Journal of Medical Virology

Molecular Characterization of Hepatitis B Virus(HBV) Strains Circulating in the Northern Coast ofthe Persian Gulf and Its Comparison withWorldwide Distribution of HBV Subgenotype D1

Mahmoud Reza Pourkarim,1,2 Valentijn Vergote,1 Samad Amini-Bavil-Olyaee,3 Zohre Sharifi,2

Steven Sijmons,1 Philippe Lemey,1 Piet Maes,1 Seyed Moayed Alavian,4 and Marc Van Ranst1*1Department of Microbiology and Immunology, Laboratory of Clinical and Epidemiological Virology, Rega Institutefor Medical Research, KU Leuven, Leuven, Belgium2Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran,Iran3Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California,Harlyne J. Norris Cancer Research Tower, Los Angeles, California4Middle East Liver Disease Clinics, Tehran, Iran

Iran is a large country that covers the northerncoast of the Persian Gulf. Iranian residents ofthis coastal region interact closely with peoplefrom neighboring countries because of histori-cal and cultural relationships, as well as eco-nomic activities. In addition, the inhabitants ofthis border region have experienced severalwars, which have affected public health infra-structures. This study characterized for the firsttime, the evolution of the full-length genomeof HBV strains in asymptomatic carrier patientsliving in this particular region. In addition, thisstudy was compared and complemented by acomprehensive evolutionary analysis of theworldwide geographical distribution of HBVsubgenotype D1. Evolutionary analysis demon-strates that patients living in the northern coastof the Persian Gulf are mainly infected withHBV subgenotype D1, subtype ayw2. Specificmutations related to advanced liver diseasewere found more frequently in these strainscompared to other strains isolated fromasymptomatic carriers from other regions ofIran. This global comprehensive analysisshowed that HBV subgenotype D1 strains havea worldwide distribution and that human mo-bility and immigration had a large impact ondispersal of HBV subgenotype D1, subtypeayw2 in Middle Eastern countries such as Iran,Syria, and Turkey. In addition to associationof subtype ayw2 with subgenotype D1, it wasdemonstrated that other HBV subtypes likeadw2, ayw1, and ayw3 are associated withHBV subgenotype D1 in different regions of

the world. This study also revealed a remark-able distribution of subgenotype D1, subtypeayw4 although this particular subtype is asso-ciated with subgenotype D4 of HBV in Europe-an countries. J. Med. Virol.# 2014 Wiley Periodicals, Inc.

KEY WORDS: hepatitis B virus; full-lengthgenome; Persian Gulf; subge-notype; subtype; Iran

INTRODUCTION

Hepatitis B virus (HBV) infection is a currentpublic health problem. Although a safe and effectivevaccine against HBV is available, the World HealthOrganization (WHO) estimates around five millionnew cases of HBV infection occur annually [Lavanchy,2005]. HBV is distributed worldwide and HBV infec-tion is geographically categorized into three differentregions of high, intermediate, and low endemicityaccording to HBsAg seroprevalence [Shepard et al.,

Grant sponsor: Fonds voor Wetenschappelijk Onderzoek(FWO) Vlaanderen

�Correspondence to: Marc Van Ranst, Laboratory of ClinicalVirology, Rega Institute for Medical Research, University ofLeuven, Minderbroedersstraat 10, BE3000 Leuven, Belgium.E-mail: marc.vanranst@uzleuven.be

Accepted 24 November 2013

DOI 10.1002/jmv.23864Published online in Wiley Online Library(wileyonlinelibrary.com).

�C 2014 WILEY PERIODICALS, INC.

2006]. Modern human mobility, especially immigra-tion, is altering the epidemiological profile of HBVprevalence [Pourkarim et al., 2010b,c, 2011a,b]. Waralso appears to have a remarkable impact on theepidemiology of HBV [Reuben, 2002]. The geneticdiversity of HBV has been classified into eight well-established genotypes (A–H). Recently two tentativegenotypes (I and J) were proposed [Tran et al., 2008;Tatematsu et al., 2009]. HBV is classified into approx-imately forty subgenotypes and nine subtypes (orserotypes) that display different geographical distribu-tions [Mulyanto et al., 2012]. Of note, the relationshipbetween particular subgenotypes and subtypes hasbeen demonstrated [Norder et al., 1994, 2004; Kram-vis et al., 2008]. Some genotypes and subgenotypesare restricted to particular geographical regions whileothers display a worldwide distribution. Genotype Dis the most widely distributed genotype throughoutthe world and is segregated into at least sevendifferent subgenotypes [Zehender et al., 2012a]. Thesediverse subgenotypes, associated with particular sub-types, are dispersed in different continents [Amini-Bavil-Olyaee et al., 2005; Lusida et al., 2008; Talloet al., 2008; Meldal et al., 2009; Kitab et al., 2011;Zehender et al., 2012b].As a large country located in the Middle East, Iran

covers the northern coast of the Persian Gulf andOman Sea and is home to a multi-ethnic population.Because of historical and cultural relationships,shared language, as well as economic activitiesincluding fishing, sailing, traveling by sea, and oil

trading, Iranian residents of this coastal regioninteract closely with people from neighboring coun-tries like Kuwait, Bahrain, Qatar, U.A.E, Iraq,Pakistan, and Afghanistan (Fig. 1). In addition, theinhabitants of this border region have experiencedseveral wars during the last 25 years, which haveaffected both economic and public health infrastruc-tures [Assarehzadegan et al., 2008]. These uniquefeatures make this region an interesting area formolecular epidemiological studies. Although someepidemiological studies on HBV in Iran are available,no specific data has been generated by systematicmolecular epidemiology studies on the circulation ofHBV in this strategic and geopolitical border. To ourknowledge, this is the first study that characterizesthe evolution of the full-length genome of HBVstrains in asymptomatic carrier patients living in thisparticular region. In addition, this study in thePersian Gulf was complemented by a comprehensiveevolutionary analysis of the worldwide geographicaldistribution of HBV subgenotype D1 [Fathimoghad-dam et al., 2011; Mohebbi et al., 2011; Omidkhodaet al., 2011; Nokhodian et al., 2012; Zahedi et al.,2012].

