB049098, AB049099, AB049100, AB080299, AB109543, AB154183, AB154184, AB191333, AB285035, AB285037, AB285038, AB285040, AB285042, AB285043, AB285046, AB285048, AB285050, AB285052, AB285054, AB285055, AB285056, AB285061, AB285068, AB285070, AB285071, AB285073, AB285078, AB285079, AB429050, AB442219, AB442220, AB442221, AB442222, AF034151, AF165045, AF165046, AF165049, AF165050, AF165051, AF165052, AF165053, AF165054, AF165055, AF165056, AF165057, AF165058, AF165059, AF165060, AF165061, AF165062, AF165063, AF165064, AF207752, AF207755, AF207757, AF207758, AF207762, AF207764, AF207765, AF207768, AF207771, AF207772, AF208024, AF265142, AF265144, AF265160, AF265162, AF356827, AF483269, AJ507157, AJ507159, AJ507160, AJ507161, AJ507165, AJ507167, AJ507171, AJ507172, AJ507174, AJ507175, AJ507189, AJ507190, AJ507192, AJ507193, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AY008261, AY587016, AY808009, AY808015, AY808017, AY808020, AY808021, AY808029, AY808033, AY808036, AY808039, AY808041, D10934, D11168, D14484, D16435, D30613, D45172, D50480, D63857, D85516, D89815, D89872, DQ244141, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ492063, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492072, DQ492073, DQ492074, DQ492075, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492110, DQ859970, EF407460, EF407462, EF407468, EF407477, EF407479, EF407480, EF407491, EF407501, EU155218, EU155222, EU155228, EU155257, EU155302, EU155306, EU155315, EU155317, EU155336, EU155356, EU155360, EU155363, EU155368, EU155370, EU155371, EU155372, EU155373, EU155382,

183

EU255962, EU256000, EU256062, EU256064, EU256075, EU256076, EU256078, EU256079, EU256082, EU256089, EU256090, EU256091, EU256099, EU256101, EU482859, EU482874, EU482875, EU660386, EU660388, EU781827, EU857431, EU862837, GQ913869, GQ913873, GU451218, GU451219, GU451220, GU451222, HM042051, HM042052, HM042054, HM042056, HM042057, HM042058, HM042061, M96362, U16362, X61596

384 gaa ggc E G AJ507188, AY808040, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957

384 gaa ggg E G AB049096, AB285041, AB285057, AB285064, AF165047, AF165048, AF207753, AF207769, AJ132997, AJ507155, AJ507164, AJ507178, AM401916, AM401917, AM401918, AM401922, AM401923, AM401924, AM401926, AM401927, AM401928, AM401930, AM401931, AM401932, AM401933, AM401936, AM401939, AM401940, AM401941, AM401943, AM401944, AM401945, AM401947, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AY045702, D50482, D50484, D90208, DQ492092, DQ492177, EU155231, EU155235, EU155325, EU155361, HM042055

384 gaa ggt E G AB285062, EU155357

384 gaa aaa E K AM402120, AY460204, AY808026, EF407467, EU155308, EU529682, HM042053

384 gaa aag E K AY808010

384 gaa atg E M AY808031

384 gaa aga E R AY808042, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, EF407470

384 gaa gta E V AB049094

385 tcg gcg S A DQ492094

385 tcg gag S E AB285073

385 tcg ttg S L EU155359

385 tcg cca S P AB285076, HM042058

385 tcg ccg S P AB285040, AJ507169, EF407470, EU155264, EU155369, EU255960, EU256083, HM042059, HM042062

385 tcg acg S T AY044867, AY808032, DQ492184

385 tcg act S T AJ507161

386 tcg gcg S A AY008261

386 tcg gag S E AF207769

386 tcg ctg S L AY240969, AY808042, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978

386 tcg cca S P AB285076, EU155376, EU482833

386 tcg ccg S P AB154196, AJ507169, AM402381, AY808024, EF407467, EF407470, EF407481, EU155359, EU256102, EU529682, GQ913873, HM042058, HM042059

386 tcg cgc S R AJ507161

386 tcg acg S T AY808033, EF407465, EU155375

387 gcc tgc A C AJ507161

387 gcc ggc A G AF165063, AF165064, AF265162, AJ507184, EU155300, EU155368, EU256083, EU482885, EU482888, EU862837

387 gcc ccc A P EU155306

387 gcc tcc A S AY240969, EU482833

387 gcc tct A S EU256102

387 gcc acc A T AB154194, AB285063, AF207769, AM402061, AM402066, AY808024, EU255960, GU451218, GU451219, GU451220, HM042062

184

387 gcc gtc A V AM401958, AM402259, AM402323, AY808034, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492016, DQ492017, DQ492018, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ859970

387 gcc gtt A V DQ492015, DQ492019, EF407468

388 gtc gca V A AB285042

388 gtc gcc V A AB049089, AB049091, AB049100, AB080299, AB154188, AB285036, AB285039, AB285044, AB285051, AB285054, AB285055, AB285062, AB285068, AB285071, AB285076, AB285077, AB285078, AB442221, AF165045, AF165046, AF207753, AF207754, AF207755, AF207768, AF207769, AF207770, AF265149, AJ238800, AJ507163, AJ507172, AJ507188, AM402096, AY240969, AY808026, AY808028, AY808038, AY808043, D14484, D50481, D50485, D85516, DQ491979, DQ491980, DQ491981, DQ491985, DQ491986, DQ491987, DQ491988, DQ491990, DQ491991, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492046, EF407463, EF407470, EF407496, EU155256, EU155263, EU155316, EU155318, EU155324, EU155335, EU155365, EU155369, EU155375, EU155376, EU255960, EU256075, EU256082, EU256091, EU256103, EU482879, GQ913871, GU451218, GU451219, GU451220, GU451222, HM042050, HM042053, HM042055, HM042059, HM042060, HM042061

388 gtc gcg V A AY808010, EF407495

388 gtc gct V A AB285040, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AY808033, D50480, D50483, DQ244140, DQ244141, EF407465, EF407472, EF407477, EF407482, EF407486, EF407494, EU155264, EU155280, EU155306, EU155370, EU155371, EU256099, EU482881, FJ390396, M84754

388 gtc gac V D AF207771

388 gtc ttc V F EU862835

388 gtc ggc V G AJ507161, AY008261, EF407467, EF407468, EU155359

388 gtc atc V I AB049098, AB285037, AY808013, EF407492, EF407499, EF638081, EU256084, GU451221, GU451223, GU451224, M96362, U16362

388 gtc att V I AY808020, EU256080

388 gtc ctt V L AB249644, AB426117

388 gtc ccc V P AF165061, AF165062

388 gtc cgc V R EF407501

388 gtc tcc V S AF207757

388 gtc acc V T AY460204, DQ491998

389 gac gct D A AJ507169

389 gac ggc D G AY808026

389 gac ggt D G M84754

389 gac aac D N AY808030, EU256080

389 gac agc D S EU529682

390 agc tgc S C DQ492170

390 agc gga S G AF207757, EU155376

390 agc ggc S G AB285040, AB285041, AM402081, AY460204, DQ492091, DQ492149, EU862835

390 agc aag S K EU155306

390 agc aac S N AB154196, AJ507169, DQ492031

390 agc aga S R D50480, D50483

390 agc acc S T EU155359

391 ggc gac G D AM402066

391 ggc aac G N EU155359

391 ggc cgc G R AF207757

185

391 ggc agc G S AB154195, AB154196, AJ507169, EU155306, EU155376

391 ggc agt G S DQ492158

392 acg gca T A AF207757, EU256080

392 acg gcg T A EF407467, EU155306, HM042056

392 acg ata T I AF207771

392 acg atg T M AB285044, AB285078, AF265147, AJ507161, AJ507169, AJ507182, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AY808042, DQ071885, DQ244140, DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ491977, DQ491978, EF407471, EU155359, EU155364

392 acg tcg T S DQ492054, DQ492055

392 acg gtg T V AB285041, AF207766, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AY808024, EU155369, EU482880

393 gca gaa A E HM042062

393 gca gag A E AJ507189, EF407501, EF407504

393 gca ggg A G DQ492006, EU256080, EU256083

393 gca tcg A S AF165053, AF165054, EU155255

393 gca acg A T AB285040, AY008261, EU155306, GQ913873

393 gca gtg A V AJ507169, AM401942, EF407467, EU482880

394 acg gca T A HM042062

394 acg gcc T A AB285040, AF176573, AM402337, DQ492076, EU155359, EU482880, HM042058

394 acg gcg T A EF407481

394 acg gct T A DQ492157, DQ492162, DQ492167

394 acg atc T I AB154195, AY808031, EU155369

394 acg att T I AB154196

394 acg agc T S AB154187, AB154188, AB442219, AF034151, AF356827, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AY808023, AY808032, EU155264, EU155332

394 acg tcc T S AB049101, AB285046, AJ507161, AJ507194, AY808017, DQ491951, EF407487, EU155281, EU155375, EU256084, EU256102, EU781825, HM042054

394 acg tct T S AB285081, AJ507160

395 gcc ggc A G AB010250, AB154183, AB154184, AB285063, AB285064, AF265152, AJ507162, AJ507182, AY587016, AY808042, D30613, D45172, D89815, D90208, DQ859970, EU256061, EU256076, EU857431, HM042059

395 gcc ggt A G AF265153, AJ507189

395 gcc atc A I AY808009, EF407478, EF407498, EU155218

395 gcc tcc A S DQ492109

395 gcc acc A T AM402355

395 gcc act A T AF207757, AM401874, AM401904

395 gcc gtc A V EU155376

396 tct ctc S L AB285077, AB429050, EU529682

396 tct ctt S L AB285065, AF333324, D63857, DQ492074, EF407470, EF407474, EU781825, M58335

186

396 tct cca S P AB249644, AB285040, AB285069, AB426117, EF407489

396 tct ccc S P AB049098, AB154194, AB154195, AB154196, AB154198, AB154200, AB285038, AB285042, AB285074, AB285076, AB442219, AF165049, AF165050, AF165059, AF165060, AF356827, AJ507167, AJ507171, AJ507189, AM402087, AM402105, AY808013, AY808014, AY808017, AY808021, AY808022, AY808033, AY808036, AY808040, AY808041, D50480, D50483, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ492021, DQ492093, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407472, EF407496, EU256045, EU256085, EU482883, FJ390397, HM042056

396 tct ccg S P EU155221, EU256084

396 tct cct S P AB010249, AB010250, AB036519, AB036520, AB036521, AB049087, AB049088, AB049089, AB049090, AB049091, AB049092, AB049094, AB049095, AB049096, AB049099, AB049100, AB080299, AB109543, AB154177, AB154178, AB154179, AB154180, AB154181, AB154182, AB154183, AB154184, AB154185, AB154186, AB154188, AB154189, AB154190, AB154193, AB154197, AB154199, AB154201, AB154202, AB154203, AB154204, AB154205, AB154206, AB191333, AB285035, AB285036, AB285037, AB285039, AB285041, AB285043, AB285044, AB285046, AB285047, AB285048, AB285050, AB285051, AB285052, AB285053, AB285054, AB285055, AB285056, AB285057, AB285058, AB285059, AB285060, AB285061, AB285062, AB285063, AB285064, AB285066, AB285067, AB285068, AB285070, AB285071, AB285072, AB285073, AB285075, AB285078, AB285079, AB285081, AB442220, AB442221, AB442222, AF033373, AF034151, AF165045, AF165046, AF165047, AF165048, AF165051, AF165052, AF165053, AF165054, AF165055, AF165056, AF165057, AF165058, AF165061, AF165062, AF165063, AF165064, AF207752, AF207753, AF207754, AF207755, AF207756, AF207757, AF207758, AF207760, AF207761, AF207762, AF207763, AF207764, AF207765, AF207766, AF207768, AF207769, AF207770, AF207771, AF207772, AF207774, AF208024, AF265142, AF265143, AF265144, AF265145, AF265146, AF265147, AF265148, AF265149, AF265150, AF265152, AF265153, AF265154, AF265155, AF265156, AF265158, AF265159, AF265160, AF265161, AF265162, AF265163, AF265164, AF265165, AF313916, AF483269, AJ000009, AJ132996, AJ132997, AJ133096, AJ238800, AJ507155, AJ507156, AJ507158, AJ507159, AJ507160, AJ507161, AJ507162, AJ507163, AJ507164, AJ507165, AJ507166, AJ507168, AJ507169, AJ507170, AJ507172, AJ507174, AJ507175, AJ507177, AJ507178, AJ507179, AJ507180, AJ507181, AJ507182, AJ507183, AJ507184, AJ507185, AJ507186, AJ507187, AJ507188, AJ507190, AJ507191, AJ507192, AJ507193, AJ507194, AJ507195, AJ507196, AJ507197, AJ507198, AJ507199, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401931, AM401932, AM401933, AM401934, AM401935, AM401936,

187

AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401944, AM401945, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402114, AM402115, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402129, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM402329, AM402330, AM402331, AM402332, AM402333, AM402334, AM402335, AM402336, AM402337, AM402338, AM402339, AM402340, AM402341, AM402342, AM402343, AM402344, AM402345, AM402346, AM402347, AM402348, AM402349, AM402350, AM402351, AM402352, AM402353, AM402354, AM402355, AM402356, AM402357, AM402358, AM402359, AM402360, AM402361, AM402362, AM402363, AM402365, AM402367, AM402369, AM402370, AM402371, AM402372, AM402373, AM402374, AM402375, AM402376, AM402377, AM402378, AM402379, AM402380, AM402381, AM402382, AM402383, AM402384, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AM494937, AY008261, AY044867, AY045702, AY240967, AY240968, AY240969, AY460204, AY587016, AY587844, AY808009, AY808010, AY808011, AY808012, AY808015, AY808016, AY808018, AY808019, AY808020, AY808023, AY808024, AY808025, AY808026, AY808027, AY808028, AY808029, AY808030, AY808032, AY808034, AY808035, AY808037, AY808038, AY808039, AY808042, AY808043, D10934, D11168, D14484, D16435, D30613, D45172, D50481, D50482, D50484, D50485, D85516, D89815, D89872, D90208, DQ071885, DQ244140, DQ244141, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492040, DQ492041,

188

DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492075, DQ492076, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, DQ492094, DQ492095, DQ492096, DQ492097, DQ492098, DQ492099, DQ492100, DQ492101, DQ492102, DQ492103, DQ492104, DQ492105, DQ492106, DQ492107, DQ492108, DQ492109, DQ492110, DQ492111, DQ492112, DQ492113, DQ492114, DQ492115, DQ492116, DQ492117, DQ492118, DQ492119, DQ492120, DQ492121, DQ492122, DQ492123, DQ492124, DQ492125, DQ492126, DQ492127, DQ492128, DQ492129, DQ492130, DQ492131, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492139, DQ492140, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492148, DQ492149, DQ492150, DQ492151, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492161, DQ492162, DQ859970, EF032892, EF032893, EF032894, EF407459, EF407460, EF407461, EF407462, EF407463, EF407464, EF407465, EF407466, EF407467, EF407468, EF407469, EF407471, EF407473, EF407475, EF407476, EF407477, EF407478, EF407479, EF407480, EF407481, EF407482, EF407483, EF407484, EF407485, EF407486, EF407487, EF407488, EF407490, EF407491, EF407492, EF407494, EF407495, EF407497, EF407498, EF407499, EF407500, EF407501, EF407502, EF407503, EF407504, EF638081, EU155217, EU155218, EU155219, EU155220, EU155222, EU155223, EU155224, EU155225, EU155226, EU155227, EU155228, EU155229, EU155230, EU155231, EU155232, EU155235, EU155253, EU155254, EU155255, EU155256, EU155257, EU155258, EU155259, EU155260, EU155261, EU155262, EU155263, EU155264, EU155279, EU155281, EU155300, EU155301, EU155302, EU155303, EU155304, EU155305, EU155306, EU155307, EU155316, EU155317, EU155318, EU155324, EU155325, EU155326, EU155327, EU155328, EU155329, EU155330, EU155331, EU155332, EU155333, EU155334, EU155335, EU155336, EU155337, EU155356, EU155357, EU155358, EU155359, EU155360, EU155361, EU155362, EU155363, EU155365, EU155366, EU155367, EU155368, EU155369, EU155370, EU155371, EU155372, EU155373, EU155374, EU155375, EU155376, EU155377, EU155381, EU155382, EU234061, EU234062, EU239714, EU255960, EU255961, EU255962, EU256000, EU256001, EU256059, EU256061, EU256062, EU256064, EU256065, EU256066, EU256075, EU256076, EU256077, EU256078, EU256079, EU256080, EU256081, EU256082, EU256083, EU256088, EU256089, EU256090, EU256091, EU256092, EU256098, EU256099, EU256100, EU256101, EU256102, EU256103, EU482833, EU482839, EU482849, EU482859, EU482860, EU482874, EU482875, EU482877, EU482879, EU482880, EU482885, EU482886, EU482888, EU660386, EU660388, EU781827, EU781830, EU781832, EU857431, EU862835, EU862837, FJ024277, FJ024279, FJ390396, FJ390398, FJ478453, FJ958890, FJ958891, FJ958892, FJ958893, FJ958894, FJ958895, FJ958896, FJ958897, FJ958898, FJ958899, FJ958900, FJ958901, FJ958902, FJ958903, FJ958904, FJ958905, FJ958906, FJ958907, FJ958909, FJ958910, FJ958911, FJ958912, FJ958913, FJ958914, FJ958915, FJ958916, FJ958917, FJ958918, FJ958919, FJ958920, FJ958921, FJ958922, FJ958923, FJ958924, FJ958925, FJ958926, FJ958927, FJ958928, FJ958929, FJ958930, FJ958931, FJ958932, FJ958933, FJ958934, FJ958935, FN435993, GQ913868, GQ913869, GQ913870, GQ913871, GQ913872, GQ913873, GU451218, GU451219, GU451220, GU451221, GU451222, GU451223, GU451224, HM042050, HM042052, HM042053, HM042054, HM042055, HM042057, HM042058, HM042059, HM042060, HM042061, HM042062, HM042063, L02836, M84754, M96362, U01214, U16362, U45476, X61596

397 cct cat P H AB154192, AB154199, AB285054, AB285055, AB285068, AF165055, AF165056, DQ492082

189

397 cct cta P L AB154197

397 cct ctc P L EU155232

397 cct ctt P L AB154198, AB285063, AB285069, AF265158, AF265159, AM402112, AM402127, AM402277, AY808032, AY808039, DQ492157, EU256077, EU256083, GU451224

397 cct tcc P S AJ507169

397 cct tct P S EU529682

398 gac gcc D A EU155306

398 gac gag D E AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915

398 gac ggc D G AB049100, AB285039, AB285041, AB285046, AB285077, AF265148, AF265149, AF265150, AF265163, AF265164, AF265165, AJ132996, AJ507159, AJ507171, AJ507174, AJ507188, AJ507198, AY808012, AY808013, AY808014, AY808020, AY808037, AY808041, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, DQ492093, DQ492123, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407467, EF407489, EF407496, EU155377, EU155382, EU256103, GU451218, GU451219, GU451220, GU451222, HM042063, U45476

398 gac ggt D G AF165045, AF165046, AF207755, AF207757, AY808026, DQ491977, DQ491978, EU155316

398 gac cac D H EU256077

398 gac aac D N AB285056, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AM494937

398 gac aat D N AB285035, AB285053, AF207764, AJ507189

398 gac agc D S EU155376

398 gac agt D S AB285073

398 gac acc D T AB285038, AF165047, AF165048

398 gac act D T AB285054, AB285055

398 gac gtt D V AB285079

398 gac tac D Y AB285075

399 cag tgc Q C GU451221, GU451223, GU451224

399 cag gag Q E AB285042, AB285053, AB442221, EU255961, EU256059, L02836

399 cag ggg Q G AB036519, AB036520, AB036521, AB109543, AB191333, AB285050

399 cag cac Q H AB285068, AY460204, EF407489, EF638081

399 cag cat Q H AY808037

399 cag cta Q L EU155316

399 cag ctg Q L AB249644, AB285035, AB285039, AB285075, AB426117, AF165055, AF165056, AF265142, AF265143, AF265161, AY808026, AY808043, EF407485, EF407496

399 cag ccg Q P AB285066, EU256090

399 cag cga Q R EU155359

190

399 cag cgg Q R AB285073, AB285076, AJ507161, AM401877, AM402057, AY008261, DQ492095

400 ccc gcc P A AB010250, AB049091, AB049094, AB049099, AB049100, AB080299, AB154185, AB154186, AB154193, AB249644, AB285035, AB285037, AB285041, AB285044, AB285046, AB285047, AB285053, AB285058, AB285059, AB285061, AB285062, AB285077, AB285078, AB426117, AB442221, AB442222, AF165049, AF165050, AF165051, AF165059, AF165060, AF165061, AF165062, AF207752, AF207754, AF207756, AF207758, AF207761, AF207762, AF207763, AF207765, AF207769, AF207770, AF207774, AF265154, AF265155, AF265156, AF333324, AJ507166, AJ507169, AJ507177, AJ507178, AJ507179, AJ507181, AJ507182, AJ507190, AJ507191, AJ507197, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AY240967, AY240968, AY460204, AY587016, AY587844, AY808010, AY808018, AY808019, AY808036, AY808040, D30613, D45172, D50480, D85516, D89815, D90208, DQ244140, DQ244141, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ492094, DQ492095, DQ492096, DQ492097, DQ492098, DQ492099, DQ492100, DQ492101, DQ492102, DQ492103, DQ492104, DQ492105, DQ492106, DQ492107, DQ492109, DQ492111, DQ492112, DQ492115, DQ492116, DQ492120, DQ492121, DQ492122, DQ492123, EF407459, EF407462, EF407465, EF407468, EF407474, EF407475, EF407478, EF407479, EF407482, EF407486, EF407494, EF407495, EF407498, EF638081, EU155232, EU155253, EU155256, EU155263, EU155302, EU155305, EU155324, EU155328, EU155336, EU155337, EU155358, EU155368, EU155373, EU155374, EU234061, EU239714, EU255961, EU256079, EU256101, EU482839, EU482874, EU482875, EU482879, EU482888, EU781827, EU857431, EU862835, GQ913869, GU451221, GU451223, GU451224, L02836, M58335, M96362, U16362

400 ccc gcg P A AB285063

400 ccc gct P A AB285051, AF165052, AF165057, AF165058, AF207772, AY045702, AY808028, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492108, DQ492110, DQ492113, DQ492114, DQ492117, DQ492118, DQ492119, DQ859970, EF407477

400 ccc ttc P F AM493506

400 ccc atc P I AJ507168

400 ccc ctc P L AB154177, AB154178, AB154189, AB154190, AB154195, AB154196, AB285056, AB285075, AF207764, AJ507156, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401867, AM401868, AM401869, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906,

191

AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AY808031, AY808034, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, EF407492, EF407499, EU155369, EU155381, EU256062, EU256075, EU256077, EU482849

400 ccc ctt P L AY808015

400 ccc cgc P R AM401865, AM401866, AM401870, EU155259

400 ccc tcc P S AB049096, AB285038, AB285048, AB285065, AB285068, AF034151, AF165047, AF165048, AF265148, AF265152, AF265164, AF483269, AJ507155, AJ507159, AJ507171, AJ507192, AJ507198, AM402121, AM402129, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493507, AM494937, AY808013, AY808020, AY808021, AY808027, AY808033, AY808035, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492050, DQ492051, DQ492052, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, EF032892, EF032893, EF032894, EF407460, EF407461, EF407472, EF407473, EF407476, EU155235, EU155366, EU155382, EU255960, EU256045, EU256080, EU256081, EU256083, EU256088, EU256103, EU482833, EU529682, EU781825, FJ958934, GU451218, GU451219, GU451220, GU451222, HM042058, U45476

400 ccc tct P S AB285072, AF265145, AF265146, AF265147, EF407500, EF407503, HM042063

400 ccc acc P T AB010249, AB049088, AB049089, AB285036, AB285040, AB285070, AF207753, AY808017, AY808022, D10934, D11168, D14484, D16435, D50482, D50483, D63857, D89872, EU155224, EU256090, EU256099, FJ390397

400 ccc act P T AB049098

400 ccc gtc P V AB285069, AF207757, EU155306, U01214

401 tcc gcc S A EU155359

401 tcc gct S A AJ507172

401 tcc ttc S F AF165054, AF207766, AF207769, AF207771, AY808025

401 tcc ttt S F AF165053

401 tcc ctc S L AB049092, AB442220, AF165057, AF165058, AF207768, AJ507161, AJ507189, AJ507196, AY808035, AY808038, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492054, DQ492055, DQ492056, DQ492057, DQ492058, DQ492059, DQ492061, DQ492062, EF407478, EF407498, EU155260, EU155301, EU155358, EU155361

401 tcc ctt S L AB285065

401 tcc ccc S P AB285042, AF265158, AF483269, AJ000009, AJ507156, AJ507169, AJ507182, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AY808015, AY808026, AY808030, AY808031, AY808043, DQ492060, DQ492152, EU155281, EU256062

401 tcc cct S P AF165061, AF165062, AY008261, EU256080

401 tcc acc S T AF034151, AM493478, EU256084

402 gac gcc D A AB010250, AY587016, D30613, D45172, D89815, DQ244141, EU857431

402 gac gaa D E AM402059, AM402062, AM402065, AM402079, AM402083, AM402086, AM402091, AM402123, AM402124, AM402350, AM402353, AM402358

402 gac gag D E AJ507190

402 gac ggc D G AB285038, AB285042, AB285072, AF207761, AJ507155, AJ507165, AJ507196, AY808015, AY808022, AY808034, DQ244140, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020,

192

DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492100, DQ492108, DQ492109, DQ492110, DQ492113, DQ492114, DQ492116, DQ492117, DQ492118, DQ492119, DQ859970, EF407486, EU155328, EU155337, EU155368, EU256075, EU781827, HM042063, L02836

402 gac ggt D G AY808027, DQ491948, EF407472, EU155256, M96362, U16362

402 gac atc D I EU256081

402 gac aaa D K AB154195, AB154196

402 gac aac D N AB285081, AF207762, AF265154, AF265155, AF265156, AJ507189, AM402258, AM402259, AM402260, AM402261, AM402262, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402270, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402277, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM402361, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AY240969, AY808017, D10934, D50485, DQ491988, DQ492112, EF032892, EF032893, EF032894, EF407495, EU155259, EU155375, EU255961, EU482860, EU482879, FJ390397, FJ958903, GQ913873

402 gac aat D N AB049101, AB249644, AB426117, AF207769, AJ507160, AY808028, D50480, D50482, D50483, D50484, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, EU256102

402 gac agc D S AB285048, AY808032, AY808041, D50481, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, EF407477, EU155257, EU155308, EU155370

402 gac agt D S EF407480, EU155376

402 gac acc D T AM494937

402 gac gtc D V EU155219

403 gac gcc D A AF207766, AF265158, AF265159, AJ507157, AJ507183, AY808009, AY808023, EF407471, EF407475, EU155318, EU256081, EU482875

403 gac tgc D C AF207755, AF207757

403 gac tgt D C AF165045, AF165046

403 gac gaa D E AB010250, AB049095, AB285056, AF207764, AY587016, D30613, D45172, D89815, EU857431

403 gac gag D E AJ507187

403 gac ttt D F AB285052

403 gac ggc D G AB285045, AB285051, AJ132997, AJ507161, AJ507179, AM401884, AM493437, AM493438, AM493439, AM493441, AM493443, AM493445, AM493446, AM493449, AM493450, AM493451, AY808011, AY808043, DQ492124, DQ492125, DQ492126, DQ492127, DQ492128, DQ492129, DQ492131, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492147, DQ492148, DQ492149, DQ492150, DQ492151, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492161, DQ492162, EF407459, EF407462, EF407468, EF407476, EF407479, EF407487, EF407502, EU155225, EU155368, EU255960, EU256045, EU482888, L02836

193

403 gac ggt D G AJ132996

403 gac cac D H DQ491982, DQ491983, DQ491984, DQ491989, DQ491992

403 gac aaa D K AB285048

403 gac aac D N AB049094, AB285078, AF034151, AF176573, AF207771, AF208024, AF265142, AF265143, AF265144, AF265160, AF265161, AF265162, AF356827, AJ000009, AJ507158, AJ507164, AJ507166, AJ507167, AJ507168, AJ507170, AJ507174, AJ507177, AJ507178, AJ507192, AJ507194, AJ507198, AJ507199, AM402337, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AY808021, AY808027, AY808035, AY808037, AY808039, DQ492040, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492046, DQ492047, DQ492048, DQ492049, DQ492050, DQ492051, DQ492052, DQ492053, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, EF032892, EF032893, EF032894, EF407460, EF407461, EF407465, EF407482, EF407483, EF407488, EF407494, EF407497, EF407500, EF407501, EF407503, EF407504, EU155220, EU155222, EU155228, EU155235, EU155263, EU155303, EU155317, EU155324, EU155325, EU155327, EU155331, EU155356, EU155360, EU155366, EU155373, EU155375, EU155376, EU234061, EU256064, EU256075, EU256076, EU256083, EU256088, EU256091, EU256092, EU256102, EU256103, EU482833, EU482874, EU482880, EU482881, EU781825, FJ390396, FJ390398, FJ958917, GQ913869, HM042051, HM042052, HM042054, HM042055, HM042056, HM042057, HM042061, HM042062, HM042063, U01214

403 gac aat D N AJ507156, EU482859, FJ024277

403 gac agc D S AB442220, AF165053, AF165054, AJ507175, AJ507186, AJ507189, AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401872, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AY808031, AY808038, DQ492054, DQ492055, DQ492139, EF407470, EF407473, EF407481, EU155254, EU155255, EU155316, EU155332, EU155334, EU155335, EU155382, EU256062, EU482885, EU781830, FJ024279, GQ913868, GQ913871, GQ913872, GQ913873

403 gac acc D T AB285068, EU256000

403 gac tac D Y AF207758

404 ggc tgc G C AB285069, EU155302

404 ggc gac G D AF207772, AJ000009, AJ507161, AJ507165, DQ492067, EF407487, EF407502, EU155364, EU155369, EU255960, EU256078, EU256081, EU482874

404 ggc gat G D AF207768, AF207774, AJ507159, AY808028, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492101, EF407462, EF407479, EU155227, EU155228, FJ390397, HM042063

404 ggc gag G E AJ507189

404 ggc aat G N EU155362

404 ggc agc G S EF407491, EU155370

404 ggc agt G S AB285038, AF208024, AM401902, AM401907, EU155368

404 ggc gtc G V U45476

405 gac gcc D A AY808026, DQ492172

194

405 gac gag D E AY808041, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970

405 gac ggc D G AB154201, AB154202, AB285036, AB285074, AF207761, AF207768, AF265145, AF265146, AF265147, AJ133096, AJ507165, AJ507169, AJ507185, AJ507187, AJ507191, AY808013, AY808018, AY808019, AY808028, AY808031, AY808034, AY808040, AY808042, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ491977, DQ491978, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492008, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492094, DQ492095, DQ492096, DQ492097, DQ492098, DQ492099, DQ492101, DQ492102, DQ492103, DQ492104, DQ492105, DQ492106, DQ492107, DQ492116, DQ492120, DQ492121, DQ492122, DQ492123, DQ492159, EF407459, EF407467, EF407492, EF407499, EU155227, EU155230, EU155253, EU155260, EU155305, EU155357, EU155364, EU155372, EU255961, EU256001, EU256059, EU256078, EU256089, GQ913869, GQ913870, HM042059

405 gac ggg D G DQ492029

405 gac ggt D G AB049092, AB285075, AF165047, AF165048, AJ507182, AY808029, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492074, DQ492075, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, EU155279, EU155369, EU256084

405 gac aac D N AB285063, AJ507197

405 gac aat D N AF207757

405 gac agc D S DQ491971, DQ491972, DQ491973, DQ491974, DQ491975, DQ491976, DQ492188

405 gac agt D S DQ492076

406 gcg gaa A E DQ859970

406 gcg gga A G AJ507182

406 gcg ata A I EU155259, EU256083, EU482879

406 gcg aaa A K AB010249, AB049088, AB049091, AB049099, AB249644, AB285038, AB285041, AB285058, AB285059, AB285061, AB285062, AB285063, AB285069, AB285070, AB285077, AB426117, AB442221, AB442222, AF165049, AF165050, AF165051, AF165052, AF165057, AF165058, AF165059, AF165060, AF165061, AF165062, AF207752, AF207753, AF207754, AF207758, AF207762, AF207770, AF207772, AF207774, AF333324, AJ507159, AJ507197, AY460204, AY587844, AY808010, D11168, D14484, D16435, D63857, D89872, D90208, DQ244140, EF407474, EF407478, EF407498, EF638081, EU155231, EU155302, EU155336, EU155362, EU155381, EU239714, EU256090, EU256099, EU256101, GU451221, GU451223, GU451224, L02836, M58335, U01214

406 gcg aag A K AB049100, AB080299, AF207756, AF207763, AJ507181, D85516

406 gcg ttg A L AJ507175

406 gcg aac A N AF207769

406 gcg cca A P DQ492008, DQ492127, DQ492130, DQ492131, DQ492139, DQ492140, GU451218, GU451219, GU451220, GU451222

406 gcg ccg A P AB285051

406 gcg caa A Q AB285037, AY045702

406 gcg aga A R AB049098, AB285036, AB285044, AF207765, D10934, M96362, U16362, X61596

195

406 gcg agg A R AB285046

406 gcg tca A S AY808011, AY808034, DQ492009, DQ492010, DQ492011, DQ492012, DQ492013, DQ492014, DQ492015, DQ492016, DQ492017, DQ492018, DQ492019, DQ492020, DQ492021, DQ492022, DQ492023, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492124, DQ492125, DQ492126, DQ492128, DQ492129, DQ492132, DQ492133, DQ492134, DQ492135, DQ492136, DQ492137, DQ492138, DQ492141, DQ492142, DQ492143, DQ492144, DQ492145, DQ492146, DQ492148, DQ492149, DQ492150, DQ492151, DQ492152, DQ492153, DQ492154, DQ492155, DQ492156, DQ492157, DQ492158, DQ492159, DQ492160, DQ492161, DQ492162

406 gcg tcg A S AJ133096, AY808015, DQ492024, DQ492147

406 gcg aca A T AB049087, AB049092, AB049094, AB049095, AB049096, AB154186, AB285042, AB285048, AB285050, AB285052, AB285054, AB285055, AB285057, AB285060, AB285064, AB285073, AB442220, AF034151, AF165045, AF165046, AF165053, AF165054, AF176573, AF207755, AF207757, AF207764, AF207768, AF207771, AF265158, AF265159, AF265160, AF265162, AJ000009, AJ132996, AJ132997, AJ507156, AJ507157, AJ507158, AJ507160, AJ507165, AJ507171, AJ507172, AJ507183, AJ507185, AJ507187, AJ507192, AM401970, AM402069, AM402258, AM402260, AM402261, AM402263, AM402264, AM402265, AM402266, AM402267, AM402268, AM402269, AM402271, AM402272, AM402273, AM402274, AM402275, AM402276, AM402278, AM402279, AM402280, AM402281, AM402282, AM402283, AM402284, AM402285, AM402286, AM402287, AM402288, AM402289, AM402290, AM402291, AM402292, AM402293, AM402294, AM402295, AM402296, AM402297, AM402298, AM402299, AM402300, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402307, AM402308, AM402309, AM402310, AM402311, AM402312, AM402313, AM402314, AM402315, AM402316, AM402317, AM402318, AM402319, AM402320, AM402321, AM402322, AM402323, AM402324, AM402325, AM402326, AM402327, AM402328, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484, AM493485, AM493486, AM493487, AM493488, AY240968, AY808009, AY808012, AY808018, AY808021, AY808022, AY808023, AY808024, AY808026, AY808027, AY808028, AY808035, AY808037, AY808039, AY808043, D50482, D50484, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492041, DQ492042, DQ492043, DQ492044, DQ492045, DQ492050, DQ492051, DQ492052, DQ492056, DQ492057, DQ492058, DQ492059, DQ492060, DQ492061, DQ492062, EF032892, EF032893, EF032894, EF407459, EF407466, EF407467, EF407471, EF407475, EF407477, EF407483, EF407488, EF407497, EF407500, EF407501, EF407503, EF407504, EU155220, EU155256, EU155260, EU155303, EU155305, EU155318, EU155327, EU155331, EU155357, EU155358, EU155360, EU155361, EU155363, EU155373, EU155375, EU155377, EU256000, EU256075, EU256079, EU256084, EU256089, EU256091, EU256092, EU256102, EU256103, EU482860, EU482880, EU482881, EU482888, EU781825, EU781827, EU862837, FJ390396, FJ390398, FJ958916, GQ913873, HM042050, HM042055, HM042061, HM042063, U45476

406 gcg acc A T EU155317

406 gcg acg A T AB429050, AJ507163, AJ507164, AJ507166, AJ507167, AJ507170, AJ507174, AJ507178, AJ507179, AJ507193, AJ507196, AJ507198, AJ507199, AM402277, AM493471, AM494937, AY808014, AY808017, AY808025, AY808038, DQ492040, DQ492047, DQ492048, DQ492049, DQ492054, DQ492055, DQ492093, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178,

196

DQ492179, DQ492180, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407461, EU155222, EU155228, EU155235, EU155325, EU155337, EU155356, EU155372, EU155374, EU256001, EU256064, EU256076, EU482859, FJ024277, HM042051, HM042052, HM042054, HM042056, HM042057, HM042058

406 gcg act A T AB285075, EU155359

406 gcg gta A V AY808041, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970, EF407465, FJ478453

406 gcg gtg A V DQ492172

407 gga gca G A AF207772

407 gga tgc G C AJ507182

407 gga gaa G E AB010249, AB049091, AB049099, AB049100, AB249644, AB285036, AB285038, AB285046, AB285058, AB285063, AB426117, AB442222, AF165057, AF165058, AF165059, AF165060, AF165061, AF165062, AF207754, AF207762, AF207763, AF207765, AF207770, AJ507196, AM402374, AY587844, AY808013, D10934, D11168, D14484, D16435, D89872, DQ491959, DQ491960, EF407489, EU155231, EU155232, EU155336, EU155376, EU155381, EU239714, EU256090, EU256099, EU256101

407 gga gag G E AB285070, AJ507175, DQ244140, M96362, U16362

407 gga aaa G K EF407491

407 gga aga G R AB285078, AM493500, DQ492067, DQ492073

407 gga agg G R HM042050

407 gga cga G R AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401931, AM401932, AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401944, AM401945, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980

408 tcc ttc S F AM401906

408 tcc ccc S P AM401890, AM402364, AM402374

408 tcc acc S T AJ507171, AJ507174, AJ507190, EU155369, EU256078

408 tcc tac S Y DQ491984

409 gac gaa D E EF407461

409 gac ggc D G AM401884, AM402072

409 gac aac D N DQ492152, EU482874

409 gac tac D Y DQ492020

410 gtt gca V A EU155362

410 gtt gcc V A AB285041, AB285060, AM401932, AM401933, AM401939, AM401945, AM401956, AM402113, AY808026, AY808031, DQ244141, EF407467, EU155361, EU155381, EU256103, EU482881, GU451220

410 gtt gcg V A AB285036

410 gtt gct V A AB010250, AB036519, AB036520, AB036521, AB049096, AB109543, AB154187, AB154188, AB191333, AB285037, AB285042, AB285047, AB285052, AB285059, AB285066, AB285068, AF165045, AF165046, AF165053, AF165054, AF207755, AF207757, AF207771, AF265152, AF265153, AF356827, AF483269, AJ132996, AJ507156, AJ507159, AJ507162, AJ507168, AJ507169, AJ507171, AJ507172, AJ507175, AJ507188, AJ507189, AJ507194, AJ507196, AJ507197, AJ507198, AM401859, AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401931, AM401934, AM401935, AM401936, AM401937, AM401938, AM401940, AM401941,