MATERIALS AND METHODS

Study Subjects

Twenty-one HBsAg-positive blood donors from fourcoastal provinces located in the northern coast of thePersian Gulf and Oman Sea (Khuzestan, Bushehr,

Fig. 1. Geographical map of Iran and its borders with Persian Gulf and neighboringcountries.

J. Med. Virol. DOI 10.1002/jmv

2 Pourkarim et al.

Hormozgan, and Sistan-Baluchistan) were enrolled inthis study (Fig. 1). None of them had a history ofHBV vaccination, HBV immunoglobulin, or antiviraltherapy. Demographic data were recorded for allpatients.

Serological Assays

Biochemical measurements like liver enzyme levelsof alanine transaminase (ALT) and aspartate amino-transferase (AST), as well as serological markers ofHBV infection including HBsAg, anti-HBc, HBeAg,anti-HBe, and anti-HBs, were tested (DIA.PRO Diag-nostic Bioprobes s.r.l, Milano, Italy) at the IranianBlood Transfusion Research Centre (Department ofVirology, High Institute for Research and Educationin Transfusion Medicine, Tehran, Iran). Possible co-infections with hepatitis D virus (HDV), hepatitis Cvirus (HCV), and human immunodeficiency virus(HIV) were tested using available ELISA kits.

Amplification, Sequencing andPhylogenetic Analysis

Viral DNA was extracted from sera using theQIAmp1 Viral DNA mini kit (QIAGEN, Hilden,Germany). Due to a low viral load among asymptom-atic carriers, a nested-PCR strategy was used toamplify the full-length HBV genome as describedpreviously [Pourkarim et al., 2011b]. Positive andnegative controls were included in each run of DNAextraction and amplification to avoid false positiveor false negative results. The PCR products werepurified with the QIAquick1 PCR purification kit(QIAGEN). Directional sequencing reactions wereperformed using the ABI PRISM Big Dye1 Termina-tor cycle sequencing kit (Applied Biosystems, FosterCity, CA) on an ABI PRISM 3130 automatedsequencer. Primer walking was performed and aconsensus sequence was generated by assemblinglarge whole genome segments using the SeqMan II1

software (DNAStar, Madison, WI). Possible genomicrecombination was investigated using SimPlot soft-ware, version 3.5.1 [Lole et al., 1999]. Phylogeneticand molecular evolutionary analyses were conductedusing the MEGA software, version 5, [Tamuraet al., 2007] in which the mean nucleotide divergence(mean�SD) over the complete genome sequencenucleotide was calculated using the Kimura 2-parameter model [Tamura et al., 2007]. Sequenceswere submitted to GenBank under accession num-bers KF471640 to KF471660.

HBV full-length genome sequences from this studyand from GenBank (as references) were aligned usingMAFFT software version 6.717b with standardsettings [Katoh and Toh, 2008]. A neighbor joiningtree was constructed under the Kimura 2-parametersubstitution model using the MEGA software, version5 [Tamura et al., 2007]. A bootstrap procedure using1,000 replicates was used to assess the robustness ofthe pairwise genetic distance comparisons and theclustering in the phylogenetic tree [Zharkikh andLi, 1995]. Only full-length genomes of HBV wereanalyzed to gain accurate subgenotyping of HBV[Pourkarim et al., 2009, 2010a; Pourkarim andVan Ranst, 2011; Amini-Bavil-Olyaee et al., 2012].In order to provide a global comparison of HBVsubgenotype D1, the strains identified in this studywere compared to all reported full-length genomes ofHBV genotype D strains submitted to GenBankbefore April 2013. Also, some other reference sequen-ces representing different HBV genotypes were usedin this analysis. HBV subtypes were assigned basedon the nucleotide sequences coding for the aminoacids in the surface antigen region [Purdy et al.,2007]. According to this algorithm, the presence ofArg122, Lys160, Pro127, and Ala159 is considered tobe subtype ayw1; presence of non-Ala159 and non-Ser140 and non-Ala159 together with Ser140 basedon the same algorithm, are considered as ayw2 andayw4, respectively. Also, detection of Arg122, Lys160,Pro127, Thr127, Ile/Leu127 indicate subtypes adw2,adw3, and adw4, respectively.

Statistical Analysis

All statistical analyses were performed with theSPSS software, version 17.0 (SPSS, Chicago, USA).Categorical data were compared using the Chi-squared test. The significance level was set to 0.05.

RESULTS

Serum samples were collected from 21 asymptom-atic carriers infected with HBV. All subjects weremales between 20 to 58 years old (with a mean age of32.8� 11.6 years). Table I summarizes the clinicaland serological results for the serological markers.HBV complete genome amplification and sequenc-

ing was successful for all 21 samples. The BLASTanalysis and online NCBI HBV genotyping tooldemonstrated that all sequenced HBV strains wereclassified as genotype D [Rozanov et al., 2004]. Thisresult was also confirmed by phylogenetic analysis.

TABLE I. Prevalence of HBV and HDV Serological Markers in the Studied Population

No (%)HBsAgpositive

Anti-HBcpositive

HBeAgpositive

Anti-HBepositive

Anti-HBspositive

AntiDelta-Agpositive ALT (IU/l) AST (IU/l)

21 (100%) 21 (100%) 21 (100%) 3 (14.3%) 18 (85.7%) 0 (0%) 2 (9.6%) 16.8� 4.07 16�4.8

J. Med. Virol. DOI 10.1002/jmv

Evolutionary Analysis of HBV Strains Circulating in Northern Coast of Persian Gulf 3

To provide a comprehensive evolutionary characteri-zation of strains from this study, a phylogeneticanalysis was conducted including all full-length HBVgenotype D strains submitted to GenBank (988strains). This analysis indicated that all strainsisolated from the patients living in the northern coastof the Persian Gulf belonged to HBV subgenotype D1(data not shown). The relevant subset of sequenceswith regards to isolated strains (n¼ 21) and allIranian strains (n¼ 138) as regional reference geneswere combined together with the most similar publi-cally available strains (n¼ 52) as global referencegenes for phylogenetic reconstruction (Fig. 2). Ofnote, all 21 HBV isolates together with other Iranianstrains (retrieved from GenBank, n¼ 138) were phy-logenetically interspersed with strains isolated fromregional countries (Turkey, Lebanon, Syria,Pakistan), Far East countries (India, Indonesia),African countries (Egypt, Tunisia), and Europeancountries (Latvia, Belgium, Italy).HBV subtypes for 21 isolates were assigned based

on the amino acid sequences at positions 122, 127,140, 159, and 160 of the surface antigen [Purdyet al., 2007]. This analysis revealed diverse HBVsubtypes including ayw2 (n¼ 19, 90.50%), ayw3(n¼ 1, 4.75%), and ayw4 (n¼ 1, 4.75%). Since thevarious subtypes among the Persian Gulf strainsclustered with similar subgenotype D1 strains fromother geographical regions, this study identified thesubtypes and analyzed the geographical distributionof HBV subtypes for all subgenotype D1 strainssubmitted to GenBank.According to phylogenetic tree analysis, 563 strains

(out of 988 full-length genomes with genotype Dretrieved from the GenBank) isolated from 29 coun-tries in different continents were classified as sub-genotype D1 (Table II). This analysis demonstratedfive different subtypes in 563 HBV D1 strains. Ofnote, ayw2 was identified as the most frequentsubtype (n¼ 522, 92.3%), followed by adw2 (n¼ 15,2.85%), ayw3 (n¼ 14, 2.6%), ayw4 (n¼ 7, 1.25%), andayw1 (n¼ 5, 1%), respectively.

Genetic Divergence

The genetic distance was calculated between D1strains circulating in different geographical regions(Table III). The mean percentage and standard devia-tion (mean�SD) of intra-subgenotype nucleotide dis-tance within HBV D1 was less than 4%. This studyshowed that the highest nucleotide divergences could