197

AM401942, AM401943, AM401944, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401978, AM401979, AM401980, AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402075, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AM402348, AY587016, AY808009, AY808016, AY808019, AY808023, AY808030, AY808036, AY808039, D10934, D30613, D45172, D89815, DQ491986, DQ491987, DQ491993, DQ492023, DQ492024, DQ492025, DQ492026, DQ492027, DQ492028, DQ492029, DQ492030, DQ492031, DQ492032, DQ492033, DQ492034, DQ492035, DQ492036, DQ492037, DQ492038, DQ492039, DQ492077, DQ492094, DQ492095, DQ492096, DQ492097, DQ492098, DQ492099, DQ492100, DQ492101, DQ492102, DQ492103, DQ492104, DQ492105, DQ492106, DQ492107, DQ492108, DQ492109, DQ492110, DQ492111, DQ492112, DQ492113, DQ492114, DQ492115, DQ492116, DQ492117, DQ492118, DQ492119, DQ492120, DQ492121, DQ492122, DQ492123, DQ492165, DQ492197, DQ859970, EF407461, EF407482, EF407500, EF407503, EU155218, EU155221, EU155259, EU155279, EU155302, EU155306, EU155307, EU155316, EU155324, EU155365, EU155367, EU155373, EU155374, EU239714, EU256045, EU256061, EU256066, EU256076, EU256077, EU256079, EU256085, EU256088, EU256101, EU482833, EU482874, EU482875, EU529682, EU660388, EU781830, EU857431, EU862835, FJ024277, FJ390396, FJ390397, GQ913870, HM042050, U01214

410 gtt gat V D DQ491988, EF407497, EU155231, X61596

410 gtt ggt V G EF407462

410 gtt att V I AB010249, AJ507170, D16435, D89872

410 gtt act V T AJ507182

411 gag gcg E A EU256079

411 gag gac E D DQ492142, EU155324

411 gag gat E D AM493503

411 gag gga E G AM402055, AM402056, AM402057, AM402058, AM402059, AM402060, AM402061, AM402062, AM402063, AM402064, AM402065, AM402066, AM402067, AM402068, AM402069, AM402070, AM402071, AM402072, AM402073, AM402074, AM402076, AM402077, AM402078, AM402079, AM402080, AM402081, AM402082, AM402083, AM402084, AM402085, AM402086, AM402087, AM402088, AM402089, AM402090, AM402091, AM402092, AM402093, AM402094, AM402095, AM402096, AM402097, AM402098, AM402099, AM402100, AM402101, AM402102, AM402103, AM402104, AM402105, AM402106, AM402107, AM402108, AM402109, AM402110, AM402111, AM402112, AM402113, AM402114, AM402115, AM402116, AM402117, AM402118, AM402119, AM402120, AM402121, AM402122, AM402123, AM402124, AM402125, AM402126, AM402127, AM402128, AM402129, AY808009, EU155381

411 gag ggg E G AB049087, AB049094, AB049095, AB049096, AB154198, AB285040, AB285050, AB285060, AB285066, AF165053, AF165054, AF165055, AF165056, AF207764, AF207771, AF207772, AF483269, AJ238800, AJ507159, AJ507160, AJ507162, AJ507163, AJ507166, AJ507168, AJ507172, AJ507175, AJ507188, AJ507196, AM493471, AM493472, AM493473, AM493474, AM493475, AM493476, AM493477, AM493478, AM493479, AM493480, AM493481, AM493482, AM493483, AM493484,

198

AM493485, AM493486, AM493487, AM493488, AM493497, AY008261, AY808016, AY808024, AY808028, AY808030, AY808032, AY808036, AY808039, D50480, D50482, D50484, DQ491979, DQ491980, DQ491981, DQ491985, DQ491986, DQ491987, DQ491988, DQ491990, DQ491991, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492111, DQ492148, DQ492170, DQ492192, EF407461, EF407468, EF407470, EF407487, EF407494, EF407497, EU155218, EU155221, EU155306, EU155307, EU155358, EU155361, EU155362, EU155363, EU155365, EU155367, EU155373, EU155374, EU155377, EU255960, EU256045, EU256061, EU256085, EU256088, EU256098, EU256103, EU529682, EU660388, EU862835, EU862837, FJ390396, HM042053


412 tcg gcg S A AJ507174

412 tcg gag S E AJ507182

412 tcg ttg S L AM402384

412 tcg ccg S P AM401886

413 tac gcc Y A AJ507182

413 tac tgc Y C AB154195, AB154196, AB285076, AB429050, AJ000009, AJ507175, AM401894, AM402267, AM402294, DQ491988, DQ491989, EF407467, EF407478, EF407489, EU155305, EU256080, EU256092, EU482875, EU529682, GQ913873, M84754

413 tac tgt Y C AB036521, AJ132996

413 tac ttc Y F EF407487, EF407502, EU155381, M96362, U16362

413 tac ttt Y F FJ390397

413 tac cac Y H AJ507178, AM402090, AY808025, AY808026, HM042056

414 tcc ttc S F DQ491979

414 tcc acc S T EU155307

416 atg ata M I AM401874, AM493443

416 atg ttg M L AM401879

416 atg acg M T AM493478

416 atg gtg M V AM401888, DQ491947


417 ccc tcc P S AM401967, AM493507, DQ492084

418 ccc gcc P A EU256077

418 ccc tcc P S AM402311

419 ctt ttt L F DQ492104

419 ctt cac L H DQ492066

419 ctt cat L H DQ492106

419 ctt cct L P AM402326, DQ492156

420 gag ggg E G AM401866, DQ492018, DQ492107

420 gag aag E K AM401956

420 gag agg E R AB285081

422 gag ggg E G AM402092, AM402329, AM493445, AM493446, DQ492072, DQ492180

423 ccg ctg P L AB285078, AM402077

423 ccg cag P Q AY808037

423 ccg tcg P S AM401851


425 gat gaa D E AB285075

425 gat ggc D G DQ492080, FJ958897

425 gat aac D N DQ491945



427 gat aat D N AM401907, AM402334

199

428 ctc ttc L F AF165045, AF165046, AF207755, AM401911, AY808040, AY808041, DQ491943, DQ491944, DQ491945, DQ491946, DQ491947, DQ491948, DQ491949, DQ491950, DQ491951, DQ491952, DQ491953, DQ491954, DQ491955, DQ491956, DQ491957, DQ491958, DQ491959, DQ491960, DQ491961, DQ491962, DQ491963, DQ491964, DQ491965, DQ491966, DQ491967, DQ491968, DQ491969, DQ491970

429 agc ggc S G AM401851, AM401898, AM401905, DQ491978

429 agc aac S N AM402262, D30613, D45172

429 agc acc S T DQ492181

430 gac gaa D E AB285078

430 gac ggc D G FJ958904

430 gac aac D N EU155305

430 gac aat D N DQ492193

431 ggg gag G E DQ492029



433 tgg ggg W G AY808033, AY808038, DQ492071, DQ492193

433 tgg cgg W R DQ492180

434 tct ttc S F AM402064, AM402094


435 acc gcc T A DQ492084

436 gta gca V A DQ491983

436 gta gcg V A AM401923

436 gta atg V M AB049100, AB285037, AB285052, AB285057, AB285058, AB285064, AB442222, AF165057, AF165058, AJ507156, AJ507185, AM402293, AM494937, AY808014, AY808015, AY808022, AY808026, DQ492064, DQ492066, DQ492069, DQ492072, DQ492076, DQ492077, DQ492081, DQ492093, DQ492127, DQ492130, DQ492131, DQ492132, DQ492135, DQ492139, DQ492140, DQ492148, DQ492163, DQ492164, DQ492165, DQ492166, DQ492167, DQ492168, DQ492169, DQ492170, DQ492171, DQ492172, DQ492173, DQ492174, DQ492175, DQ492176, DQ492177, DQ492178, DQ492179, DQ492180, DQ492181, DQ492182, DQ492183, DQ492184, DQ492185, DQ492186, DQ492187, DQ492188, DQ492189, DQ492190, DQ492191, DQ492192, DQ492193, DQ492194, DQ492195, DQ492196, DQ492197, EF407471, EU155235, EU155257, EU155264, EU482859, HM042061

437 agc tgt S C DQ492141

437 agc gac S D EF407464

437 agc ggc S G AJ507189, DQ491978

437 agc ggt S G DQ492095

437 agc aac S N AF207765, DQ491970, DQ492061

437 agc acc S T DQ492181

438 gag gac E D D63857, EU155223, EU155253

438 gag gat E D EU256084, HM042059

438 gag gga E G EF407470

438 gag ggg E G AB010249, AB285054, AB285055, AF207757, AM401931, AY240969, D11168, D16435, D89872, D90208, DQ492179, EU155359

438 gag aaa E K AM402290, AM402293, AM402294, AM402296, AM402297, AM402299, AM402301, AM402302, AM402303, AM402304, AM402305, AM402306, AM402308


438 gag gtg E V AM402347

439 gag gac E D AB285046, EU529682

439 gag gat E D FJ390398

439 gag ggg E G AM402291, AM402364, AY808026

439 gag cag E Q AF165053

200


440 gct gac A D AJ507169, EU155359

440 gct gat A D AF165053, AY808026, M84754

440 gct ggc A G EF407470

440 gct ggt A G AB049092, AF165054, EU256090

440 gct cat A H EU256077

440 gct ccc A P AY808029, DQ492064, DQ492066, DQ492069, DQ492072, DQ492076, DQ492077, DQ492081

440 gct cct A P AB442222, AF165057, AF165058

440 gct acc A T AB285061, AM493497, DQ492071, DQ492073, DQ492078, DQ492079, DQ492083, DQ492088, DQ492092, EU256088

440 gct act A T AB285054, AB285055, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, DQ492063, DQ492065, DQ492067, DQ492068, DQ492070, DQ492074, DQ492075, DQ492080, DQ492082, DQ492084, DQ492085, DQ492086, DQ492087, DQ492089, DQ492090, DQ492091, FJ390398

440 gct gtc A V AF265164, AM402339, AY808014, EF407460

440 gct gtt A V AB049089, AF207760, AF265148, AF265149, AF265150, AF265152, AF265153, AF265163, AF265165, DQ244141, DQ492056, EU155280, GQ913868

441 agt gcc S A AF207770

441 agt gct S A EU155306

441 agt tgt S C DQ492017

441 agt gac S D AB442221, AF207760, AM401978

441 agt gat S D AM401916, AM401917, AM401918, AM401919, AM401920, AM401921, AM401922, AM401923, AM401924, AM401925, AM401926, AM401927, AM401928, AM401929, AM401930, AM401931, AM401932, AM401933, AM401934, AM401935, AM401936, AM401937, AM401938, AM401939, AM401940, AM401941, AM401942, AM401943, AM401944, AM401945, AM401946, AM401947, AM401948, AM401949, AM401950, AM401951, AM401952, AM401953, AM401954, AM401955, AM401956, AM401957, AM401958, AM401959, AM401960, AM401961, AM401962, AM401963, AM401964, AM401965, AM401966, AM401967, AM401968, AM401969, AM401970, AM401971, AM401972, AM401973, AM401974, AM401975, AM401976, AM401977, AM401979, AM401980, GQ913873

441 agt gga S G EF407467

441 agt ggc S G AB010249, AB285038, AB285039, AB442220, AF165061, AJ507157, AJ507167, AJ507173, D16435, D50480, D50483, D89872, EF407496, EU482877, EU482885, FJ390398

441 agt ggt S G AB049087, AB049088, AB049089, AB049092, AB049094, AB049098, AB049100, AB154180, AB154189, AB154190, AB154196, AB249644, AB285036, AB285041, AB285042, AB285044, AB285047, AB285051, AB285052, AB285054, AB285055, AB285057, AB285058, AB285060, AB285061, AB285064, AB285065, AB285067, AB285071, AB285076, AB426117, AB442222, AF034151, AF165053, AF165054, AF165057, AF165058, AF165062, AF207753, AF207756, AF207764, AF207765, AF207768, AF207771, AF207772, AJ238800, AJ507163, AJ507168, AJ507169, AJ507171, AJ507181, AJ507184, AJ507186, AJ507189, AJ507194, AJ507195, AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, AY008261, AY044867, AY045702, AY240967, AY240968, AY240969, AY808026, AY808028, AY808029, AY808033, AY808034, AY808036, AY808039, D50481, D50482, D50484, D50485, D90208, DQ244140, DQ244141, DQ491979, DQ491980, DQ491981, DQ491982, DQ491983, DQ491984, DQ491985, DQ491986, DQ491987, DQ491988, DQ491989, DQ491990, DQ491991, DQ491992, DQ491993, DQ491994, DQ491995, DQ491996, DQ491997, DQ491998, DQ491999, DQ492000, DQ492001, DQ492002, DQ492003, DQ492004, DQ492005, DQ492006, DQ492007, DQ492047, DQ492048,

201

DQ492054, DQ492055, DQ492063, DQ492064, DQ492065, DQ492066, DQ492067, DQ492068, DQ492069, DQ492070, DQ492071, DQ492072, DQ492073, DQ492074, DQ492075, DQ492076, DQ492077, DQ492078, DQ492079, DQ492080, DQ492081, DQ492082, DQ492083, DQ492084, DQ492085, DQ492086, DQ492087, DQ492088, DQ492089, DQ492090, DQ492091, DQ492092, DQ492125, EF407470, EF407472, EF407475, EF407477, EF407480, EF407481, EF407485, EF407490, EF407491, EF407495, EF407500, EF407501, EF407503, EF407504, EU155232, EU155257, EU155261, EU155305, EU155315, EU155318, EU155335, EU155359, EU155363, EU155364, EU155368, EU155370, EU256080, EU256081, EU256082, EU256083, EU256090, EU482849, EU781827, EU862835, FN435993, GQ913869, GQ913872, HM042050, HM042052, HM042054, HM042056, HM042057, HM042059, HM042061, M84754

441 agt aac S N AM493450, U45476

441 agt aat S N AB285043, HM042051, HM042058

441 agt cgc S R AF207769

441 agt act S T AB049091, HM042062

442 gag gac E D AB010249, D11168, D16435, D89872

442 gag gat E D AM493489, AM493490, AM493491, AM493492, AM493493, AM493494, AM493495, AM493496, AM493497, AM493498, AM493499, AM493500, AM493501, AM493502, AM493503, AM493504, AM493505, AM493506, AM493507, EF407481

442 gag ggg E G AM402112, AM402352, D10934, EU155358, EU256080

442 gag aag E K FJ958919

442 gag cag E Q AB285061, AY240967, AY240968, AY240969

443 gac gcc D A EF407468

443 gac gaa D E EU155358

443 gac ggc D G AB285040, AJ507169, AM402318, AM494937, M84754

443 gac cac D H AM493450

443 gac aac D N AB285041, AJ507161, EF407461, EF407481, EU529682, EU862835, GQ913873, HM042058, HM042059

443 gac agc D S AB285076, EF407467, EU155306, HM042056

444 gtc gct V A AJ507164

444 gtc atc V I AB049098, AB285041, AJ507169, D11168, D16435, D89872, EF407461, GQ913873, HM042059

444 gtc att V I AB285040, EF407481, EU529682

444 gtc ctt V L AF165056

445 gtc gcc V A AM401848, AM401849, AM401850, AM401851, AM401852, AM401853, AM401854, AM401855, AM401856, AM401857, AM401858, AM401859, AM401860, AM401861, AM401862, AM401863, AM401864, AM401865, AM401866, AM401867, AM401868, AM401869, AM401870, AM401871, AM401873, AM401874, AM401875, AM401876, AM401877, AM401878, AM401879, AM401880, AM401881, AM401882, AM401883, AM401884, AM401885, AM401886, AM401887, AM401888, AM401889, AM401890, AM401891, AM401892, AM401893, AM401894, AM401895, AM401896, AM401897, AM401898, AM401899, AM401900, AM401901, AM401902, AM401903, AM401904, AM401905, AM401906, AM401907, AM401908, AM401909, AM401910, AM401911, AM401912, AM401913, AM401914, AM401915, AM402111, AM493436, AM493437, AM493438, AM493439, AM493440, AM493441, AM493442, AM493443, AM493444, AM493445, AM493446, AM493447, AM493448, AM493449, AM493450, AM493451

445 gtc gct V A X61596

445 gtc atc V I AB285045, AB285053, AF165054, AJ507189, AM494937, AY045702, EU256083, M84754

445 gtc acc V T AM401872

446 tgc cgc C R AM401864, AM401944, AM402115, AM402264

447 tgc tac C Y AM401856, AM401889

202

CHAPTER 5: SUMMARY AND CONCLUSIONS

203

In these studies, we demonstrated that ethanol increases complete HCV

replication in vitro as previously reported in patient and subgenomic replicon studies,

and that ethanol as well as reactive species can act as chemical mutagen to the HCV

RNA to increase its sequence heterogeneity that could result in the emergence of

specific viral sequence variants implicated in immune evasion, viral replication, and

antiviral resistance. Studying the effects on HCV caused by changes in its

biochemical environment has presented a series of challenges in the HCV field. For

example, concerns with physiological dosage, relevance, and kinetics of reactive

species often plague researchers trying to study the effects of reactive species in vitro

(208,209,262,344). This problem is compounded by the fact that HCV itself induces

the production of reactive species and that many antioxidants under certain conditions

can act as a pro-oxidant (22,258,326). In addition, these chemical species can have a

multitude of effects via signaling or damage on both HCV and the host cells, which

could make the isolation of a single mechanism or process for study difficult,

requiring multiple experimental approaches to confirm trends and cautious

interpretation of results. Furthermore, until recently, there was a lack of robust cell

culture and animal models for studying HCV infection. Development of the replicon

cell culture systems and more recently the JFH1 genotype 2a infective cell culture

system have allowed for the study of the HCV viral life cycle in vitro. In terms of

other genotypes, infective cell culture and robust systems using noncancer-derived

cells are still in development (246,249). In vivo studies have required the use of

human patients or experimentally infected chimpanzees, due to lack of nonprimate

204

small animal model for HCV infection (12). Difficulty of finding appropriate control

subjects, unreliability of self-reporting of certain factors such as ethanol intake, and

ethical issues all present major challenges to human patient studies. Chimpanzee

studies have to deal with the ethical issues as well as the high cost of primate research.

Recently, the generation of chimeric mice with humanized livers has opened up new

doors for studying HCV in vivo, although the model has limitations, such as the mice

are immune-suppressed in order to accept the human liver cells, and therefore might

not show the same pathologies (389). With the race for more realistic cell culture and

in vivo animal models going strong, the studies presented in this thesis will need to be

confirmed in a genotype 1 infective cell culture system and eventually in an in vivo

animal model. Despite the challenges, research advances in determining the effects of

ethanol and reactive species on HCV continue to be made.

Previously, the effect of ethanol on HCV replication was only studied in the

context of the HCV replicon system (does not produce virus) and patient studies, in

which alcohol intake was self-reported (144,191,197,244,258). A study by Zhang, et

al. (197) suggested that downstream ethanol metabolites were responsible for the

potentiation of HCV subgenomic replication, whereas another study by McCartney et

al. (244) used NAC to suggest that ROS was responsible for the elevation of HCV

replication in subgenomic and genomic replicons. Our data, using the JFH1 genotype

2a infectious cell culture system, agreed with Zhang et al. that acetaldehyde and

acetate could increase HCV replication and that an elevation of relative NADH levels

was required. On the contrary, we found that endogenous and exogenous ROS

205

suppressed rather than increase HCV replication in the context of the complete viral

life cycle, which is consistent with previous replicon studies from our lab and others

(208,209,214,260,261,279). In our study, NAC slightly exacerbated the ethanol-

induced increase in HCV replication (Fig. 3.4G), rather than suppressing it as seen in

the McCartney et al. study (244).

There are several possible explanations for the inconsistencies, including

differences in the Huh7 clones, HCV construct, and the conditions inside the cells.