JF754617-TURKEY-ayw2JN040782-IRAN-ayw2

JN040777-IRAN-ayw2AB104711-EGYPT-ayw2

AB104709-EGYPT-ayw2AB674407-TURKEY-ayw2

GU456653-IRAN-ayw2GU456654-IRAN-ayw2

GU456655-IRAN-ayw2JN040774-IRAN-ayw2

X59795-ITALY-ayw2JN040784-IRAN-ayw2

JF754606-TURKEY-ayw2GU456684-IRAN-ayw2

AB104710-EGYPT-ayw2JN257176-SYRIA-ayw2JF754590-TURKEY-ayw1

JN040779-IRAN-ayw2JN642153-LEBANON-ayw2

JN040778-IRAN-ayw2TUNISIAJN040791-IRAN-ayw2

JN040793-IRAN-ayw2JN040792-IRAN-ayw2

JN040775-IRAN-ayw2JN642129-LEBANON-ayw2

AB583680-PAKISTAN-ayw2FJ349228-BELGIUM-ayw2JN040786-IRAN-ayw2

JN040788-IRAN-ayw2JN040787-IRAN-ayw2

JN040785-IRAN-ayw2JN040789-IRAN-ayw2

GQ358159-INDONESIA-ayw2JN040783-IRAN-ayw2

AB674403-TURKEY-ayw2JN040790-IRAN-ayw2

JF754588-TURKEY-ayw2GU456657-IRAN-ayw2

GU456656-IRAN-ayw2JN040757-IRAN-ayw2

JN040756-IRAN-ayw2GU456677-IRAN-ayw2

JN040750-IRAN-ayw2AJ344116-FRANCE-ayw2

GU456682-IRAN-ayw2GU456683-IRAN-ayw2PG-25-ayw2JF754599-TURKEY-ayw3

AY721608-TURKEY-ayw2AY721605-TURKEY-ayw2

JN040751-IRAN-ayw4EF103281-INDIA-ayw2

JN040794-IRAN-ayw2JN040817-IRAN-ayw2

JN040772-IRAN-ayw2JN040773-IRAN-ayw2

JN040752-IRAN-ayw2D7-FJ904424-TUNISIA-ayw2

AY161157-INDIA-ayw2JN664931-INDIA-ayw2

JN040781-IRAN-ayw2JN040780-IRAN-ayw2

PG-8-ayw2GU563549-BELGIUM-ayw2

JN040776-IRAN-ayw2GU456680-IRAN-ayw2

GU456681-IRAN-ayw2JN642136-LEBANON-ayw2

GU456679-IRAN-ayw2JN040754-IRAN-ayw2

AY161162-INDIA-ayw2AY161159-INDIA-ayw2

PG-37-ayw2JN040753-IRAN-ayw2

GU456662-IRAN-ayw2GU456665-IRAN-ayw2

GU456663-IRAN-ayw2GU456664-IRAN-ayw2GU456661-IRAN-ayw2

JN040769-IRAN-ayw2JN040771-IRAN-ayw2

JN040770-IRAN-ayw2JN040762-IRAN-ayw2

GU456676-IRAN-ayw2JN040761-IRAN-ayw2

GU456669-IRAN-ayw2JN040763-IRAN-ayw2

JN040764-IRAN-ayw2JN040765-IRAN-ayw2

PG-52-ayw4GU456668-IRAN-ayw2

PG-2-ayw2PG 4-ayw2

JN040766-IRAN-ayw2JN257173-SYRIA-ayw2GU456666-IRAN-ayw2

PG-3-ayw2PG-50-ayw2PG-18-ayw2GU456667-IRAN-ayw2

AY741795-IRAN-ayw2AY741796-IRAN-ayw2AY741794-IRAN-ayw2JN040758-IRAN-ayw2

JN040759-IRAN-ayw2GU456670-IRAN-ayw2

PG-14-ayw2GU456671-IRAN-ayw2PG-7-ayw3

PG-65-ayw2GU456658-IRAN-ayw2

GU456678-IRAN-ayw1GU456659-IRAN-ayw2

JN040760-IRAN-ayw2GU456660-IRAN-ayw2

JN257214-SYRIA-2009-ayw2JN040767-IRAN-ayw2

JN040768-IRAN-ayw2PG-66-ayw2

GU456674-IRAN-ayw2GU456675-IRAN-ayw2

GU456673-IRAN-ayw2AB674421-TURKEY-ayw2

GU456672-IRAN-ayw2JN040827-IRAN-ayw2

JN040822-IRAN-ayw2JN040823-IRAN-ayw2

JF754630-TURKEY-ayw2GU456639-IRAN-ayw2

PG-86-ayw2GU456638-IRAN-ayw2

GU456643-IRAN-ayw2AY741798-IRAN-ayw2

JN642142-LEBANON-ayw2JN040818-IRAN-ayw2

GU456650-IRAN-ayw2PG-60-ayw2

JF754603-TURKEY-ayw2PG-11-ayw2

JN040796-IRAN-ayw2JN040803-IRAN-ayw2

GU456651-IRAN-ayw2GU456652-IRAN-ayw2

JN040813-IRAN-ayw2GU456648-IRAN-ayw2

JN040814-IRAN-ayw2JN040820-IRAN-ayw4GU456642-IRAN-ayw2

JN040805-IRAN-ayw2JN040806-IRAN-ayw2

JN040804-IRAN-ayw3JN040811-IRAN-ayw2JN040810-IRAN-ayw2

GU456641-IRAN-ayw2GU456645-IRAN-ayw2

GU456646-IRAN-ayw2GU456647-IRAN-ayw2

GU456640-IRAN-ayw2GU456644-IRAN-ayw2

JN040808-IRAN-ayw2JN040815-IRAN-ayw2JN040816-IRAN-ayw2

JN040798-IRAN-ayw2JN040797-IRAN-ayw2

JN040821-IRAN-ayw2JN040800-IRAN-ayw3

JN040801-IRAN-ayw3AB222713-Uzbekistan-ayw2JN040809-IRAN-ayw2

JN040802-IRAN-ayw2JN040819-IRAN-ayw2

PG-35-ayw2JN040812-IRAN-ayw2

JN040795-IRAN-ayw2GU456649-IRAN-ayw2

JN257152-SYRIA-ayw2AY721612-TURKEY-ayw2

JN040799-IRAN-ayw2PG-32-ayw2

JN040807-IRAN-ayw2PG-31-ayw2PG-22-ayw2PG-20-ayw2JN040828--IRAN-ayw2

JN040829-IRAN-ayw2FJ349235-BELGIUM-ayw2

AY796030TURKEY-ayw2JN257191-SYRIA-ayw2

FJ349231-BELGIUM-ayw2JN257154-SYRIA-ayw2

JF754613-TURKEY-ayw2JF754586-TURKEY-ayw2

JN040830-IRAN-ayw2JN040825-IRAN-ayw2

GU456636-IRAN-ayw2GU456637-IRAN-ayw2

JN040826-IRAN-ayw2JF754633-TURKEY-ayw2

AB104712-EGYPT-ayw2JF754620-TURKEY-ayw2JN040824-IRAN-ayw2AY741797-IRAN-ayw2JN040755-IRAN-ayw2

D2-GQ477453

D3-GQ922000D4-GQ922003

D7-FJ904394D6-KF170740

D5-GQ205378E-HM363565

C-JX026887B-JX026883

G-GU563556F-JN792918

H-AB51639599

96

99 93

84

99

99

99

96

99

93

99

99

99

99

96

96

91

99

83

61

68

63

96

69

66

74

64

70

79

0.01

Fig. 2. Neighbor-joining phylogenetic tree based on HBV full-length genomes from 21 HBV-D1 Iranian strains isolated fromthe northern coast of Persian Gulf and 198 HBV-D1 referencestrains isolates from different geographical locations retrievedfrom GenBank. The HBV Persian Gulf isolates are marked by�, other Iranian strains by �, Turkish strains by ~,Egyptian strains by 5, Indian and Indonesian strains by&. The numbers at the nodes represent the bootstrapsupport values (% obtained for 1,000 replicates).

J. Med. Virol. DOI 10.1002/jmv

4 Pourkarim et al.

be observed between South African strains andEuropean strains, including Italy (2.6� 0.3%), France(2.3� 0.2%), Germany (2.2� 0.3%), and the UK(2.1� 0.2%). In contrast, the minimum nucleotidedivergence was identified between isolates fromPakistan and New Zealand (0.6� 0.1%). HBV isolatesfrom Iran (submitted previously to GenBank) and theisolates from this study were considered as separategroups in this calculation. The nucleotide divergencebetween the Iranian group and subjects from othercountries was almost the same. Interestingly, isolatesfrom Uzbekistan, Belarus, and Mongolia showed thesame nucleotide difference with the other groups(Table III).

Mutational Patterns of Persian Gulf Strains

Nucleotide deletions and insertions. In thissurvey, 2 out of 21 Persian Gulf strains showeddeletions. Strain PG-18 showed a 27-nucleotide (9amino acids) deletion in the pre-S2 region. In the PG-22 strain, a nine-nucleotide deletion was detectedbetween positions 1762 and 1770 in the core region.None of the isolates showed insertions or intra-genotype recombination.HBV S and P open reading frames (ORFs).

Table IV illustrates amino acid substitutions in T-cellepitopes and B-cell epitopes from HBV pre-S1 andpre-S2 regions in the isolated strains. Furthermore,

several medically important mutations were detectedin the major hydrophilic region (MHR) of the HBsAgof the studied strains (Fig. 3). In total, 9 out of 21strains (42.8%) carried 11 substitutions in the MHRregion. Of note, eight strains showed a single muta-tion (88.8%) and one strain carried four mutations(11.2%) at the MHR region. Three out of elevenvariations were detected within the “a determinant”motif. There was no mutational change in the cyste-ine residues of the disulphide bonds in the “adeterminant” domain. Interestingly, one strain car-ried the sP120S mutation, which is one of the mostrelevant substitutions in the HBsAg with respect tovaccines and diagnostic assays. In this study, twostop codons were detected in the HBsAg: one strainharbored a stop codon mutation at the position 163(PG-32) and the other at position 172 (PG-50).Evaluation of the P ORFs revealed two naı̈ve-strainscarrying adefovir resistant mutations (rtA181V/T). Incase of rtA181T mutation at the P ORF, a stop codonmight be introduced at the sW172� in the S ORF.C and X ORFs. In this study, the mutation

frequency in the core region was relatively high; 10out of 21 strains (47.6%) contained a G1896A muta-tion that introduced a stop codon resulting in prema-ture precore protein and HBeAg-negativity. Threeout of 21 strains (14.3%) harbored a mutation atG1899A in the precore region. Also, this high rate ofmutation was observed in the basal core promoter(BCP) region. Of note, more than 50% of strains (12/21) showed the most common double BCP mutation(A1762T and G1764A/T) and 29% of strains (6/21)carried another BCP double mutation (G1764A/T andC1766G/T). Interestingly, in one strain, a triplemutation at the BCP region was detected (A1762G,G1764C, and C1766A). Also, a strain that carriedboth G1896A and G1899A had an additional C1766Gmutation. In the present study, no T1768A mutationswere identified (Fig. 4). There were no significantdifferences in liver enzyme levels between patientsinfected by wild type or precore and basal coremutant variants (P> 0.05).Because of the overlap between the HBV X and

Core gene, three hotspot substitutions (xI127, xK130,and xV131) were observed in several cases. In detail,the mutations A1752T/C, T1753C/G/A, A1762T/G,G1764A/T, and C1766G/T in the Core gene led toxI127T/N/S/F/M, xK130M/C/E/Q, and xV131I/L/Calterations in the X protein, respectively. Five strainsproduced wild type HBV X protein (Table V). Inter-estingly, in two strains (PG-2 and P-G 4) thexI127SþxK130CþxV131L and xI127FþxK130MþxV131C combination of mutations were observed as aunique arrangement. In addition, one strain (P-G 20)with a stop codon at xG135� was identified.

DISCUSSION

Iran is a vast country with a multi-ethnic popula-tion. It is located in the Middle East region and

TABLE II. Geographical Distribution of HBV SubtypesWithin 563 Reported D1 Subgenotype of HBV in GenBank

Subtypecountry

ayw1No.

ayw2No.

ayw3No.

ayw4No.

adw2No.

TotalNo.

Belarus 3 3Belgium 14 14China 28 1 29Egypt 4 4France 2 1 3Gabon 1 1Germany 1 1 2India 44 6 50Indonesia 1 1Iran 1 130 3 2 2 138Iran (this study) 19 1 1 21Italy 10 10Japan 1 1Kazakhstan 1 1Lebanon 33 2 35Mongolia 12 12New Zealand 42 42Pakistan 2 2Poland 1 1 2Russia 1 1Serbia 1 1South Africa 3 3Spain 1 1Sweden 4 4Syria 62 2 1 65Tunisia 1 15 16Turkey 3 79 3 1 4 90United States 2 2England 2 2Uzbekistan 6 1 7Total 5 522 14 7 15 563

J. Med. Virol. DOI 10.1002/jmv

Evolutionary Analysis of HBV Strains Circulating in Northern Coast of Persian Gulf 5

TABLE

III.

Mea

nPercentageof

Nucleotidedivergen

ces(M

ean�SD;Over

theEntire

Gen

ome)

ofallRep

ortedHBV

Subgen

otypeD1Submittedinto

theGen

Bank

from

DifferentCou

ntriesand21StrainsBelon

gto

theCurren

tStudy(M

ark

edasIran� )