Our Huh7 cells endogenously expressed CYP2E1 at levels comparable to human liver

tissue and we used the JFH1 construct (genotype 2a), which produces infectious virus,

instead of a different Huh7 clone that required transfection of an overexpression

vector of CYP2E1 and a genotype 1 replicon system, lacking production of infectious

virus, respectively. Since CYP2E1 generates acetaldehyde as well as ROS, both of

which can react with thiols generated from NAC, the overexpression of CYP2E1

could possibly alter cellular conditions enough to cause the NAC to behave

differently. NAC can alter the pH of the cells and in some conditions act as a pro-

oxidant, so careful monitoring of the pH and comparison with other antioxidants and

pro-oxidants are necessary to draw consistent conclusions from experiments using

NAC.

If ROS suppresses rather than elevates HCV replication and abolishing ethanol

metabolism with inhibitors abrogates the ethanol-induced increase of HCV RNA

levels, then other products of ethanol metabolism must be responsible. We found,

indeed, that both acetaldehyde and acetate, as well as isopropanol and its metabolite,

206

acetone, elevated HCV replication levels, whereas t-butanol, which is poorly

metabolized by human cells, did not (Fig. 3.6). The treatments that increased HCV

replication, but not t-butanol, also increased NADH/NAD+ ratios, suggesting that

NADH plays a role in the ethanol-induced elevation of HCV replication (Fig. 3.6).

Pyruvate, which oxidizes NADH to NAD+, abrogated the increase of HCV

replication by ethanol and its metabolites, suggesting that elevation of NADH levels is

required (Fig. 3.6C). To determine if increased levels of NADH alone could increase

HCV replication, lactate was used to increase the NADH/NAD+ ratio without ethanol

metabolism, and it did not cause a corresponding increase in HCV replication (Fig

3.6D). Increased levels of NADH indicates a more reduced state in cells, but NADH

also plays a role in cellular bioenergetics, can modulate fatty acid synthesis, and

suppresses β-oxidation (153,170). Though elevated NADH levels are not sufficient to

increase HCV replication by itself, its requirement for the elevation of HCV

replication by ethanol and its metabolites is likely to involve a combination of more

specific processes, such as driving fatty acid synthesis and suppressing β-oxidation of

fatty acids, and more general effects, such as causing a more reduced rather than

oxidized state in the cell or acting as an energy source to drive various cellular

reactions, interacting with other effects that ethanol and its metabolites have on the

cellular environment.

Acetate, from ethanol metabolism, and acetone, from isopropanol metabolism,

can both be converted to acetyl-CoA in one or more steps in mammalian cells

(153,170,268). Just like NADH, acetyl-CoA has multiple roles in the cell, including

207

entering the tricarboxylic acid (TCA) cycle and acting as the building blocks for fatty

acid synthesis. Generally, acetyl-CoA would likely enter the TCA cycle, and the

resulting NADH, as well as the NADH from ethanol metabolism, would support

oxidative phosphorylation resulting in additional ATP production. However, ethanol

metabolism has also been shown to suppress glycolysis and the TCA cycle, in addition

to fatty acid ethyl esters, produced by non-oxidative ethanol metabolism, potentially

interfering with oxidative phosphorylation, leading to no significant elevation of

cellular metabolism as evidenced by the lack of increases in ATP levels (Fig. 3.1E)

(174,390,391).

Alternatively, acetyl-CoA, NADH, and fatty acid ethyl esters can also affect

fatty acid synthesis and oxidation pathways. Excess amounts of acetyl-CoA can be

used to synthesize fatty acids or ketone bodies. With the inhibition of the TCA cycle,

acetyl-CoA will likely be shunted to fatty acid synthesis rather than ketogenesis due to

the elevation of antiketogenic lactate from the suppression of glycolysis caused by

ethanol metabolism(153,170,268,390). The NADH produced by metabolism of

physiological concentrations of ethanol do not significantly alter cellular ATP levels,

so it is more likely driving fatty acid synthesis while inhibiting -oxidation, leading to

a pro-lipogenic state in the cell. Fatty acid ethyl esters have also been suggested to

induce intracellular lipid accumulation and reorganization (174). Additionally, HCV

proteins cause significant intracellular membrane alterations within the host cell,

creating lipid-rich regions conducive to replicating HCV RNA and assembling

infectious virions (17,270,392,393). The lipid-rich environment induced by ethanol

208

metabolism is likely to synergize with the effect of HCV proteins on intracellular lipid

metabolism to favor HCV replication and virus production.

To explore this, we used inhibitors of cholesterol and fatty acid synthesis and

were able to abolish the elevation of HCV replication by ethanol and its metabolites

(Fig. 3.8). Measurement of cholesterol levels indicated that ethanol and its

metabolites increase cholesterol synthesis that could be inhibitied by lovastatin (Fig.

3.8D). These inhibitors may cause reductions in HCV replication via differing

mechanisms and need to be investigated further, but it is clear that changes in

cholesterol or fatty acid synthesis and HCV replication are correlated (270,392,394).

There has been considerable interest in using lipid modulation in anti-HCV therapy,

especially since targeting host factors necessary for HCV replication is less likely to

lead to treatment-resistant HCV mutant strains. Statins, which inhibit 3-hydroxy-3-

methyl-glutaryl-CoA reductase in the treatment of hypercholesteremia, are suggested

to reduce HCV RNA levels both in vitro and in vivo (274,392,395,396). Inhibitors of

sphingolipid metabolism and VLDL secretion also show potential as anti-HCV

therapy (395-397). Inhibiting the activity of diacylglycerol acyltransferase-1, which is

required in trafficking HCV core to lipid droplets, also significantly impairs infectious

HCV production (398). Furthermore, the activity of AMP-activated protein kinase,

which plays a role in the regulation of both lipid and glucose metabolism, is inhibited

during HCV infection, and if restored inhibits viral replication (399). The use of these

agents that modulate lipid metabolism and trafficking, in addition to dietary changes,

are likely to play an important role in the control of HCV infection in patients (400).

209

In this study, we concluded that physiological concentrations of ethanol

elevated HCV replication by the modulation of host lipid metabolism, and required

increased NADH levels. ROS alone, on the other hand, suppressed HCV replication

and is not likely to be involved in the increased HCV replication caused by ethanol

and its metabolites but instead, likely to counter the potentiating effect of ethanol.

Indeed, ADH-transfected cells that would rely less heavily on CYP2E1-dependent

ethanol metabolism that generates ROS showed an even higher increase in HCV

replication with ethanol (Fig. 3.7C). Also, exogenous H2O2 and BSO, added together

with ethanol, could suppress the potentiation of HCV replication by ethanol (Fig.

3.4F). Therefore, while ethanol metabolism can generate ROS, how HCV responds to

ethanol and/or ROS appears to be a complex biochemical decision weighing what

appears to be two clearly opposing signals acting on HCV, where the decision is

governed by the metabolic state of the cell, not unlike that of lysogeny versus latency

of bacteriophage lambda responding differently to the metabolic state of the host.

Although ROS may also contribute to the lipid-rich environment of the cell by

activating sterol regulatory element binding proteins (401), in our studies the rapid

effect that H2O2 and BSO have on the HCV replication complex seems to override any

other favorable effects that they might have on HCV. In our Huh7 cells expressing

endogenous CYP2E1, local concentrations of H2O2 may also be held in check by

catalase-dependent ethanol metabolism, which uses H2O2 to convert ethanol to

acetaldehyde, possibly reducing the suppressive impact of ROS produced during

CYP2E1-dependent ethanol metabolism and HCV infection (173). Further study of

210

the interactions between ethanol, lipogenesis, and HCV replication may lead to the

development of novel treatments that target both the virus and its pathogenic

interactions with ethanol in chronic hepatitis C patients.

Other than repeating this study in a genotype 1 infective system and an in vivo

animal model of HCV, there are several research directions that can be taken to

expand upon these findings. Live-cell confocal microscopy using an HCV construct

with a fluorescent-tagged NS5A, and oxidant-sensitive probe, such as HyPER, could

be used together to determine if the HCV replication complexes are exposed to the

ROS produced during ethanol metabolism and HCV infection, since both location and

kinetics are crucial determinants of the action of ROS in cells (90). In this regard, it is

interesting to note that recent studies from this and other labs have shown that

hepatocyte Nox1 and Nox4 are important sources of ROS in HCV-replicating cells.

Nox1 showed primarily cytosolic localization whereas Nox4 colocalized to both ER

and nucleus (326,327). It would also be important to determine if BSO has any effect

on the cell cycle in our experiments, which can affect HCV replication. HCV-

replicating cells exposed to ethanol also need to be tested for the expression and

activity levels of sterol regulatory element binding proteins and other important lipid-

related genes to determine the effect of ethanol on these genes and their role in

creating a lipid-rich environment favorable to HCV replication. By doing so, the

combined lipogenic effects of HCV and ethanol in the development of hepatic

steatosis (fatty liver) can also be studied. To further examine which components of

the fatty acid and cholesterol synthesis pathways are responsible for the elevation of

211

HCV replication by ethanol, the same inhibitors from the study and potentially others

can be used in conjunction with downstream products of the pathway to determine if

the effect can be rescued. Host gene microarray data could also be used to identify

additional host genes that may be involved in the effect of ethanol and its metabolites

on HCV replication. Once the contributing factors have been determined and

analyzed, specific therapeutic targets can be identified to abrogate the effects of

ethanol on HCV replication and possibly the development of steatosis or other HCV-

related pathogenesis.

Additional environmental effects of ethanol metabolism also need to be

considered, such as changes in pH of the cell. Discovering that acetone can increase

HCV replication also opens up the ketogenic pathway to investigation of its effects on

the HCV life cycle and possible involvement in HCV-related diabetes, especially since

the concentration of acetone used in the study is physiologically relevant in diabetics,

even those without ketoacidosis.

Another fundamental question of how alcohol intake decreases the efficacy of

antiviral therapy and increases HCV sequence heterogeneity (115,148,237,238,305)

while antioxidants typically improve therapy outcome (356) was broached in the

second part of this thesis. Although, increased HCV replication is associated with

lower treatment efficacy and may lead to more NS5B polymerase errors through

increased activity, it is not likely to account for the entirety of these changes,

suggesting the involvement of other causes. In addition to increasing HCV

replication, ethanol metabolism leads to the generation of various genotoxins that can

212

synergize with HCV-induced reactive species to oxidatively damage DNA or RNA

potentially causing mutations (7,65,302,402-404) While DNA damage caused by

ROS is well characterized, especially in the context of carcinogenesis, RNA is

generally considered an expendable and temporary, so there has been little interest in

mutations caused by oxidative RNA damage. RNA, however, is the permanent

genetic information of RNA viruses, in which any mutations caused by oxidative RNA

damage could alter the viral genetic sequence, potentially leading to functional

changes. For instance, HCV interferon resistance is sequence-dependent, though the

precise effects of mutations in the viral RNA on treatment resistance are unclear.

Since HCV already exists as a quasispecies, an increased mutation rate would likely

provide the virus with an even greater genetic repertoire to adapt to changes in the

environment and develop resistance to therapy (234).

In our study, we demonstrated that ethanol and reactive species could increase

HCV sequence heterogeneity of NS5A, within 48 hrs (Table 4.1). The increased nt.

and amino acid substitution rates of HCV RNA were associated with oxidative RNA

damage (Fig. 4.1). Mimicing in vivo GSH depletion in patients with BSO was also

sufficient to increase the HCV mutation rate and oxidative RNA damage, suggesting

that the HCV RNA sequence is likely affected by the genotoxic effects of reactive

species in vivo, in patients. Inhibiting the oxidative RNA damage with iron chelators,

GSH ester, and inhibitors of ROS/RNS generating enzymes abrogated the increased

nt. and amino acid substitution rates of HCV RNA induced by ethanol and BSO (Fig.

4.2). Additionally, infectious JFH1 HCV induced RNA damage even in the absence

213

of ethanol or BSO and this was exacerbated by these treatments (Fig. 4.3). It is

important to note that the nt. substitution rates of HCV RNA in our study were close

to those observed in patients (346). These data suggest that endogenous reactive

species induced by HCV infection are capable of causing mutations in the viral

genome via oxidative RNA damage, and this effect is amplified by GSH depletion

occurring in patients and by exogenous sources of genotoxins, such as ethanol. These

findings suggest an alternative mechanism of increasing viral sequence heterogeneity

in the development of quasispecies, and a role of human behavior (drinking alcohol) in

the evolution of viruses.

This study could also have broad impacts on the treatment and control of

viruses with a diverse yet targeted biochemical approach to attenuate the mutation rate

of a virus. The use of iron chelators, GSH ester, and other inhibitors of reactive

species to reduce the mutation rate of HCV has the potential to enhance the efficacy of

existing antiviral therapy and facilitate the development of future antivirals and

vaccines through the reduction of potential escape mutations. Additionally, some of

the endogenous sources of genotoxins activated by HCV, which include hepatocyte

NAD(P)H oxidases, inducible nitric oxide synthase, and CYP2E1, can be targeted

pharmacologically (73,298,326,327,344). Since reactive species can modify both

RNA and DNA, and oxidative/nitrosative stress occurs in many different virus

infections (332,340,341), these findings could relevant to other viral infections.

As for how the mutations effect the virus, the increased sequence heterogeneity

of the viral RNA genomes is likely to facilitate antiviral resistance and immune

214

escape, as evidenced by patients who attain SVR having lower NS5A quasispecies

complexity than non-responders and those experiencing viral breakthrough during

treatment (377,405-407). Whether the mutations induced by ethanol and reactive

species affect the virulence of HCV remains to be determined, however, comparison

of our data to other studies of NS5A may point to functional changes that are likely to

occur with the mutations found in our study (Table 5.1). NS5A is a viral

phosphoprotein that has been suggested to have diverse functions, including

participation in viral RNA replication, alteration of cell metabolism, virion assembly,

and interferon resistance. Depending on the phosphorylation status of NS5A, it can act

as a molecular switch that regulates HCV replication, translation, assembly, and

secretion (315,357-359).

As shown in our previous study, ethanol and acetaldehyde increased HCV

RNA through lipid metabolism and by elevating relative NADH levels in the cell

(197,344). Mutations in NS5A induced by ethanol could potentially play a part in the

modulation of this effect. Patients, infected from a single HCV source, with an

increasing viral load had significantly more accumulated mutations in NS5A than

those with decreasing viral loads, suggesting that the NS5A mutation rate could affect

patient viral load (408). Three of the mutations (T21A, M133V, and R318H)

identified in chimpanzees infected with homogenous HCV inoculum corresponded to

mutations and sites undergoing positive selection in our study (409). Additionally, a

study by Kohashi et al. demonstrated several mutations within the ISDR that could, in

combination, enhance HCV RNA replication (410). Interestingly, four of the sites

215

described by Kohashi et al. had a dN/dS ratio >1 in our study, suggesting that

increased amino acid substitutions at these sites could possibly lead to the

development of strains with enhanced replication. Mutation sites in other regions of

NS5A that when combined with other mutations are associated with enhanced

replication also correspond to mutations in our study (321,411-413).

NS5A phosphorylation also may affect HCV RNA replication and virion

assembly. The fact that 44.8 % of all mutations in our study involved serine,

threonine, or tyrosine residues suggests that ethanol and reactive species can alter both

cell signaling and mutations to change the phosphorylation status of NS5A. Removal

of serine residues within the basal phosphorylation region of NS5A in ethanol and

BSO groups further suggests that the basal phosphorylation has a significant effect on

HCV so that the removal of these sites allowed those sequences to persist. In a recent

study, CKII-induced basal phosphorylation of NS5A decreased the activity of NS5B

and its association with viral RNA templates (249,315,361-364). On the other hand,

previously, it was reported that removing NS5A basal phosphorylation sites besides

S457 of genotype 2a JFH1 had no effect on HCV replication, though virion secretion

was inhibited (363,365,366). However, the equivalent site in Con1, S437, and other

critical regions, including hyperphosphorylation sites and the zinc coordination motif,

did not change in our study. How these mutations involving serine, threonine, and

tyrosine, especially the most frequent ones (Table 4.3), and cellular signaling changes

induced by ethanol and reactive species affect HCV replication and secretion remains

to be tested (363,367-369).

216

We found other nonsynonymous mutations in various structural and functional

domains of NS5A, including the double-stranded RNA-activated interferon induced

PKR, binding regions for various viral and host proteins, and putative ISDR.

Regarding therapy resistance, more than two mutations in the ISDR is correlated with

improved SVR in patients (319,414); however, no single sequence in our study had

more than two amino acid substitutions within the ISDR. Additionally, accumulation

of mutations in NS3, NS4B, and NS5A of HCV replicon RNA is suggested to be

important to the development of interferon resistance (413,415).

Though the overall complexity of quasispecies in HCV is typically more

important in the success of viral adaptive response to selection pressures (416), there

are several individual or combinations of sites that can confer resistance to various

anti-HCV treatments (Table 5.1). F37L, which occurs four times in our study with a

dN/dS ratio >1, was found to augment the binding of NS5A to 2',5'-oligoadenylate

synthetase, thereby inhibiting its action during IFN response (417). Two other

mutations, E442G and G404S, were found to to confer resistance to ribavirin in HCV

replicon cells (418). E442G occurred in our study with a dN/dS ratio >1, and though

G404S did not occur in our study, G404D did. The negative charge on the aspartic

acid (D) can mimic a phosphorylated serine (S), so there is a possibility of the

mutation in our study conferring similar resistance to ribavirin. These mutations

demonstrate specific changes to the HCV genome that increase its resistance to

existing interferon-ribavarin cotreatments.

217

During IFN treatment, CD8+ T cells specific to HCV play an important role in

the control of the virus infection. Queiróz et al. mapped 21 CD8+ HCV-specific

epitopes in NS5A and three of these were highly conserved in patients who attained

SVR, located at codons 15-23, 219-238, and 225-238 (419). Mutations in these three

regions would likely result in a lack of treatment response, especially since the first

epitope was conserved in 100 % of the patients who attained SVR (419). In our study,

ethanol and reactive species caused mutations in all three of these epitopes, with a

dN/dS ratio >1 for sites 17 and 21 within the first epitope. On the other hand, our

study also caused mutations in the single epitope that was conserved in 98 % of non-

responders. This suggests that the destruction of these HCV-specific CD8+ T cell

epitopes by mutations, depending on the epitope, can either improve or worsen

treatment efficacy, although since susceptible strains would likely die off during

treatment, it is more likely to lead to antiviral resistance. It should be noted that, when

challenged with a selection pressure (i.e., antiviral treatment), sequence(s) containing

advantageous mutations would then be selected.

Cyclophilin inhibitors are a relatively new class of anti-HCV agents that show

promise. Individually, Y321N and D320E, as well as combinations of other

mutations, can confer resistance to cyclosporine A (420-423). Y321N did not show up

specifically in our study, but Y321C occurred four times with a dN/dS ratio >1.

D320E also did not show up in our study, but a D320H mutation did. Due to the

importance of Y321 and D320 to the effectiveness of cyclophilin inhibitors, it is likely

the two mutations that occurred in our study would also render the HCV sequence

218

resistant, but it would be interesting to determine if mutations other than Y321N and

D320E could have the same effect. Amidst the combinations of mutations that also

conferred cyclosporine A resistance, F284S occurred twice with a dN/dS ratio >1.

V445A confers resistance to another cyclophilin inhibitor, Debio 025, and occurs in

our study with a dN/dS ratio >1 (420). It has been suggested that these mutations

cause a conformational change to NS5A reducing its dependency on cyclophilin

binding for HCV replication and therefore reducing its susceptibility to cyclophilin

inhibitors (423). It is interesting to note that so many of these mutations that are

known to affect antiviral sensitivity, immune response, and viral replication cycle

were generated by treating cells either with ethanol or BSO in our study. Here, it

should be noted that cyclosporine was recently reported to inhibit HCV replication

through oxidative stress (424); ribavirin can act as a mutagen to the HCV RNA but it

also induces hemolytic anemia with signs of increased oxidative stress (425); and

protein kinases implicated in NS5A phosphorylation (e.g, MAPK) respond to ethanol

and/or oxidative stress (315,426).