SD

Mea

n

Egypt

Iran �

New Zealand

Turkey

China

Lebanon

India

Syria

Iran

France

Pakistan

Belgium

Poland

S. Africa

Tunisia

Uzbekistan

Belarus

Mongolia

United States

Italy

Sweden

Germany

England

Egypt

0.2

0.2

0.2

0.2

0.2

0.2

0.1

0.2

0.2

0.2

0.2

0.2

0.3

0.2

0.2

0.2

0.1

0.2

0.2

0.2

0.2

0.2

Iran�

1.6

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.2

0.1

0.1

0.2

0.3

0.2

0.1

0.1

0.1

0.2

0.2

0.2

0.2

0.2

New

Zea

land

1.2

1.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.2

0.3

0.1

0.1

0.1

0.1

0.2

0.2

0.2

0.2

0.2

Turk

ey1.5

1.5

1.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.3

0.1

0.1

0.1

0.1

0.2

0.2

0.2

0.2

0.2

China

1.3

1.3

0.8

1.3

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.3

0.1

0.1

0.1

0.1

0.2

0.2

0.2

0.2

0.2

Leb

anon

1.5

1.4

1.1

1.4

1.2

0.1

0.1

0.1

0.2

0.1

0.1

0.2

0.3

0.1

0.1

0.1

0.1

0.2

0.2

0.2

0.2

0.2

India

1.5

1.4

1.1

1.5

1.2

1.4

0.1

0.1

0.2

0.1

0.1

0.2

0.3

0.1

0.1

0.1

0.1

0.2

0.2

0.2

0.2

0.2

Syria

1.3

1.3

0.9

1.3

1.1

1.2

1.3

0.1

0.1

0.1

0.1

0.1

0.2

0.1

0.1

0.1

0.1

0.2

0.2

0.2

0.2

0.2

Iran

1.5

1.4

1.1

1.5

1.3

1.4

1.4

1.3

0.2

0.1

0.1

0.1

0.2

0.1

0.1

0.1

0.1

0.2

0.2

0.2

0.2

0.2

France

1.7

1.7

1.3

1.7

1.4

1.6

1.7

1.5

1.7

0.1

0.2

0.2

0.2

0.2

0.1

0.2

0.1

0.2

0.2

0.2

0.2

0.2

Pakistan

1.1

1.1

0.6

1.0

0.7

1.0

1.0

0.8

1.1

1.2

0.1

0.2

0.3

0.1

0.1

0.1

0.1

0.2

0.2

0.2

0.2

0.2

Belgium

1.4

1.4

1.0

1.4

1.1

1.3

1.3

1.2

1.4

1.6

0.9

0.2

0.3

0.1

0.1

0.1

0.1

0.2

0.2

0.2

0.2

0.2

Poland

1.5

1.4

1.1

1.4

1.2

1.3

1.4

1.2

1.4

1.6

1.0

1.2

0.3

0.2

0.1

0.2

0.2

0.2

0.2

0.2

0.2

0.2

S.Africa

2.2

2.2

1.7

2.1

1.9

2.1

2.1

1.9

2.1

2.3

1.6

2.0

2.0

0.3

0.3

0.2

0.2

0.3

0.3

0.3

0.3

0.3

Tunisia

1.5

1.4

1.0

1.4

1.2

1.3

1.4

1.2

1.4

1.6

0.9

1.3

1.4

2.0

0.1

0.1

0.1

0.2

0.2

0.2

0.2

0.2

Uzb

ekistan

1.3

1.3

0.9

1.2

1.0

1.2

1.2

1.1

1.3

1.4

0.7

1.1

1.2

1.9

1.0

0.1

0.1

0.2

0.2

0.2

0.2

0.2

Belaru

s1.3

1.3

0.9

1.2

1.0

1.2

1.2

1.0

1.3

1.4

0.7

1.0

1.1

1.8

1.1

1.0

0.1

0.2

0.2

0.2

0.2

0.2

Mon

golia

1.3

1.3

0.9

1.3

1.0

1.2

1.2

1.1

1.3

1.5

0.7

1.1

1.2

1.9

1.1

1.0

0.9

0.2

0.2

0.2

0.2

0.2

United

States

1.3

1.6

1.1

1.5

1.3

1.4

1.5

1.3

1.5

1.7

1.0

1.4

1.4

2.1

1.4

1.3

1.3

1.3

0.2

0.2

0.2

0.2

Italy

2.1

2.1

1.7

2.0

1.8

2.0

2.0

1.9

2.0

2.2

1.6

1.9

2.0

2.6

1.9

1.8

1.8

1.8

2.0

0.2

0.2

0.2

Swed

en1.3

1.3

0.7

1.3

1.0

1.2

1.2

1.1

1.3

1.2

0.8

1.1

1.2

1.8

1.2

1.0

1.0

1.0

1.3

1.9

0.2

0.3

Germany

1.6

1.6

1.2

1.5

1.3

1.5

1.5

1.3

1.6

1.7

1.1

1.4

1.4

2.2

1.5

1.3

1.3

1.3

1.5

2.1

1.3

0.2

England

1.4

1.5

1.0

1.4

1.2

1.4

1.4

1.3

1.5

1.6

1.0

1.3

1.5

2.1

1.4

1.2

1.2

1.2

1.4

2.0

1.2

1.4

Lightgraycolorindicatesthenucleotides

divergen

ceof

D1strainsfrom

Iranianisolatespresentedin

this,withtheD1strainsfrom

other

geographicalregions.

J. Med. Virol. DOI 10.1002/jmv

6 Pourkarim et al.

covers the whole northern part of the Persian Gulfcoast [Doosti et al., 2009; Zidan et al., 2012]. Al-though the implementation of infant vaccination in1992 has dramatically decreased the prevalence ofHBV in Iran, the virus is still considered to be themain infectious cause of liver disease in this country[Merat et al., 2009; Smolle et al., 2012]. Because ofdifferent ethnicities, habits and lifestyle, the preva-

lence and route of transmission of HBV amongIranian population varies from region to region[Alavian et al., 2007; Zidan et al., 2012]. Severalprevious studies cover the epidemiological profile ofHBV in the northern, western, and central provincesof Iran [Mohebbi et al., 2008, 2012; Amini-Bavil-Olyaee et al., 2009].According to few available sero-epidemiological

studies, the prevalence of HBsAg among blood donorsis 0.47% in Bushehr, 3.4% in Sistan-Baluchistan, and2.4% in Bandar Abbas (capital of Hormozgan prov-ince) [Alavian et al., 2008; Kafi-abad et al., 2009;Maneshi et al., 2010]. There are a few sero-epidemio-logical reports showing the prevalence of HBsAg inthis region among general population. These limitedinvestigations have demonstrated the prevalence ofHBsAg among the healthy population to be 3.4% inmales in Bandar Abbas and 2.0% in females [Meratet al., 2009] while in Sistan-Baluchistan it was 3.4%[Karimabad et al., 2012]. Also, the prevalence ofhepatitis viral markers among high risk groups likehemodialysis cases was 5.1% in Khuzestan province,6.7% in Bushehr, in drug abusers in Bandar Abbas,this was found in 4.7% of subjects [Mostaghniet al., 2011; Zahedi et al., 2012; Noroozi Karimabadet al., 2013].In addition, the inhabitants of this border region

have experienced the burden of wars during the last25 years, which have destroyed public health infra-structures [Assarehzadegan et al., 2008; Salehiet al., 2012]. The long-lasting armed conflict hascrippled the medical system, destroyed the socialstructure and brought poverty [Ali et al., 2012]. Sev-eral reports demonstrate that mass casualty and poor