In this study, we have shown that ethanol and reactive species could increase

oxidative RNA damage and the mutation frequency of HCV in Huh7 cells. Both the

oxidative RNA damage and increased mutation frequency could be reversed by

chemical intervention. These findings suggest another potential key mechanism of the

adverse interaction between ethanol and HCV. Not only could the elevated mutation

rated induced by ethanol contribute to increased HCV replication and resistance to

anti-HCV therapy, but it could also contribute to pathogenesis. For instance, increased

219

NS5A sequence heterogeneity is associated with hepatocellular carcinoma in cirrhotic

patients (427). Our findings also suggest another important consequence of chronic

oxidative/nitrosative stress induced by HCV other than just pathogenesis (258).

Additionally, there is the possibility that, by understanding the nature of these

chemical-biological interactions, it may be possible to attenuate the mutation rate of

HCV, thereby improving the chances for successful treatment.

Literature queries led to the identification of specific mutations and

combinations of mutations that may confer advantages to HCV for developing

persistent infection (Table 5.1). Studies are in progress to confirm the most important

functions by adding the corresponding mutation(s) to the wild-type sequence via site-

directed mutagenesis and testing for functional changes. In addition to performing

functional tests on mutations that were found in literature, the mutations involving

serine, threonine, and tyrosine that occurred frequently in our study and the database

(Table 4.3) will also need to be tested. By transfecting these constructs or HCV RNA

from treated cells into naïve cells and exposing them to various selection pressures,

such as interferon, we can test if any of our mutations allow for the development of

escape mutants. Replication or colony formation assays can be used to determine

differences between the strains. Pre-oxidizing the viral RNA in vitro can act as a

control or be used in conjunction with oxidized NS5B polymerase to determine if

ethanol and reactive species are causing mutations by directly modifying RNA bases

or by oxidizing the polymerase, increasing its error rate. In addition to determining

the function of the mutations that are induced by ethanol and reactive species, we can

220

use other aspects of the study. For instance, ethanol can be used as a mutagen or

selection agent to manipulate the sequence heterogeneity of HCV or other viruses in

vitro. We can also use this system to screen for candidates that could potentially

decrease the mutation rate of HCV and perhaps other viruses, as well. We also need

to assess how the mutation rates/trends of other HCV genes in the presence of ethanol

and reactive species. All of these then should ultimately be confirmed in an infective

genotype 1b in vitro system and an in vivo animal model.

In conclusion, the work in this thesis elucidated important mechanisms

involving the synergy between ethanol and HCV. We found that ethanol increases

HCV replication by lipogenesis and increasing relative NADH levels. We also found

that these increases could be abrogated by inhibitors of lipogenic pathways. In the

second half of the thesis, we describe another mechanism of how ethanol could be

affecting HCV replication, viral persistence, and pathogenesis by increasing the

mutation frequency of the HCV RNA. We also show that the mutation rate could be

increased by HCV-induced reactive species in the absence of ethanol or BSO,

suggesting that this is a typical consequence of HCV replication cycle. We were able

to use agents that could reduce the oxidative RNA damage to reduce the mutation

rates of HCV as well. The studies in this thesis both point out novel methods for

disrupting the synergy between ethanol and HCV that can potentially improve

treatment success rates in alcoholics. These studies may even have farther-reaching

consequences that may lead to novel therapeutic targets that could treat HCV and

221

possibly also other viruses, particularly those showing high rates of mutability and/or

redox modulation.

222

Table 5.1. Mutations that altered function found in literature that occurred in our studya

Mutation in Literature Functionb

Occurrences in Studyc

dN/dS>1 at codond Referencee

F37L Increases NS5A binding and inhibition of 2’,5’-oligoadenylate synthetase during IFN response

F37L (4) Yes (417)

Y321N

Causes a conformational change in a major cyclophilin A binding region of NS5A and confers resistance to cyclosporine

Y321C (4) Yes (421,423)

D320E Confers resistance to cyclosporine

D320H (1) No (420,423)

E442G Confers resistance to ribavirin and found in IFN-resistant clones

E442G (1) E422K (2) E422V (1)

Yes (415,418)

G404S Confers resistance to ribavirin G404D (1) (D mimics

phosphoserine) No (418)

V410A Found in IFN-resistant clones V410A (2) Yes (415) I302V Found in IFN-resistant clones I302V (1) Yes (415) S414P Found in IFN-resistant clones S414P (1) No (415)

W433R Found in IFN-resistant clones W433S (1) W433C (1)

Yes (415)

S390R Found in IFN-resistant clones S390N (1) No (415) K277E Found in IFN-resistant clones K277R (1) No (415)

F284S Found in cyclosporine-resistant HCV strains

F284S (2) F284L (1)

Yes (420,422)

V279A Found in cyclosporine-resistant HCV strains

V279A (2) No (420,422)

V445A Found in Debio 025-resistant HCV strains

V445A (1) Yes (420)

T21A Occurred during in vivo infection of chimpanzees with homogeneous strain of HCV

T21A (2) Yes (409)

M133V Occurred during in vivo infection of chimpanzees with homogeneous strain of HCV

M133V (1) Yes (409)

R318H Occurred during in vivo infection of chimpanzees with homogeneous strain of HCV

R318H (1) Yes (409)

N69T Occurred during passage of HCV in HEK 293 cells

N69T (3) Yes (412)

223

expressing Sip-L

V177I Occurred during passage of HCV in HEK 293 cells expressing Sip-L

V177I (1) No (412)

N105Y Occurred during passage of HCV in HEK 293 cells expressing Sip-L

N105S (1) N105D (1) N105G (1)

Yes (412)

A211E Occurred during passage of HCV in HEK 293 cells expressing Sip-L

A211V (1) No (412)

A252V/F Can increase HCV replication, combined with other mutations

A252V (3) Yes (410)

T244I Can increase HCV replication, combined with other mutations

T244A (2) Yes (410)

H247Y Can increase HCV replication, combined with other mutations

H247Y (2) Yes (410)

R246Q Can increase HCV replication, combined with other mutations

R246L (1) Yes (410)

K68R Can increase HCV replication, combined with other mutations

K68R (2) No (413)

L226S Can increase HCV replication, combined with other mutations

L226S (1) No (413)

S264P Can increase HCV replication, combined with other mutations

E264G (1) (E can mimic

phosphoserine) No (413)

M313A Can increase HCV replication with other mutations in Con1

M313V (1) Yes (321)

G341A Can increase HCV replication with other mutations in Con1

G341R (1) No (321)

a All mutations in this table were aligned to the numbering in our study. b Function of the mutation that occurred in the literature. c Type and frequency of mutations in our study that occurred at the same site as the

mutation from the literature. d Is positive selection occurring at the mutation site in our study? e References for HCV mutations from the literature

224

REFERENCES 1. Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., and

Houghton, M. (1989) Science 244, 359-362 2. Alter, M. J., Kruszon-Moran, D., Nainan, O. V., McQuillan, G. M., Gao, F.,

Moyer, L. A., Kaslow, R. A., and Margolis, H. S. (1999) N Engl J Med 341, 556-562

3. (1998) MMWR 47 (RR19), 1-39 4. (2000) Weekly epidemiological record 75, 18 -19 5. Di Bisceglie, A. M., Order, S. E., Klein, J. L., Waggoner, J. G., Sjogren, M.

H., Kuo, G., Houghton, M., Choo, Q. L., and Hoofnagle, J. H. (1991) Am J Gastroenterol 86, 335-338

6. Tong, M. J., el-Farra, N. S., Reikes, A. R., and Co, R. L. (1995) N Engl J Med 332, 1463-1466

7. McKillop, I. H., and Schrum, L. W. (2005) Alcohol 35, 195-203 8. Davis, G. L., Albright, J. E., Cook, S. F., and Rosenberg, D. M. (2003) Liver

Transpl 9, 331-338 9. Fried, M. W., Shiffman, M. L., Reddy, K. R., Smith, C., Marinos, G.,

Goncales, F. L., Jr., Haussinger, D., Diago, M., Carosi, G., Dhumeaux, D., Craxi, A., Lin, A., Hoffman, J., and Yu, J. (2002) N Engl J Med 347, 975-982

10. Manns, M. P., McHutchison, J. G., Gordon, S. C., Rustgi, V. K., Shiffman, M., Reindollar, R., Goodman, Z. D., Koury, K., Ling, M., and Albrecht, J. K. (2001) Lancet 358, 958-965

11. Robertson, B., Myers, G., Howard, C., Brettin, T., Bukh, J., Gaschen, B., Gojobori, T., Maertens, G., Mizokami, M., Nainan, O., Netesov, S., Nishioka, K., Shin i, T., Simmonds, P., Smith, D., Stuyver, L., and Weiner, A. (1998) Arch Virol 143, 2493-2503

12. Lindenbach, B. D., and Rice, C. M. (2005) Nature 436, 933-938 13. Choi, J., Xu, Z., and Ou, J. H. (2003) Mol Cell Biol 23, 1489-1497 14. Varaklioti, A., Vassilaki, N., Georgopoulou, U., and Mavromara, P. (2002) J

Biol Chem 277, 17713-17721 15. Walewski, J. L., Keller, T. R., Stump, D. D., and Branch, A. D. (2001) Rna 7,

710-721 16. Xu, Z., Choi, J., Yen, T. S., Lu, W., Strohecker, A., Govindarajan, S., Chien,

D., Selby, M. J., and Ou, J. (2001) EMBO J 20, 3840-3848. 17. Egger, D., Wolk, B., Gosert, R., Bianchi, L., Blum, H. E., Moradpour, D., and

Bienz, K. (2002) J Virol 76, 5974-5984 18. Moradpour, D., Gosert, R., Egger, D., Penin, F., Blum, H. E., and Bienz, K.

(2003) Antiviral Res 60, 103-109 19. Miyanari, Y., Atsuzawa, K., Usuda, N., Watashi, K., Hishiki, T., Zayas, M.,

Bartenschlager, R., Wakita, T., Hijikata, M., and Shimotohno, K. (2007) Nat Cell Biol 9, 1089-1097

20. Moradpour, D., Penin, F., and Rice, C. M. (2007) Nat Rev Microbiol 5, 453-463

225

21. Wang, X. W., Hussain, S. P., Huo, T. I., Wu, C. G., Forgues, M., Hofseth, L. J., Brechot, C., and Harris, C. C. (2002) Toxicology 181-182, 43-47

22. Choi, J., and Ou, J. H. (2006) Am J Physiol Gastrointest Liver Physiol 290, G847-851

23. Poli, G. (2000) Mol Aspects Med 21, 49-98 24. Benhamou, Y., Di Martino, V., Bochet, M., Colombet, G., Thibault, V., Liou,

A., Katlama, C., Poynard, T., and Group., M. (2001) Hepatology 34, 283-287. 25. Di Martino, V., Rufat, P., Boyer, N., Renard, P., Degos, F., Martinot-

Peignoux, M., Matheron, S., Le Moing, V., Vachon, F., Degott, C., Valla, D., and Marcellini, P. (2001) Hepatology 34, 1193-1199.

26. Ishak, K. G., Zimmerman, H. J., and Ray, M. B. (1991) Alcohol Clin Exp Res 15, 45-66

27. Poynard, T., Bedossa, P., and Opolon, P. (1997) Lancet 349, 825-832. 28. Barbaro, G., Di Lorenzo, G., Asti, A., Ribersani, M., Belloni, G., Grisorio, B.,

Filice, G., and Barbarini, G. (1999) Am J Gastroenterol 94, 2198-2205 29. Barbaro, G., Di Lorenzo, G., Ribersani, M., Soldini, M., Giancaspro, G.,

Bellomo, G., Belloni, G., Grisorio, B., and Barbarini, G. (1999) J Hepatol 30, 774-782

30. Farinati, F., Cardin, R., De Maria, N., Della Libera, G., Marafin, C., Lecis, E., Burra, P., Floreani, A., Cecchetto, A., and Naccarato, R. (1995) J Hepatol 22, 449-456

31. Farinati, F., Cardin, R., Degan, P., De Maria, N., Floyd, R. A., Van Thiel, D. H., and Naccarato, R. (1999) Free Radic Biol Med 27, 1284-1291

32. Higueras, V., Raya, A., Rodrigo, J. M., Serra, M. A., Roma, J., and Romero, F. J. (1994) Free Radic Biol Med 16, 131-133

33. Kageyama, F., Kobayashi, Y., Kawasaki, T., Toyokuni, S., Uchida, K., and Nakamura, H. (2000) Am J Gastroenterol 95, 1041-1050

34. Loguercio, C., De Girolamo, V., Federico, A., Feng, S. L., Cataldi, V., Del Vecchio Blanco, C., and Gialanella, G. (1997) J Trace Elem Med Biol 11, 158-161

35. Mutlu-Turkoglu, U., Ademoglu, E., Turkoglu, S., Badur, S., Uysal, M., and Toker, G. (1997) Res Commun Mol Pathol Pharmacol 96, 357-361

36. Swietek, K., and Juszczyk, J. (1997) J Viral Hepat 4, 139-141 37. Konishi, M., Iwasa, M., Araki, J., Kobayashi, Y., Katsuki, A., Sumida, Y.,

Nakagawa, N., Kojima, Y., Watanabe, S., Adachi, Y., and Kaito, M. (2006) J Gastroenterol Hepatol 21, 1821-1825

38. Jain, S. K., Pemberton, P. W., Smith, A., McMahon, R. F., Burrows, P. C., Aboutwerat, A., and Warnes, T. W. (2002) J Hepatol 36, 805-811

39. Mahmood, S., Kawanaka, M., Kamei, A., Izumi, A., Nakata, K., Niiyama, G., Ikeda, H., Hanano, S., Suehiro, M., Togawa, K., and Yamada, G. (2004) Antioxid Redox Signal 6, 19-24

40. Izumi, N., Enomoto, N., Uchihara, M., Murakami, T., Ono, K., Noguchi, O., Miyake, S., Nouchi, T., Fujisawa, K., Marumo, F., and Sato, C. (1996) Dig Dis Sci 41, 989-994

226

41. Hara, Y., Hino, K., Okuda, M., Furutani, T., Hidaka, I., Yamaguchi, Y., Korenaga, M., Li, K., Weinman, S. A., Lemon, S. M., and Okita, K. (2006) J Gastroenterol 41, 257-268

42. Paradis, V., Mathurin, P., Kollinger, M., Imbert-Bismut, F., Charlotte, F., Piton, A., Opolon, P., Holstege, A., Poynard, T., and Bedossa, P. (1997) J Clin Pathol 50, 401-406

43. Salem, T. A., El-Refaei, M. F., and Badra, G. A. (2003) Egypt J Immunol 10, 37-45

44. Hayashi, J., Aoki, H., Arakawa, Y., and Hino, O. (1999) Intervirology 42, 205-210

45. Reynolds, W. F., Patel, K., Pianko, S., Blatt, L. M., Nicholas, J. J., and McHutchison, J. G. (2002) Genes Immun 3, 345-349

46. Roberts, J. M., Searle, J. W., and Cooksley, W. G. (1993) Gastroenterol Jpn 28 Suppl 5, 37-41

47. Toro, F., Conesa, A., Garcia, A., Bianco, N. E., and De Sanctis, J. B. (1998) Clin Immunol Immunopathol 88, 169-175

48. Winterbourn, C. C. (1995) Toxicol Lett 82-83, 969-974. 49. Bacon, B. R., and Britton, R. S. (1989) Chem Biol Interact 70, 183-226 50. Hezode, C., Cazeneuve, C., Coue, O., Roudot-Thoraval, F., Lonjon, I., Bastie,

A., Duvoux, C., Pawlotsky, J. M., Zafrani, E. S., Amselem, S., and Dhumeaux, D. (1999) J Hepatol 31, 979-984.

51. Thorburn, D., Curry, G., Spooner, R., Spence, E., Oien, K., Halls, D., Fox, R., McCruden, E. A., MacSween, R. N., and Mills, P. R. (2002) Gut 50, 248-252.

52. Emerit, J., Beaumont, C., and Trivin, F. (2001) Biomed Pharmacother 55, 333-339

53. Caltagirone, A., Weiss, G., and Pantopoulos, K. (2001) J Biol Chem 276, 19738-19745

54. Pantopoulos, K., and Hentze, M. W. (1995) EMBO J 14, 2917-2924 55. Pantopoulos, K., and Hentze, M. W. (1998) Proc Natl Acad Sci U S A 95,

10559-10563 56. Fujita, N., Horiike, S., Sugimoto, R., Tanaka, H., Iwasa, M., Kobayashi, Y.,

Hasegawa, K., Ma, N., Kawanishi, S., Adachi, Y., and Kaito, M. (2007) Free Radic Biol Med 42, 353-362

57. Vrba, J., and Modriansky, M. (2002) Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 146, 15-20

58. Koike, N., Takamura, T., and Kaneko, S. (2007) Life Sci 59. Garcia-Trevijano, E. R., Iraburu, M. J., Fontana, L., Dominguez-Rosales, J. A.,

Auster, A., Covarrubias-Pinedo, A., and Rojkind, M. (1999) Hepatology 29, 960-970

60. Friedman, S. L. (2000) J Biol Chem 275, 2247-2250 61. Kota, R. S., Ramana, C. V., Tenorio, F. A., Enelow, R. I., and Rutledge, J. C.

(2005) J Biol Chem 280, 31076-31084 62. Liu, C., Gaca, M. D., Swenson, E. S., Vellucci, V. F., Reiss, M., and Wells, R.

G. (2003) J Biol Chem 278, 11721-11728

227

63. Korenaga, M., Wang, T., Li, Y., Showalter, L. A., Chan, T., Sun, J., and Weinman, S. A. (2005) J Biol Chem 280, 37481-37488

64. Abdalla, M. Y., Ahmad, I. M., Spitz, D. R., Schmidt, W. N., and Britigan, B. E. (2005) J Med Virol 76, 489-497

65. Moriya, K., Nakagawa, K., Santa, T., Shintani, Y., Fujie, H., Miyoshi, H., Tsutsumi, T., Miyazawa, T., Ishibashi, K., Horie, T., Imai, K., Todoroki, T., Kimura, S., and Koike, K. (2001) Cancer Res 61, 4365-4370

66. Okuda, M., Li, K., Beard, M. R., Showalter, L. A., Scholle, F., Lemon, S. M., and Weinman, S. A. (2002) Gastroenterology 122, 366-375

67. Li, K., Prow, T., Lemon, S. M., and Beard, M. R. (2002) Hepatology 35, 1237-1246

68. Perlemuter, G., Letteron, P., Carnot, F., Zavala, F., Pessayre, D., Nalpas, B., and Brechot, C. (2003) J Hepatol 39, 1020-1027

69. Li, Y., Boehning, D. F., Qian, T., Popov, V. L., and Weinman, S. A. (2007) Faseb J

70. Gochee, P. A., Jonsson, J. R., Clouston, A. D., Pandeya, N., Purdie, D. M., and Powell, E. E. (2003) J Gastroenterol Hepatol 18, 386-392

71. de Lucas, S., Bartolome, J., Amaro, M. J., and Carreno, V. (2003) Antiviral Res 60, 117-124

72. Garcia-Mediavilla, M. V., Sanchez-Campos, S., Gonzalez-Perez, P., Gomez-Gonzalo, M., Majano, P. L., Lopez-Cabrera, M., Clemente, G., Garcia-Monzon, C., and Gonzalez-Gallego, J. (2005) J Hepatol 43, 606-613

73. Machida, K., Cheng, K. T., Sung, V. M., Lee, K. J., Levine, A. M., and Lai, M. M. (2004) J Virol 78, 8835-8843

74. Miller, K., McArdle, S., Gale, M. J., Jr., Geller, D. A., Tenoever, B., Hiscott, J., Gretch, D. R., and Polyak, S. J. (2004) J Interferon Cytokine Res 24, 391-402

75. Nunez, O., Fernandez-Martinez, A., Majano, P. L., Apolinario, A., Gomez-Gonzalo, M., Benedicto, I., Lopez-Cabrera, M., Bosca, L., Clemente, G., Garcia-Monzon, C., and Martin-Sanz, P. (2004) Gut 53, 1665-1672

76. Waris, G., and Siddiqui, A. (2005) J Virol 79, 9725-9734 77. Choi, J., Lu, W., and Ou, J. (2001) Recent Res. Devel. Virol. 3, 105-120 78. Irshad, M., and Dhar, I. (2006) Med Princ Pract 15, 405-416 79. Ray, R. B., and Ray, R. (2001) FEMS Microbiol Lett 202, 149-156 80. Gong, G., Waris, G., Tanveer, R., and Siddiqui, A. (2001) Proc Natl Acad Sci

U S A 98, 9599-9604 81. Qadri, I., Iwahashi, M., Capasso, J. M., Hopken, M. W., Flores, S., Schaack, J.,

and Simon, F. R. (2004) Biochem J 378, 919-928 82. Thoren, F., Romero, A., Lindh, M., Dahlgren, C., and Hellstrand, K. (2004) J

Leukoc Biol 76, 1180-1186 83. Bureau, C., Bernad, J., Chaouche, N., Orfila, C., Beraud, M., Gonindard, C.,

Alric, L., Vinel, J. P., and Pipy, B. (2001) J Biol Chem 276, 23077-23083 84. Ghaziani, T., Shan, Y., Lambrecht, R. W., Donohue, S. E., Pietschmann, T.,

Bartenschlager, R., and Bonkovsky, H. L. (2006) J Hepatol 45, 5-12

228

85. Klaunig, J. E., Xu, Y., Isenberg, J. S., Bachowski, S., Kolaja, K. L., Jiang, J., Stevenson, D. E., and Walborg, E. F., Jr. (1998) Environ Health Perspect 106 Suppl 1, 289-295

86. Loguercio, C., and Federico, A. (2003) Free Radic Biol Med 34, 1-10 87. Parola, M., and Robino, G. (2001) J Hepatol 35, 297-306 88. Capocaccia, L., Merli, M., Piat, C., Servi, R., Zullo, A., and Riggio, O. (1991)

Ital J Gastroenterol 23, 386-391 89. Vendemiale, G., Grattagliano, I., Portincasa, P., Serviddio, G., Palasciamo, G.,

and Altomare, E. (2001) Eur J Clin Invest 31, 54-63 90. Forman, H. J., and Cadenas, E. (1997) Oxidative stress and signal

transduction, Chapman & Hall, New York 91. Furutani, T., Hino, K., Okuda, M., Gondo, T., Nishina, S., Kitase, A.,

Korenaga, M., Xiao, S. Y., Weinman, S. A., Lemon, S. M., Sakaida, I., and Okita, K. (2006) Gastroenterology 130, 2087-2098

92. Machida, K., Cheng, K. T., Sung, V. M., Shimodaira, S., Lindsay, K. L., Levine, A. M., Lai, M. Y., and Lai, M. M. (2004) Proc Natl Acad Sci U S A 101, 4262-4267

93. Moriya, K., Fujie, H., Shintani, Y., Yotsuyanagi, H., Tsutsumi, T., Ishibashi, K., Matsuura, Y., Kimura, S., Miyamura, T., and Koike, K. (1998) Nat Med 4, 1065-1067

94. Hahn, C. S., Cho, Y. G., Kang, B. S., Lester, I. M., and Hahn, Y. S. (2000) Virology 276, 127-137

95. Honda, M., Kaneko, S., Shimazaki, T., Matsushita, E., Kobayashi, K., Ping, L. H., Zhang, H. C., and Lemon, S. M. (2000) Hepatology 31, 1351-1359

96. Koike, K. (2006) J Gastroenterol 41, 292-294 97. Marusawa, H., Hijikata, M., Chiba, T., and Shimotohno, K. (1999) J Virol 73,

4713-4720 98. Otsuka, M., Kato, N., and Omata, M. (2004) Nippon Rinsho 62 Suppl 7, 185-

190 99. Otsuka, M., Kato, N., Taniguchi, H., Yoshida, H., Goto, T., Shiratori, Y., and

Omata, M. (2002) Virology 296, 84-93 100. Ray, R. B., Steele, R., Meyer, K., and Ray, R. (1998) Gene 208, 331-336 101. Ruggieri, A., Harada, T., Matsuura, Y., and Miyamura, T. (1997) Virology

229, 68-76 102. Sacco, R., Tsutsumi, T., Suzuki, R., Otsuka, M., Aizaki, H., Sakamoto, S.,

Matsuda, M., Seki, N., Matsuura, Y., Miyamura, T., and Suzuki, T. (2003) Virology 317, 24-35

103. Saito, K., Meyer, K., Warner, R., Basu, A., Ray, R. B., and Ray, R. (2006) J Virol 80, 4372-4379

104. Zhu, N., Khoshnan, A., Schneider, R., Matsumoto, M., Dennert, G., Ware, C., and Lai, M. M. (1998) J Virol 72, 3691-3697

105. Brune, B. (2005) Antioxid Redox Signal 7, 497-507 106. Davies, K. J. (1999) IUBMB Life 48, 41-47

229

107. Hagen, T. M., Huang, S., Curnutte, J., Fowler, P., Martinez, V., Wehr, C. M., Ames, B. N., and Chisari, F. V. (1994) Proc Natl Acad Sci U S A 91, 12808-12812

108. Saito, I., Miyamura, T., Ohbayashi, A., Harada, H., Katayama, T., Kikuchi, S., Watanabe, Y., Koi, S., Onji, M., Ohta, Y., and et al. (1990) Proc Natl Acad Sci U S A 87, 6547-6549

109. Melhem, A., Stern, M., Shibolet, O., Israeli, E., Ackerman, Z., Pappo, O., Hemed, N., Rowe, M., Ohana, H., Zabrecky, G., Cohen, R., and Ilan, Y. (2005) J Clin Gastroenterol 39, 737-742

110. Lieber, C. S. (2001) Am J Addict 10 Suppl, 29-50 111. Seitz, H. K., Poschl, G., and Simanowski, U. A. (1998) Recent Dev Alcohol 14,

67-95 112. Hurwitz, E. S., Holman, R. C., Strine, T. W., and Chorba, T. L. (1995) Am J

Public Health 85, 1256-1260 113. Balasekaran, R., Bulterys, M., Jamal, M. M., Quinn, P. G., Johnston, D. E.,

Skipper, B., Chaturvedi, S., and Arora, S. (1999) Am J Gastroenterol 94, 1341-1346

114. Bhattacharya, R., and Shuhart, M. C. (2003) J Clin Gastroenterol 36, 242-252. 115. Jamal, M. M., and Morgan, T. R. (2003) Best Pract Res Clin Gastroenterol 17,

649-662 116. Szabo, G. (2003) Curr Gastroenterol Rep 5, 86-92 117. Coelho-Little, M. E., Jeffers, L. J., Bernstein, D. E., Goodman, J. J., Reddy, K.

R., de Medina, M., Li, X., Hill, M., La Rue, S., and Schiff, E. R. (1995) Alcohol Clin Exp Res 19, 1173-1176

118. Mendenhall, C. L., Seeff, L., Diehl, A. M., Ghosn, S. J., French, S. W., Gartside, P. S., Rouster, S. D., Buskell-Bales, Z., Grossman, C. J., Roselle, G. A., and et al. (1991) Hepatology 14, 581-589

119. Takase, S., Takada, N., Sawada, M., Tsutsumi, M., and Takada, A. (1993) Alcohol Alcohol Suppl 1A, 77-84

120. Aizawa, Y., Shibamoto, Y., Takagi, I., Zeniya, M., and Toda, G. (2000) Cancer 89, 53-59.

121. Cooksley, W. (1996) J Gastroenterol Hepatol 11, 187-192 122. Corrao, G., and Arico, S. (1998) Hepatology 27, 914-919. 123. Donato, F., Tagger, A., Gelatti, U., Parrinello, G., Boffetta, P., Albertini, A.,

Decarli, A., Trevisi, P., Ribero, M. L., Martelli, C., Porru, S., and Nardi, G. (2002) Am J Epidemiol 155, 323-331.

124. Harris, D. R., Gonin, R., Alter, H. J., Wright, E. C., Buskell, Z. J., Hollinger, F. B., Seeff, L. B., and National Heart, L., and Blood Institute Study Group. (2001) Ann Intern Med 134, 120-124.

125. Ikeda, K., Saitoh, S., Suzuki, Y., Kobayashi, M., Tsubota, A., Koida, I., Arase, Y., Fukuda, M., Chayama, K., Murashima, N., and Kumada, H. (1998) J Hepatol 28, 930-938.

126. Kuwana, K., Ichida, T., Kamimura, T., Ohkoshi, S., Ogata, N., Harada, T., Endoh, K., and Asakura, H. (1997) J Gastroenterol Hepatol 12, 149-155.

230

127. Matsuda, Y., Amuro, Y., Higashino, K., Hada, T., Yamamoto, T., Fujikura, M., Yamaguchi, K., Shimomura, S., Iijima, H., and Nakano, T., et al. (1995) Hepatogastroenterology 42, 151-154.

128. Monto, A., Alonzo, J., Watson, J. J., Grunfeld, C., and Wright, T. L. (2002) Hepatology 36, 729-736.

129. Niederau C, L. S., Heintges T, Erhardt A, Buschkamp M, Hurter D, Nawrocki M, Kruska L, Hensel F, Petry W, Haussinger D. (1998) Hepatology 28, 1687-1695.

130. Ostapowicz, G., Watson, K. J., Locarnini, S. A., and Desmond, P. V. (1998) Hepatology 27, 1730-1735

131. Pianko, S., Patella, S., Ostapowicz, G., Desmond, P., and Sievert, W. (2001) J Viral Hepat 8, 406-413.

132. Roudot-Thoraval, F., Bastie, A., Pawlotsky, J. M., and Dhumeaux, D. (1997) Hepatology 26, 485-490.

133. Sachithanandan, S., Kay, E., Leader, M., and Fielding, J. F. (1997) Biomed Pharmacother 51, 295-297.

134. Serfaty, L., Chazouilleres, O., Poujol-Robert, A., Morand-Joubert, L., Dubois, C., Chretien, Y., Poupon, R. E., Petit, J. C., and Poupon, R. (1997) Hepatology 26, 776-779.

135. Thomas, D. L., Astemborski, J., Rai, R. M., Anania, F. A., Schaeffer, M., Galai, N., Nolt, K., Nelson, K. E., Strathdee, S. A., Johnson, L., Laeyendecker, O., Boitnott, J., Wilson, L. E., and Vlahov, D. (2000) JAMA 284, 450-456.

136. Westin, J., Lagging, L. M., Spak, F., Aires, N., Svensson, E., Lindh, M., Dhillon, A. P., Norkrans, G., and Wejstal, R. (2002) J Viral Hepat 9, 235-241.

137. Wiley, T. E., McCarthy, M., Breidi, L., McCarthy, M., and Layden, T. J. (1998) Hepatology 28, 805-809

138. Kim, W. H., Hong, F., Jaruga, B., Hu, Z., Fan, S., Liang, T. J., and Gao, B. (2001) Faseb J 15, 2551-2553

139. Fukushima, W., Tanaka, T., Ohfuji, S., Habu, D., Tamori, A., Kawada, N., Sakaguchi, H., Takeda, T., Nishiguchi, S., Seki, S., Shiomi, S., and Hirota, Y. (2006) Hepatol Res 34, 141-149

140. Jamal, M. M., Saadi, Z., and Morgan, T. R. (2005) Dig Dis 23, 285-296 141. Befrits, R., Hedman, M., Blomquist, L., Allander, T., Grillner, L., Kinnman,

N., Rubio, C., and Hultcrantz, R. (1995) Scand J Gastroenterol 30, 1113-1118 142. Caldwell, S. H., Li, X., Rourk, R. M., Millar, A., Sosnowski, K. M., Sue, M.,

Barritt, A. S., McCallum, R. W., and Schiff, E. R. (1993) Am J Gastroenterol 88, 1016-1021

143. Pares, A., Barrera, J. M., Caballeria, J., Ercilla, G., Bruguera, M., Caballeria, L., Castillo, R., and Rodes, J. (1990) Hepatology 12, 1295-1299

144. Sata, M., Fukuizumi, K., Uchimura, Y., Nakano, H., Ishii, K., Kumashiro, R., Mizokami, M., Lau, J. Y., and Tanikawa, K. (1996) J Viral Hepat 3, 143-148.

145. Yamauchi, M., Nakahara, M., Maezawa, Y., Satoh, S., Nishikawa, F., Ohata, M., Mizuhara, Y., Hirakawa, J., Nakajima, H., and Fujisawa, K., et al. (1993) Am J Gastroenterol 88, 39-43.

231

146. Szabo, G., Dolganiuc, A., Mandrekar, P., and White, B. (2004) Alcohol 33, 241-249

147. Geissler, M., Gesien, A., and Wands, J. R. (1997) J Immunol 159, 5107-5113 148. Mochida, S., Ohnishi, K., Matsuo, S., Kakihara, K., and Fujiwara, K. (1996)

Alcohol Clin Exp Res 20, 371A-377A 149. Di Bona, D., Cippitelli, M., Fionda, C., Camma, C., Licata, A., Santoni, A.,

and Craxi, A. (2006) J Hepatol 45, 271-279 150. Lin, W., Kim, S. S., Yeung, E., Kamegaya, Y., Blackard, J. T., Kim, K. A.,

Holtzman, M. J., and Chung, R. T. (2006) J Virol 80, 9226-9235 151. Plumlee, C. R., Lazaro, C. A., Fausto, N., and Polyak, S. J. (2005) Virol J 2, 89 152. Bondy, S. C. (1992) Toxicol Lett 63, 231-241 153. Cederbaum, A. I. (1991) Alcohol Alcohol Suppl 1, 291-296 154. Cederbaum, A. I., Wu, D., Mari, M., and Bai, J. (2001) Free Radic Biol Med

31, 1539-1543 155. Devi, B. G., Henderson, G. I., Frosto, T. A., and Schenker, S. (1993)

Hepatology 18, 648-659 156. Dianzani, M. U. (1985) Alcohol Alcohol 20, 161-173 157. Husain, K., Scott, B. R., Reddy, S. K., and Somani, S. M. (2001) Alcohol 25,

89-97 158. Ishii, H., Kurose, I., and Kato, S. (1997) J Gastroenterol Hepatol 12, S272-282 159. Kanbagli, O., Balkan, J., Aykac-Toker, G., and Uysal, M. (2002) Biol Pharm

Bull 25, 1482-1484 160. Lauterburg, B. H., Davies, S., and Mitchell, J. R. (1984) J Pharmacol Exp

Ther 230, 7-11 161. Montoliu, C., Valles, S., Renau-Piqueras, J., and Guerri, C. (1994) J

Neurochem 63, 1855-1862 162. Moss, M., and Burnham, E. L. (2003) Crit Care Med 31, S207-212 163. Nordmann, R., Ribiere, C., and Rouach, H. (1992) Free Radic Biol Med 12,

219-240 164. Oh, S. I., Kim, C. I., Chun, H. J., and Park, S. C. (1998) J Nutr 128, 758-763 165. Arteel, G. E. (2003) Gastroenterology 124, 778-790 166. Venkatraman, A., Landar, A., Davis, A. J., Ulasova, E., Page, G., Murphy, M.

P., Darley-Usmar, V., and Bailey, S. M. (2004) Am J Physiol Gastrointest Liver Physiol 286, G521-527

167. Cunningham, C. C., and Bailey, S. M. (2001) Biol Signals Recept 10, 271-282 168. Lieber, C. S. (1991) Alcohol Alcohol Suppl 1, 283-290 169. Lieber, C. S. (1997) Adv Pharmacol 38, 601-628 170. Lieber, C. S. (2004) Alcohol 34, 9-19 171. Dupont, I., Lucas, D., Clot, P., Menez, C., and Albano, E. (1998) J Hepatol 28,

564-571 172. Morimoto, M., Hagbjork, A. L., Nanji, A. A., Ingelman-Sundberg, M.,

Lindros, K. O., Fu, P. C., Albano, E., and French, S. W. (1993) Alcohol 10, 459-464

173. Crabb, D. W., Bosron, W. F., and Li, T. K. (1987) Pharmacol Ther 34, 59-73

232

174. Laposata, M. (1999) Lipids 34 Suppl, S281-285 175. Rigamonti, C., Mottaran, E., Reale, E., Rolla, R., Cipriani, V., Capelli, F.,

Boldorini, R., Vidali, M., Sartori, M., and Albano, E. (2003) Hepatology 38, 42-49

176. Otani, K., Korenaga, M., Beard, M. R., Li, K., Qian, T., Showalter, L. A., Singh, A. K., Wang, T., and Weinman, S. A. (2005) Gastroenterology 128, 96-107

177. Wen, F., Abdalla, M. Y., Aloman, C., Xiang, J., Ahmad, I. M., Walewski, J., McCormick, M. L., Brown, K. E., Branch, A. D., Spitz, D. R., Britigan, B. E., and Schmidt, W. N. (2004) J Med Virol 72, 230-240

178. Wheeler, M. (2005) Gastroenterology 128, 232-234 179. Albano, E. (2002) Free Radic Biol Med 32, 110-114 180. Dreosti, I. E., Ballard, F. J., Belling, G. B., Record, I. R., Manuel, S. J., and

Hetzel, B. S. (1981) Alcohol Clin Exp Res 5, 357-362 181. Sun, A. Y., Ingelman-Sundberg, M., Neve, E., Matsumoto, H., Nishitani, Y.,

Minowa, Y., Fukui, Y., Bailey, S. M., Patel, V. B., Cunningham, C. C., Zima, T., Fialova, L., Mikulikova, L., Popov, P., Malbohan, I., Janebova, M., Nespor, K., and Sun, G. Y. (2001) Alcohol Clin Exp Res 25, 237S-243S

182. Purohit, V., and Brenner, D. A. (2006) Hepatology 43, 872-878 183. Tsutsumi, T., Suzuki, T., Moriya, K., Shintani, Y., Fujie, H., Miyoshi, H.,

Matsuura, Y., Koike, K., and Miyamura, T. (2003) Hepatology 38, 820-828 184. Tamai, T., Seki, T., Shiro, T., Nakagawa, T., Wakabayashi, M., Imamura, M.,

Nishimura, A., Yamashiki, N., Takasu, M., Inoue, K., and Okamura, A. (2000) Alcohol Clin Exp Res 24, 106S-111S

185. Uchimura, Y., Sata, M., Kage, M., Abe, H., and Tanikawa, K. (1995) Liver 15, 300-306

186. Oshita, M., Hayashi, N., Kasahara, A., Hagiwara, H., Mita, E., Naito, M., Katayama, K., Fusamoto, H., and Kamada, T. (1994) Hepatology 20, 1115-1120.

187. Oshita, M., Takei, Y., Kawano, S., Hijioka, T., Masuda, E., Goto, M., Nishimura, Y., Nagai, H., Iio, S., Tsuji, S., and et al. (1994) Hepatology 20, 961-965

188. Pessione, F., Degos, F., Marcellin, P., Duchatelle, V., Njapoum, C., Martinot-Peignoux, M., Degott, C., Valla, D., Erlinger, S., and Rueff, B. (1998) Hepatology 27, 1717-1722

189. Romero-Gomez, M., Grande, L., Nogales, M. C., Fernandez, M., Chavez, M., and Castro, M. (2001) Dig Liver Dis 33, 698-702

190. Sawada, M., Takada, A., Takase, S., and Takada, N. (1993) Alcohol Alcohol Suppl 1B, 85-90

191. Cromie, S. L., Jenkins, P. J., Bowden, D. S., and Dudley, F. J. (1996) J Hepatol 25, 821-826

192. Anand, B. S., and Thornby, J. (2005) Gut 54, 1468-1472 193. Anand, B. S., and Velez, M. (2000) Dig Dis 18, 168-171

233

194. Haydon, G. H., Jarvis, L. M., Blair, C. S., Simmonds, P., Harrison, D. J., Simpson, K. J., and Hayes, P. C. (1998) Gut 42, 570-575

195. Khan, K. N., and Yatsuhashi, H. (2000) Alcohol Alcohol 35, 286-295 196. Carriere, M., Rosenberg, A. R., Conti, F., Chouzenoux, S., Terris, B., Sogni,

P., Soubrane, O., Calmus, Y., and Podevin, P. (2006) J Viral Hepat 13, 633-642

197. Zhang, T., Li, Y., Lai, J. P., Douglas, S. D., Metzger, D. S., O'Brien, C. P., and Ho, W. Z. (2003) Hepatology 38, 57-65

198. Kakizaki, S., Takagi, H., Horiguchi, N., Toyoda, M., Takayama, H., Nagamine, T., Mori, M., and Kato, N. (2000) Liver 20, 125-128.