TABLE IV. Mutational Pattern of the pre-S1/pre-S2 Region of the 21 HBV Strains Isolated in this Study

IDGenotype/subtype

PreS1 N-terminal (1–57) PreS1 C-terminal (58–119) PreS2 C-terminal (120–174)

B-cellepitope

T-cellepitope

B-cellepitope

T-cellepitope

B-cellepitope

T-cellepitope

PG-65 D1/ayw2 N103D N103D R124K, S151L, F171L R124K, S151L, F171LPG-14 D1/ayw2 L54P L54P N103D N103D F130S, D159V, P160L F130S, D159V, P160LPG-50 D1/ayw2 F130L F130LPG-18 D1/ayw2 G91V G91V I150T I150TPG-3 D1/ayw2 R24K R24K I150T, D159V I150T, D159VPG-7 D1/ayw3 L54P L54P Y129F Y129FPG-52 D1/ayw4PG-2 D1/ayw2 K7T K7TPG-4 D1/ayw2 T40P T40PPG-66 D1/ayw2 T119N T119N R126K R126KPG-37 D1/ayw2 A37V, T40P A37V, T40PPG-25 D1/ayw2 R126K R126KPG-8 D1/ayw2PG-22 D1/ayw2PG-20 D1/ayw2PG-31 D1/ayw2 R24K, T40P R24K, T40PPG-35 D1/ayw2 F171L F171LPG-32 D1/ayw2 T40P T40PPG-11 D1/ayw2 S67�, N103D S67�, N103D F171L F171LPG-60 D1/ayw2 F56L F56L N103D N103DPG-86 D1/ayw2 R124K, R126K,

G127D, F154LR124K, R126K,G127D, F154L

Fig. 3. Distribution of amino acid mutations detected withinthe MHR region of HBsAg among asymptomatic carriersinfected with HBV, who live in the northern coast of thePersian Gulf. The amino acid sequences represent positions 99-170 of the HBsAg protein. The amino acid variations in the “adeterminant” motif are framed in a yellow box.

J. Med. Virol. DOI 10.1002/jmv

Evolutionary Analysis of HBV Strains Circulating in Northern Coast of Persian Gulf 7

socioeconomic conditions resulting from war acceler-ate the transmission rate of HBV by forcing closecontact of injured people, necessitating emergencyblood transfusions from non-tested donors, and forc-ing the administration of non-hygienic injections[Hakre et al., 2011; Italiano et al., 2011]. In addition,war has led to the restriction of access to basic healthservices, such as vaccination, safe injections, andobstetric health care. Also, a high prevalence of HBVamong people displaced by war has been reported

[Khan et al., 2011]. Temporary immigration out ofwar-affected could also have a significant impact onthe epidemiological profile of infectious agents like HBV.In the present study, full-length genomic sequences

of 21 isolates were successfully sequenced and ana-lyzed from blood donors residing in this geographicalregion. BLAST analysis, the NCBI online genotypingtool, and phylogenetic analysis revealed the predomi-nance of subgenotype D1 in this part of Iran. Thisresult is in accordance with previous studies in the

1653 (C)

1654 (A)

1655 (T)

1665 (A)

1678 (T)

1679 (A)

1727 (A)

1728 (G)

1741 (T)

1745 (G)

1752 (A)

1753 (T)

1757 (A)

1762 (A)

1764 (G)

1766 (C)

1809 (G)

1810 (C)

1811 (A)

1812 (C)

1858 (T)

1862 (G)

1896 (G)

1899 (G)

T 2 0 19 1 20 0 0 0 20 0 1 16 0 6 6 0 0 0 0 1 21 0 0 0

G 0 0 0 0 0 0 9 19 1 20 0 1 2 1 8 6 20 0 0 0 0 21 11 18

C 18 1 1 19 1 1 0 0 0 0 2 3 0 0 1 13 0 21 0 20 0 0 0 0

A 1 20 1 1 0 20 12 2 0 1 18 1 19 13 5 1 1 0 21 0 0 0 10 3

10

20

num

ber o

f str

ains

(WT)

0

Fig. 4. Distribution of nucleotide diversity in the core region of 21 HBV strains isolated fromthe northern coast of Persian Gulf. WT, wild type nucleotide.

TABLE V. Mutational Patterns Detected in the BCP and X Gene Regions among 21 Isolated HBV

Strain Genotype/subtype

Nucleotide divergence of precore and BCP region X protein diversity

A1752 T1753 A1762 G1764 C1766 I127 K130 V131

PG-65 D1/ayw2 G1764T C1766G V131LPG-14 D1/ayw2 A1752C A1762T G1764A I127L K130M V131IPG-50 D1/ayw2 I127MPG-18 D1/ayw2 A1762T G1764A K130M V131IPG-3 D1/ayw2PG-7 D1/ayw3 A1752C I127LPG-52 D1/ayw4PG-2 D1/ayw2 T1753G A1762G G1764C C1766A I127S K130C V131LPG-4 D1/ayw2 A1752T A1762T G1764T I127F K130M V131CPG-66 D1/ayw2 T1753C C1766G I127T K130EPG-37 D1/ayw2PG-25 D1/ayw2 T1753C A1762T G1764A I127T K130M V131IPG-8 D1/ayw2PG-22 D1/ayw2 Deletion Deletion Deletion Deletion DeletionPG-20 D1/ayw2PG-31 D1/ayw2 G1764T C1766G V131LPG-35 D1/ayw2 T1753C G1764T C1766G I127T K130Q V131LPG-32 D1/ayw2 A1762T G1764A K130M V131IPG-11 D1/ayw2 T1753A A1762T G1764A I127N K130M V131IPG-86 D1/ayw2 G1764T C1766G V131LPG-60 D1/ayw2 G1764T C1766G V131L