199. Morbitzer, M., and Herget, T. (2005) J Biol Chem 280, 8831-8841 200. Tardif, K. D., Waris, G., and Siddiqui, A. (2005) Trends Microbiol 13, 159-

163 201. Waris, G., Turkson, J., Hassanein, T., and Siddiqui, A. (2005) J Virol 79,

1569-1580 202. Fillebeen, C., Rivas-Estilla, A. M., Bisaillon, M., Ponka, P., Muckenthaler, M.,

Hentze, M. W., Koromilas, A. E., and Pantopoulos, K. (2005) J Biol Chem 280, 9049-9057

203. Ferrari, E., Wright-Minogue, J., Fang, J. W., Baroudy, B. M., Lau, J. Y., and Hong, Z. (1999) J Virol 73, 1649-1654

204. Hardy, R. W., Marcotrigiano, J., Blight, K. J., Majors, J. E., and Rice, C. M. (2003) J Virol 77, 2029-2037.

205. Lai, V. C., Dempsey, S., Lau, J. Y., Hong, Z., and Zhong, W. (2003) J Virol 77, 2295-2300

206. Zhu, H., Nelson, D. R., Crawford, J. M., and Liu, C. (2005) J Interferon Cytokine Res 25, 528-539

207. Zhu, H., Shang, X., Terada, N., and Liu, C. (2004) Biochem Biophys Res Commun 324, 518-528

208. Choi, J., Forman, H. J., Ou, J. H., Lai, M. M., Seronello, S., and Nandipati, A. (2006) Free Radic Biol Med 41, 1488-1498

209. Choi, J., Lee, K. J., Zheng, Y., Yamaga, A. K., Lai, M. M., and Ou, J. H. (2004) Hepatology 39, 81-89

210. Frese, M., Schwarzle, V., Barth, K., Krieger, N., Lohmann, V., Mihm, S., Haller, O., and Bartenschlager, R. (2002) Hepatology 35, 694-703.

211. Inoue, K., Sekiyama, K., Yamada, M., Watanabe, T., Yasuda, H., and Yoshiba, M. (2003) J Gastroenterol 38, 567-572

212. Nakagawa, M., Sakamoto, N., Tanabe, Y., Koyama, T., Itsui, Y., Takeda, Y., Chen, C. H., Kakinuma, S., Oooka, S., Maekawa, S., Enomoto, N., and Watanabe, M. (2005) Gastroenterology 129, 1031-1041

213. Watashi, K., Hijikata, M., Hosaka, M., Yamaji, M., and Shimotohno, K. (2003) Hepatology 38, 1282-1288

214. Yano, M., Ikeda, M., Abe, K., Dansako, H., Ohkoshi, S., Aoyagi, Y., and Kato, N. (2007) Antimicrob Agents Chemother 51, 2016-2027

234

215. Segal, B. H., Leto, T. L., Gallin, J. I., Malech, H. L., and Holland, S. M. (2000) Medicine (Baltimore) 79, 170-200

216. Schreck, R., Rieber, P., and Baeuerle, P. A. (1991) Embo J 10, 2247-2258 217. Zheng, Y. W., and Yen, T. S. (1994) J Biol Chem 269, 8857-8862. 218. Bar-Peled, O., Korkotian, E., Segal, M., and Groner, Y. (1996) Proc Natl Acad

Sci U S A 93, 8530-8535 219. Lei, X. G., and Cheng, W. H. (2005) J Nutr 135, 2295-2298 220. Morliere, P., and Santus, R. (1998) Eur J Biochem 256, 184-189 221. Wenk, J., Brenneisen, P., Wlaschek, M., Poswig, A., Briviba, K., Oberley, T.

D., and Scharffetter-Kochanek, K. (1999) J Biol Chem 274, 25869-25876 222. Wild, A. C., and Mulcahy, R. T. (1999) Biochem J 338 ( Pt 3), 659-665 223. Gronbaek, K., Sonne, J., Ring-Larsen, H., Poulsen, H. E., Friis, H., and Bygum

Krarup, H. (2005) Scand J Infect Dis 37, 686-689 224. Ko, W. S., Guo, C. H., Yeh, M. S., Lin, L. Y., Hsu, G. S., Chen, P. C., Luo, M.

C., and Lin, C. Y. (2005) World J Gastroenterol 11, 4697-4702 225. Cheung, R. C., Chan, R. T., and Lok, A. S. (1993) J Med Virol 41, 338-342 226. Grant, P. R., Black, A., Garcia, N., Prieto, J., and Garson, J. A. (2000) J Med

Virol 61, 439-442 227. Look, M. P., Gerard, A., Rao, G. S., Sudhop, T., Fischer, H. P., Sauerbruch, T.,

and Spengler, U. (1999) Antiviral Res 43, 113-122 228. Mahmood, S., Yamada, G., Niiyama, G., Kawanaka, M., Togawa, K., Sho, M.,

Ito, T., Sasagawa, T., Okita, M., Nakamura, H., and Yodoi, J. (2003) Free Radic Res 37, 781-785

229. Neri, S., Ierna, D., Antoci, S., Campanile, E., D'Amico, R. A., and Noto, R. (2000) Panminerva Med 42, 187-192

230. Shedlofsky, S. I. (1998) Hepatogastroenterology 45, 349-355 231. De Franceschi, L., Fattovich, G., Turrini, F., Ayi, K., Brugnara, C., Manzato,

F., Noventa, F., Stanzial, A. M., Solero, P., and Corrocher, R. (2000) Hepatology 31, 997-1004.

232. Morisco, F., Vitaglione, P., Carbone, A., Stingo, S., Scarpati, S., Ascione, A., Marmo, R., Fogliano, V., and Caporaso, N. (2004) J Clin Gastroenterol 38, S118-120

233. Buettner, G. R., and Jurkiewicz, B. A. (1996) Radiat Res 145, 532-541 234. Bukh, J., Miller, R. H., and Purcell, R. H. (1995) Semin Liver Dis 15, 41-63 235. Biebricher, C. K., and Eigen, M. (2006) Curr Top Microbiol Immunol 299, 1-

31 236. Manrubia, S. C., Escarmis, C., Domingo, E., and Lazaro, E. (2005) Gene 347,

273-282 237. Sherman, K. E., Rouster, S. D., Mendenhall, C., and Thee, D. (1999)

Hepatology 30, 265-270 238. Takahashi, K., Takahashi, T., Takahashi, S., Watanabe, K., Boku, S., Matsui,

S., Arai, F., and Asakura, H. (2001) J Gastroenterol Hepatol 16, 416-423 239. Conaway, C. C., Nie, G., Hussain, N. S., and Fiala, E. S. (1991) Cancer Res

51, 3143-3147

235

240. Mehta, S., Levey, J. M., and Bonkovsky, H. L. (2001) Clin Liver Dis 5, 979-1008

241. Cardin, R., Saccoccio, G., Masutti, F., Bellentani, S., Farinati, F., and Tiribelli, C. (2001) J Hepatol 34, 587-592

242. Chen, J., Clemens, D. L., Cederbaum, A. I., and Gao, B. (2001) Clin Biochem 34, 203-209

243. Li, Z., Wu, J., and Deleo, C. J. (2006) IUBMB Life 58, 581-588 244. McCartney, E. M., Semendric, L., Helbig, K. J., Hinze, S., Jones, B.,

Weinman, S. A., and Beard, M. R. (2008) J Infect Dis 245. Lindenbach, B. D., Evans, M. J., Syder, A. J., Wolk, B., Tellinghuisen, T. L.,

Liu, C. C., Maruyama, T., Hynes, R. O., Burton, D. R., McKeating, J. A., and Rice, C. M. (2005) Science 309, 623-626

246. Wakita, T., Pietschmann, T., Kato, T., Date, T., Miyamoto, M., Zhao, Z., Murthy, K., Habermann, A., Krausslich, H. G., Mizokami, M., Bartenschlager, R., and Liang, T. J. (2005) Nat Med 11, 791-796

247. Zhong, J., Gastaminza, P., Cheng, G., Kapadia, S., Kato, T., Burton, D. R., Wieland, S. F., Uprichard, S. L., Wakita, T., and Chisari, F. V. (2005) Proc Natl Acad Sci U S A 102, 9294-9299

248. Kato, T., Date, T., Miyamoto, M., Sugiyama, M., Tanaka, Y., Orito, E., Ohno, T., Sugihara, K., Hasegawa, I., Fujiwara, K., Ito, K., Ozasa, A., Mizokami, M., and Wakita, T. (2005) J Clin Microbiol 43, 5679-5684

249. Blight, K. J., Kolykhalov, A. A., and Rice, C. M. (2000) Science 290, 1972-1974

250. Kicic, A., Chua, A. C., and Baker, E. (2001) Cancer 92, 3093-3110 251. Brittenham, G. M. (1990) Ann N Y Acad Sci 612, 315-326 252. Ponka, P., Borova, J., Neuwirt, J., and Fuchs, O. (1979) FEBS Lett 97, 317-321 253. Edward, J. T., Gauthier, M., Chubb, F. L., and Ponka, P. (1988) J Chem Eng

Data 33, 538-540 254. Blight, K. J., McKeating, J. A., and Rice, C. M. (2002) J Virol 76, 13001-

13014 255. Ali, N., Tardif, K. D., and Siddiqui, A. (2002) J Virol 76, 12001-12007 256. Chang, T. K., Crespi, C. L., and Waxman, D. J. (1998) Methods Mol Biol 107,

147-152 257. Kuiken, C., Yusim, K., Boykin, L., and Richardson, R. (2005) Bioinformatics

21, 379-384 258. Seronello, S., Sheikh, M. Y., and Choi, J. (2007) Free Radic Biol Med 43, 869-

882 259. Trujillo-Murillo, K., Alvarez-Martinez, O., Garza-Rodriguez, L., Martinez-

Rodriguez, H., Bosques-Padilla, F., Ramos-Jimenez, J., Barrera-Saldana, H., Rincon-Sanchez, A. R., and Rivas-Estilla, A. M. (2007) J Viral Hepat 14, 608-617

260. Huang, H., Chen, Y., and Ye, J. (2007) Proc Natl Acad Sci U S A 104, 18666-18670

236

261. Kuroki, M., Ariumi, Y., Ikeda, M., Dansako, H., Wakita, T., and Kato, N. (2009) J Virol 83, 2338-2348

262. Eysseric, H., Gonthier, B., Soubeyran, A., Bessard, G., Saxod, R., and Barret, L. (1997) Alcohol 14, 111-115

263. Shin, H. R. (2006) Intervirology 49, 18-22 264. Marshall, D. J., Heisler, L. M., Lyamichev, V., Murvine, C., Olive, D. M.,

Ehrlich, G. D., Neri, B. P., and de Arruda, M. (1997) J Clin Microbiol 35, 3156-3162

265. Honda, M., Kaneko, S., Matsushita, E., Kobayashi, K., Abell, G. A., and Lemon, S. M. (2000) Gastroenterology 118, 152-162

266. Adachi, J., Mizoi, Y., Fukunaga, T., Ogawa, Y., and Imamichi, H. (1989) Alcohol Clin Exp Res 13, 601-604

267. Clemens, D. L., Forman, A., Jerrells, T. R., Sorrell, M. F., and Tuma, D. J. (2002) Hepatology 35, 1196-1204

268. Kalapos, M. P. (1999) Med Hypotheses 53, 236-242 269. Ye, J. (2007) PLoS Pathog 3, e108 270. Sagan, S. M., Rouleau, Y., Leggiadro, C., Supekova, L., Schultz, P. G., Su, A.

I., and Pezacki, J. P. (2006) Biochem Cell Biol 84, 67-79 271. Gretch, D., Corey, L., Wilson, J., dela Rosa, C., Willson, R., Carithers, R., Jr.,

Busch, M., Hart, J., Sayers, M., and Han, J. (1994) J Infect Dis 169, 1219-1225 272. Hosogaya, S., Ozaki, Y., Enomoto, N., and Akahane, Y. (2006) Transl Res

148, 79-86 273. Gale, M., Jr., and Foy, E. M. (2005) Nature 436, 939-945 274. Bader, T., Fazili, J., Madhoun, M., Aston, C., Hughes, D., Rizvi, S., Seres, K.,

and Hasan, M. (2008) Am J Gastroenterol 103, 1383-1389 275. Sheikh, M. Y., Choi, J., Qadri, I., Friedman, J. E., and Sanyal, A. J. (2008)

Hepatology 47, 2127-2133 276. Badawy, A. A. B. (1977) Alcohol Alcohol. 12, 120-136 277. Adiels, M., Boren, J., Caslake, M. J., Stewart, P., Soro, A., Westerbacka, J.,

Wennberg, B., Olofsson, S. O., Packard, C., and Taskinen, M. R. (2005) Arterioscler Thromb Vasc Biol 25, 1697-1703

278. Towle, H. C. (1995) J Biol Chem 270, 23235-23238 279. Yano, M., Ikeda, M., Abe, K. I., Kawai, Y., Kuroki, M., Mori, K., Dansako,

H., Ariumi, Y., Ohkoshi, S., Aoyagi, Y., and Kato, N. (2009) Hepatology 280. Holland, J. J. (2006) Curr Top Microbiol Immunol 299, 371-401 281. Feld, J. J., and Hoofnagle, J. H. (2005) Nature 436, 967-972 282. Hadziyannis, S. J., Sette, H., Jr., Morgan, T. R., Balan, V., Diago, M.,

Marcellin, P., Ramadori, G., Bodenheimer, H., Jr., Bernstein, D., Rizzetto, M., Zeuzem, S., Pockros, P. J., Lin, A., and Ackrill, A. M. (2004) Ann Intern Med 140, 346-355

283. Hofmann, W. P., Zeuzem, S., and Sarrazin, C. (2005) J Clin Virol 32, 86-91 284. Duncan, M., and Younossi, Z. (2003) Cleve Clin J Med 70 Suppl 4, S21-26 285. Yoshioka, K., Kakumu, S., Wakita, T., Ishikawa, T., Itoh, Y., Takayanagi, M.,

Higashi, Y., Shibata, M., and Morishima, T. (1992) Hepatology 16, 293-299

237

286. Mita, E., Hayashi, N., Hagiwara, H., Ueda, K., Kanazawa, Y., Kasahara, A., Fusamoto, H., and Kamada, T. (1994) Dig Dis Sci 39, 977-982

287. Kanai, K., Kako, M., Aikawa, T., Kumada, T., Kawasaki, T., Hatahara, T., Oka, Y., Mizokami, M., Sakai, T., Iwata, K., and et al. (1995) Liver 15, 185-188

288. Pawlotsky, J. M. (2003) Curr Opin Infect Dis 16, 587-592 289. Taylor, D. R., Shi, S. T., and Lai, M. M. (2000) Microbes Infect 2, 1743-1756 290. Gale, M. J., Jr., Korth, M. J., and Katze, M. G. (1998) Clin Diagn Virol 10,

157-162 291. Martell, M., Esteban, J. I., Quer, J., Genesca, J., Weiner, A., Esteban, R.,

Guardia, J., and Gomez, J. (1992) J Virol 66, 3225-3229 292. Kurosaki, M., Enomoto, N., Marumo, F., and Sato, C. (1994) Virology 205,

161-169 293. Okamoto, H., and Mishiro, S. (1994) Intervirology 37, 68-76 294. Cristina, J. (2005) J Clin Virol 34 Suppl 2, S1-7 295. Valko, M., Izakovic, M., Mazur, M., Rhodes, C. J., and Telser, J. (2004) Mol

Cell Biochem 266, 37-56 296. Honda, S., Sugita, I., Miki, K., and Saito, I. (2004) Free Radic Res 38, 481-485 297. Burney, S., Caulfield, J. L., Niles, J. C., Wishnok, J. S., and Tannenbaum, S.

R. (1999) Mutat Res 424, 37-49 298. Machida, K., Cheng, K. T., Lai, C. K., Jeng, K. S., Sung, V. M., and Lai, M.

M. (2006) J Virol 80, 7199-7207 299. Wilson, D. M., 3rd, Sofinowski, T. M., and McNeill, D. R. (2003) Front Biosci

8, d963-981 300. Weimann, A., Belling, D., and Poulsen, H. E. (2002) Nucleic Acids Res 30, E7 301. Zhang, J., Perry, G., Smith, M. A., Robertson, D., Olson, S. J., Graham, D. G.,

and Montine, T. J. (1999) Am J Pathol 154, 1423-1429 302. Martinet, W., de Meyer, G. R., Herman, A. G., and Kockx, M. M. (2004) Eur J

Clin Invest 34, 323-327 303. Hassett, D. J., Bean, K., Biswas, G., and Cohen, M. S. (1989) Free Radic Res

Commun 7, 83-87 304. Jezowska-Bojczuk, M., Szczepanik, W., Lesniak, W., Ciesiolka, J.,

Wrzesinski, J., and Bal, W. (2002) Eur J Biochem 269, 5547-5556 305. Ohnishi, K., Matsuo, S., Matsutani, K., Itahashi, M., Kakihara, K., Suzuki, K.,

Ito, S., and Fujiwara, K. (1996) Am J Gastroenterol 91, 1374-1379 306. Barbaro, G., Di Lorenzo, G., Soldini, M., Parrotto, S., Bellomo, G., Belloni,

G., Grisorio, B., and Barbarini, G. (1996) Am J Gastroenterol 91, 2569-2573 307. Fong, T. L., Han, S. H., Tsai, N. C., Morgan, T. R., Mizokami, M., Qian, D.,

Phan, C., Goad, K., and Redeker, A. G. (1998) J Hepatol 28, 369-374 308. Fargion, S., Fracanzani, A. L., Sampietro, M., Molteni, V., Boldorini, R.,

Mattioli, M., Cesana, B., Lunghi, G., Piperno, A., Valsecchi, C., and Fiorelli, G. (1997) Eur J Gastroenterol Hepatol 9, 497-503

309. Siegmund, S. V., and Brenner, D. A. (2005) Alcohol Clin Exp Res 29, 102S-109S

238

310. Wong, R. H., Yeh, C. Y., Hsueh, Y. M., Wang, J. D., Lei, Y. C., and Cheng, T. J. (2003) Mutat Res 535, 181-186

311. Griffith, O. W., and Meister, A. (1979) J Biol Chem 254, 7558-7560 312. Brakmann, S., and Grzeszik, S. (2001) Chembiochem 2, 212-219 313. Sooknanan, R., Howes, M., Read, L., and Malek, L. T. (1994) Biotechniques

17, 1077-1080, 1083-1075 314. Malet, I., Belnard, M., Agut, H., and Cahour, A. (2003) J Virol Methods 109,

161-170 315. Huang, Y., Staschke, K., De Francesco, R., and Tan, S. L. (2007) Virology

364, 1-9 316. Pawlotsky, J. M., Germanidis, G., Neumann, A. U., Pellerin, M., Frainais, P.

O., and Dhumeaux, D. (1998) J Virol 72, 2795-2805 317. Rispeter, K., Lu, M., Behrens, S. E., Fumiko, C., Yoshida, T., and Roggendorf,

M. (2000) Virus Genes 21, 179-188 318. Enomoto, N., Sakuma, I., Asahina, Y., Kurosaki, M., Murakami, T.,

Yamamoto, C., Ogura, Y., Izumi, N., Marumo, F., and Sato, C. (1996) N Engl J Med 334, 77-81

319. Chayama, K., Tsubota, A., Kobayashi, M., Okamoto, K., Hashimoto, M., Miyano, Y., Koike, H., Kobayashi, M., Koida, I., Arase, Y., Saitoh, S., Suzuki, Y., Murashima, N., Ikeda, K., and Kumada, H. (1997) Hepatology 25, 745-749

320. Kurosaki, M., Enomoto, N., Murakami, T., Sakuma, I., Asahina, Y., Yamamoto, C., Ikeda, T., Tozuka, S., Izumi, N., Marumo, F., and Sato, C. (1997) Hepatology 25, 750-753

321. Tellinghuisen, T. L., Foss, K. L., Treadaway, J. C., and Rice, C. M. (2008) J Virol 82, 1073-1083

322. Smith, D. B., McAllister, J., Casino, C., and Simmonds, P. (1997) J Gen Virol 78 ( Pt 7), 1511-1519

323. Shan, X., Tashiro, H., and Lin, C. L. (2003) J Neurosci 23, 4913-4921 324. deRojas-Walker, T., Tamir, S., Ji, H., Wishnok, J. S., and Tannenbaum, S. R.

(1995) Chem Res Toxicol 8, 473-477 325. Meister, A., and Anderson, M. E. (1983) Annu Rev Biochem 52, 711-760 326. de Mochel, N. S., Seronello, S., Wang, S. H., Ito, C., Zheng, J. X., Liang, T. J.,

Lambeth, J. D., and Choi, J. (2010) Hepatology 52, 47-59 327. Boudreau, H. E., Emerson, S. U., Korzeniowska, A., Jendrysik, M. A., and

Leto, T. L. (2009) J Virol 83, 12934-12946 328. Piccoli, C., Scrima, R., Quarato, G., D'Aprile, A., Ripoli, M., Lecce, L.,

Boffoli, D., Moradpour, D., and Capitanio, N. (2007) Hepatology 46, 58-65 329. Korber, B. (2000) HIV Signature and Sequence Variation Analysis. in

Computational Analysis of HIV Molecular Sequences (Learn, A. G. R. a. G. H. ed.), Kluwer Academic Publishers, Dordrecht, Netherlands. pp 55-72

330. Hennet, T., Peterhans, E., and Stocker, R. (1992) J Gen Virol 73 ( Pt 1), 39-46 331. Eck, H. P., Gmunder, H., Hartmann, M., Petzoldt, D., Daniel, V., and Droge,

W. (1989) Biol Chem Hoppe Seyler 370, 101-108 332. Pace, G. W., and Leaf, C. D. (1995) Free Radic Biol Med 19, 523-528

239

333. Baruchel, S., and Wainberg, M. A. (1992) J Leukoc Biol 52, 111-114 334. Ciriolo, M. R., Palamara, A. T., Incerpi, S., Lafavia, E., Bue, M. C., De Vito,

P., Garaci, E., and Rotilio, G. (1997) J Biol Chem 272, 2700-2708 335. Beck, M. A., Kolbeck, P. C., Rohr, L. H., Shi, Q., Morris, V. C., and Levander,

O. A. (1994) J Med Virol 43, 166-170 336. Beck, M. A., Levander, O. A., and Handy, J. (2003) J Nutr 133, 1463S-1467S 337. Beck, M. A., Nelson, H. K., Shi, Q., Van Dael, P., Schiffrin, E. J., Blum, S.,

Barclay, D., and Levander, O. A. (2001) Faseb J 15, 1481-1483 338. Nelson, H. K., Shi, Q., Van Dael, P., Schiffrin, E. J., Blum, S., Barclay, D.,

Levander, O. A., and Beck, M. A. (2001) Faseb J 15, 1846-1848 339. Akaike, T., Fujii, S., Kato, A., Yoshitake, J., Miyamoto, Y., Sawa, T.,

Okamoto, S., Suga, M., Asakawa, M., Nagai, Y., and Maeda, H. (2000) Faseb J 14, 1447-1454

340. Akaike, T. (2001) Rev Med Virol 11, 87-101 341. Peterhans, E. (1997) Biol Trace Elem Res 56, 107-116 342. Adachi, J., Mizoi, Y., Fukunaga, T., Ogawa, Y., Ueno, Y., and Imamichi, H.

(1991) J Stud Alcohol 52, 448-453 343. Zhang, X., Li, S. Y., Brown, R. A., and Ren, J. (2004) Alcohol 32, 175-186 344. Seronello, S., Ito, C., Wakita, T., and Choi, J. (2010) J Biol Chem 285, 845-

854 345. Faraonio, R., Vergara, P., Di Marzo, D., Pierantoni, M. G., Napolitano, M.,

Russo, T., and Cimino, F. (2006) J Biol Chem 281, 39776-39784 346. Ogata, N., Alter, H. J., Miller, R. H., and Purcell, R. H. (1991) Proc Natl Acad

Sci U S A 88, 3392-3396 347. Kong, Q., and Lin, C. L. (2010) Cell Mol Life Sci 67, 1817-1829 348. Kamiya, H., Suzuki, A., Yamaguchi, Y., Handa, H., and Harashima, H. (2009)

Free Radic Biol Med 46, 1703-1707 349. Bebenek, K., Boyer, J. C., and Kunkel, T. A. (1999) Mutat Res 429, 149-158 350. Freistadt, M. S., Vaccaro, J. A., and Eberle, K. E. (2007) Virol J 4, 44 351. Wiseman, H., and Halliwell, B. (1996) Biochem J 313 ( Pt 1), 17-29 352. Vo, N. V., Tuler, J. R., and Lai, M. M. (2004) Biochemistry 43, 10579-10591 353. Waris, G., Sarker, S., and Siddiqui, A. (2004) RNA 10, 321-329 354. Herrera, M., Garcia-Arriaza, J., Pariente, N., Escarmis, C., and Domingo, E.

(2007) PLoS Pathog 3, e53 355. Sanz-Ramos, M., Diaz-San Segundo, F., Escarmis, C., Domingo, E., and

Sevilla, N. (2008) J Virol 82, 10465-10476 356. Beloqui, O., Prieto, J., Suarez, M., Gil, B., Qian, C. H., Garcia, N., and

Civeira, M. P. (1993) J Interferon Res 13, 279-282 357. Macdonald, A., and Harris, M. (2004) J Gen Virol 85, 2485-2502 358. Pawlotsky, J. M., and Germanidis, G. (1999) J Viral Hepat 6, 343-356 359. Reyes, G. R. (2002) J Biomed Sci 9, 187-197 360. Hoek, J. B., and Pastorino, J. G. (2004) Semin Liver Dis 24, 257-272 361. Dal Pero, F., Di Maira, G., Marin, O., Bortoletto, G., Pinna, L. A., Alberti, A.,

Ruzzene, M., and Gerotto, M. (2007) J Hepatol 47, 768-776

240

362. Appel, N., Pietschmann, T., and Bartenschlager, R. (2005) J Virol 79, 3187-3194

363. Tellinghuisen, T. L., Foss, K. L., and Treadaway, J. (2008) PLoS Pathog 4, e1000032

364. McCormick, C. J., Brown, D., Griffin, S., Challinor, L., Rowlands, D. J., and Harris, M. (2006) J Gen Virol 87, 93-102

365. Huang, L., Hwang, J., Sharma, S. D., Hargittai, M. R., Chen, Y., Arnold, J. J., Raney, K. D., and Cameron, C. E. (2005) J Biol Chem 280, 36417-36428

366. Chen, Y. C., Su, W. C., Huang, J. Y., Chao, T. C., Jeng, K. S., Machida, K., and Lai, M. M. (2010) J Virol 84, 7983-7993

367. Reed, K. E., Xu, J., and Rice, C. M. (1997) J Virol 71, 7187-7197 368. Hirota, M., Satoh, S., Asabe, S., Kohara, M., Tsukiyama-Kohara, K., Kato, N.,

Hijikata, M., and Shimotohno, K. (1999) Virology 257, 130-137 369. Kaul, A., Woerz, I., Meuleman, P., Leroux-Roels, G., and Bartenschlager, R.

(2007) J Virol 81, 13168-13179 370. Chevaliez, S., Brillet, R., Lazaro, E., Hezode, C., and Pawlotsky, J. M. (2007)

J Virol 81, 7732-7741 371. Donlin, M. J., Cannon, N. A., Yao, E., Li, J., Wahed, A., Taylor, M. W., Belle,

S. H., Di Bisceglie, A. M., Aurora, R., and Tavis, J. E. (2007) J Virol 81, 8211-8224

372. El-Shamy, A., Sasayama, M., Nagano-Fujii, M., Sasase, N., Imoto, S., Kim, S. R., and Hotta, H. (2007) Microbiol Immunol 51, 471-482

373. Kuntzen, T., Timm, J., Berical, A., Lennon, N., Berlin, A. M., Young, S. K., Lee, B., Heckerman, D., Carlson, J., Reyor, L. L., Kleyman, M., McMahon, C. M., Birch, C., Schulze Zur Wiesch, J., Ledlie, T., Koehrsen, M., Kodira, C., Roberts, A. D., Lauer, G. M., Rosen, H. R., Bihl, F., Cerny, A., Spengler, U., Liu, Z., Kim, A. Y., Xing, Y., Schneidewind, A., Madey, M. A., Fleckenstein, J. F., Park, V. M., Galagan, J. E., Nusbaum, C., Walker, B. D., Lake-Bakaar, G. V., Daar, E. S., Jacobson, I. M., Gomperts, E. D., Edlin, B. R., Donfield, S. M., Chung, R. T., Talal, A. H., Marion, T., Birren, B. W., Henn, M. R., and Allen, T. M. (2008) Hepatology 48, 1769-1778

374. Nagayama, K., Kurosaki, M., Enomoto, N., Miyasaka, Y., Marumo, F., and Sato, C. (2000) Hepatology 31, 745-750

375. Nousbaum, J., Polyak, S. J., Ray, S. C., Sullivan, D. G., Larson, A. M., Carithers, R. L., Jr., and Gretch, D. R. (2000) J Virol 74, 9028-9038

376. Veillon, P., Payan, C., Gaudy, C., Goudeau, A., and Lunel, F. (2004) Pathol Biol (Paris) 52, 505-510

377. Veillon, P., Payan, C., Le Guillou-Guillemette, H., Gaudy, C., and Lunel, F. (2007) World J Gastroenterol 13, 1195-1203

378. Kumthip, K., Pantip, C., Chusri, P., Thongsawat, S., O'Brien, A., Nelson, K. E., and Maneekarn, N. (2010) J Viral Hepat

379. Sarrazin, C., Herrmann, E., Bruch, K., and Zeuzem, S. (2002) J Virol 76, 11079-11090

241

380. Jain, M. K., Yuan, H. J., Adams-Huet, B., Reeck, A., Shelton, J., Attar, N., Zhang, S., Neumann, A. U., Carney, D. S., Gale, M., Jr., and Lee, W. M. (2009) J Infect Dis 200, 866-876

381. Nagayama, K., Kurosaki, M., Enomoto, N., Maekawa, S. Y., Miyasaka, Y., Tazawa, J., Izumi, N., Marumo, F., and Sato, C. (1999) Virology 263, 244-253

382. Piodi, A., Chouteau, P., Lerat, H., Hezode, C., and Pawlotsky, J. M. (2008) Hepatology 48, 16-27

383. Yagi, S., Mori, K., Tanaka, E., Matsumoto, A., Sunaga, F., Kiyosawa, K., and Yamaguchi, K. (2005) J Med Virol 77, 399-413

384. Polyak, S. J., Khabar, K. S., Paschal, D. M., Ezelle, H. J., Duverlie, G., Barber, G. N., Levy, D. E., Mukaida, N., and Gretch, D. R. (2001) J Virol 75, 6095-6106

385. Trowbridge, R., and Gowans, E. J. (1998) Arch Virol 143, 501-511 386. Yildiz, E., Oztan, A., Sar, F., Pinarbasi, E., Cetin-Atalay, R., Akkiz, H., and

Ozturk, M. (2002) Virus Genes 25, 169-177 387. Mao, H. X., Hu, Y. W., Wu, Y., Lan, S. Y., and Yuan, Z. H. (2004) Zhonghua

Shi Yan He Lin Chuang Bing Du Xue Za Zhi 18, 122-126 388. Takahashi, K., Iwata, K., Matsumoto, M., Matsumoto, H., Nakao, K.,

Hatahara, T., Ohta, Y., Kanai, K., Maruo, H., Baba, K., Hijikata, M., and Mishiro, S. (2001) Hepatol Res 20, 161-171

389. Turrini, P., Sasso, R., Germoni, S., Marcucci, I., Celluci, A., Di Marco, A., Marra, E., Paonessa, G., Eutropi, A., Laufer, R., Migliaccio, G., and Padron, J. (2006) Transplant Proc 38, 1181-1184

390. Berry, M. N., Gregory, R. B., Grivell, A. R., Phillips, J. W., and Schon, A. (1994) Eur J Biochem 225, 557-564

391. Thurman, R. G. (1977) Fed Proc 36, 1640-1646 392. Kapadia, S. B., and Chisari, F. V. (2005) Proc Natl Acad Sci U S A 102, 2561-

2566 393. Ogawa, K., Hishiki, T., Shimizu, Y., Funami, K., Sugiyama, K., Miyanari, Y.,

and Shimotohno, K. (2009) Proc Jpn Acad Ser B Phys Biol Sci 85, 217-228 394. Wang, C., Gale, M., Jr., Keller, B. C., Huang, H., Brown, M. S., Goldstein, J.

L., and Ye, J. (2005) Mol Cell 18, 425-434 395. Ikeda, M., and Kato, N. (2007) Adv Drug Deliv Rev 59, 1277-1289 396. Khattab, M. A. (2009) World J Gastroenterol 15, 3472-3479 397. Goldwasser, J., Cohen, P. Y., Yang, E., Balaguer, P., Yarmush, M. L., and

Nahmias, Y. (2010) PLoS One 5 398. Herker, E., Harris, C., Hernandez, C., Carpentier, A., Kaehlcke, K., Rosenberg,

A. R., Farese, R. V., Jr., and Ott, M. (2010) Nat Med 399. Mankouri, J., Tedbury, P. R., Gretton, S., Hughes, M. E., Griffin, S. D., Dallas,

M. L., Green, K. A., Hardie, D. G., Peers, C., and Harris, M. (2010) Proc Natl Acad Sci U S A 107, 11549-11554

400. Loguercio, C., Federico, A., Masarone, M., Torella, R., Blanco Cdel, V., and Persico, M. (2008) Am J Gastroenterol 103, 3159-3166

242

401. Waris, G., Felmlee, D. J., Negro, F., and Siddiqui, A. (2007) J Virol 81, 8122-8130

402. Fiala, E. S., Conaway, C. C., and Mathis, J. E. (1989) Cancer Res 49, 5518-5522

403. Cooke, M. S., Evans, M. D., Dizdaroglu, M., and Lunec, J. (2003) Faseb J 17, 1195-1214

404. Yu, T. W., and Anderson, D. (1997) Mutat Res 379, 201-210 405. Torres-Puente, M., Cuevas, J. M., Jimenez-Hernandez, N., Bracho, M. A.,

Garcia-Robles, I., Wrobel, B., Carnicer, F., Del Olmo, J., Ortega, E., Moya, A., and Gonzalez-Candelas, F. (2008) J Viral Hepat 15, 188-199

406. Puig-Basagoiti, F., Forns, X., Furcic, I., Ampurdanes, S., Gimenez-Barcons, M., Franco, S., Sanchez-Tapias, J. M., and Saiz, J. C. (2005) J Gen Virol 86, 1067-1075

407. Xu, Z., Fan, X., Xu, Y., and Di Bisceglie, A. M. (2008) J Virol 82, 9417-9424 408. Itakura, J., Nagayama, K., Enomoto, N., Hamano, K., Sakamoto, N., Fanning,

L. J., Kenny-Walsh, E., Shanahan, F., and Watanabe, M. (2005) J Viral Hepat 12, 594-603

409. Thomson, M., Nascimbeni, M., Gonzales, S., Murthy, K. K., Rehermann, B., and Liang, T. J. (2001) Gastroenterology 121, 1226-1233

410. Kohashi, T., Maekawa, S., Sakamoto, N., Kurosaki, M., Watanabe, H., Tanabe, Y., Chen, C. H., Kanazawa, N., Nakagawa, M., Kakinuma, S., Yamashiro, T., Itsui, Y., Koyama, T., Enomoto, N., and Watanabe, M. (2006) J Viral Hepat 13, 582-590

411. Bungyoku, Y., Shoji, I., Makine, T., Adachi, T., Hayashida, K., Nagano-Fujii, M., Ide, Y. H., Deng, L., and Hotta, H. (2009) J Gen Virol 90, 1681-1691

412. Hwang, D. R., Lai, H. Y., Chang, M. L., Hsu, J. T., and Yeh, C. T. (2005) J Virol Methods 129, 170-177

413. Sumpter, R., Jr., Wang, C., Foy, E., Loo, Y. M., and Gale, M., Jr. (2004) J Virol 78, 11591-11604

414. Hayashi, K., Katano, Y., Honda, T., Ishigami, M., Itoh, A., Hirooka, Y., Nakano, I., Urano, F., Yoshioka, K., Toyoda, H., Kumada, T., and Goto, H. (2009) J Med Virol 81, 459-466

415. Noguchi, T., Otsubaki, T., Ando, I., Ogura, N., Ikeda, S., and Shimotohno, K. (2008) Virology 375, 424-432

416. Cuevas, J. M., Torres-Puente, M., Jimenez-Hernandez, N., Bracho, M. A., Garcia-Robles, I., Carnicer, F., Olmo, J. D., Ortega, E., Gonzalez-Candelas, F., and Moya, A. (2009) J Med Virol 81, 650-656

417. Taguchi, T., Nagano-Fujii, M., Akutsu, M., Kadoya, H., Ohgimoto, S., Ishido, S., and Hotta, H. (2004) J Gen Virol 85, 959-969

418. Pfeiffer, J. K., and Kirkegaard, K. (2005) J Virol 79, 2346-2355 419. de Queiroz, A. T., Maracaja-Coutinho, V., Jardim, A. C., Rahal, P., de

Carvalho-Mello, I. M., and Matioli, S. R. (2010) J Viral Hepat

243

420. Chatterji, U., Lim, P., Bobardt, M. D., Wieland, S., Cordek, D. G., Vuagniaux, G., Chisari, F., Cameron, C. E., Targett-Adams, P., Parkinson, T., and Gallay, P. A. (2010) J Hepatol 53, 50-56

421. Fernandes, F., Ansari, I. U., and Striker, R. (2010) PLoS One 5, e9815 422. Fernandes, F., Poole, D. S., Hoover, S., Middleton, R., Andrei, A. C., Gerstner,

J., and Striker, R. (2007) Hepatology 46, 1026-1033 423. Yang, F., Robotham, J. M., Grise, H., Frausto, S., Madan, V., Zayas, M.,

Bartenschlager, R., Robinson, M., Greenstein, A. E., Nag, A., Logan, T. M., Bienkiewicz, E., and Tang, H. (2010) PLoS Pathog 6

424. Yano, M., Ikeda, M., Abe, K., Kawai, Y., Kuroki, M., Mori, K., Dansako, H., Ariumi, Y., Ohkoshi, S., Aoyagi, Y., and Kato, N. (2009) Hepatology 50, 678-688

425. De Franceschi, L., Fattovich, G., Turrini, F., Ayi, K., Brugnara, C., Manzato, F., Noventa, F., Stanzial, A. M., Solero, P., and Corrocher, R. (2000) Hepatology 31, 997-1004

426. Koike, K., Tsutsumi, T., Miyoshi, H., Shinzawa, S., Shintani, Y., Fujie, H., Yotsuyanagi, H., and Moriya, K. (2008) J Gastroenterol Hepatol 23 Suppl 1, S87-91

427. Gimenez-Barcons, M., Franco, S., Suarez, Y., Forns, X., Ampurdanes, S., Puig-Basagoiti, F., Sanchez-Fueyo, A., Barrera, J. M., Llovet, J. M., Bruix, J., Sanchez-Tapias, J. M., Rodes, J., and Saiz, J. C. (2001) Hepatology 34, 158-167

UNIVERSITY OF CALIFORNIA, MERCED Effects of Ethanol and ...

Documents

Transcript of UNIVERSITY OF CALIFORNIA, MERCED Effects of Ethanol and ...