J. Med. Virol. DOI 10.1002/jmv

8 Pourkarim et al.

Middle East and Iran [Amini-Bavil-Olyaee et al.,2005; Bozdayi et al., 2005; Mohebbi et al., 2008;Veazjalali et al., 2009] though other subgenotypes ofgenotype D have previously been reported in Iran[Garmiri et al., 2011; Mumtaz et al., 2011; Mohebbiet al., 2012]. This study, together with previousstudies from Iran, compares the epidemiologicalprofile of HBV in northern and southern regions ofthe Persian Gulf. For instance, recent investigationidentified HBV genotypes A and D in Kuwait [Aliet al., 2010], and genotypes A, C and D were found inthe U.A.E [Alfaresi et al., 2010]. Also, both genotypesA and D have been reported in Yemen [Sallam andWilliam Tong, 2004]. The most recent study showedgenotype A, C, and D in Saudi Arabia. This compre-hensive study reported the prevalence of HBsAg tobe 4.25% in Saudi Arabia, 5.1% in Yemen, 2.7% inOman, Bahrain, Qatar, U.A.E, and 3.5% in Kuwait[Gasim, 2013].In line with recent investigations, which demon-

strated a relationship between particular genotype/subgenotypes and subtypes [Hollinger et al., 2007;Kramvis et al., 2008] subtype ayw2 was detected in90.5% (n¼ 19) of HBV D1 isolates in this survey andthe rest (n¼ 2) had subtypes ayw3 and ayw4. Thedominance of HBV subtype ayw2 had previously beenreported in Iran [Mohebbi et al., 2008, 2012; Moradiet al., 2012]. Of note, the detection of rare subtypeslike ayw3 and ayw4 in the current study was also inline with other investigators’ findings [Mohebbi et al.,2008, 2012; Veazjalali et al., 2009; Garmiri et al.,2011].Subtype ayw4 was originally thought to be a

subtype restricted to HBV genotype E strains isolatedfrom African patients [Hollinger, 2007; Kay andZoulim, 2007]. However, more recent epidemiologicalstudies identified this subtype among subgenotypesD7, D4, D3, and D1 strains in different geographicalregions [Tallo et al., 2004; Echevarria et al., 2005;Kramvis et al., 2005; Mohebbi et al., 2008; Kitabet al., 2011; Mohebbi et al., 2012; Yousif andKramvis, 2013]. Subtype adw2 was similarly thoughtto be exclusively of genotype A but was also discov-ered in subgenotype D1 strains [Veazjalali et al.,2009; Yousif and Kramvis, 2013]. Even though noneof the strains in this study were of subtype adw2,this particular subtype has been identified togetherwith subgenotype D1 in our most recent study amongblood donor isolates in Iran [Pourkarim et al., 2014]as well as in other studies [Veazjalali et al., 2009;Garmiri et al., 2011]. In a previous study, due to amutation detected at serine-127, one untypable sub-type strain was detected in genotype D1 strainsisolated from HBV carriers living in central, north-ern, and western Iran [Mohebbi et al., 2008]. Also,there is a report, which shows that an unusualsubtype (ayr) in genotype D was isolated from Irani-an HBV carriers [Mohebbi et al., 2009]. This unusualsubtype in genotype D has been reported in Spain[Echevarria et al., 2005]. The present study did not

identify these strains in this particular geographicalregion.To provide the most detailed phylogenetic analysis

possible, the 21 isolates from this study were com-pared to all full-length genome HBV strains withgenotype D submitted to GenBank. Phylogeneticanalysis demonstrated that 563 strains (including 21strains of the present study) belong to subgenotypeD1. Interestingly, this analysis revealed the widedispersal of subgenotype D1 that was shown previ-ously [Zehender et al., 2012a]. Clustering of PersianGulf strains and other Iranian strains with isolatesfrom particular countries like Turkey, Syria, andLebanon corroborated the previous finding that thisregion might be the location for the common ancestorof HBV D1 strains [Zehender et al., 2012a]. Since thepreliminary phylogenetic tree (based on almost 1,000HBV strains) was too large to clearly illustrate andonly part of it was relevant to the identified strains,only the most similar strains to the Persian Gulfstrains were used to generate the phylogeny ofinterest. Calculation of genetic distances betweendifferent strains with subgenotype D1 (563 full-lengthgenomes) isolated from different geographical regionsshowed that the Persian Gulf strains and otherIranian strains have the same genetic distance withother D1 strains from other parts of the world. Thisanalysis showed that D1 strains from South Africahad the highest genetic distance with D1 strainsfrom other parts of the world (see Table III). Theseresults support the recent hypothesis that demon-strated that central Asia was the source of dispersalof genotype D, and that it translocated to SouthAfrica during recent decades [Zehender et al., 2012a].In this analysis, D1 strains from Tunisia clusteredmonophyletically and their genetic distance to Irani-an strains does not support the conclusion of onereport from Iran, which considered Tunisian strainsto be closely related to Iranian ones [Garmiriet al., 2011]. However, the result of genetic distanceanalysis in the present study is in agreement with arecent study, which showed a closer relationship withcountries neighboring Iran, such Turkey [Mohebbiet al., 2012].Interesting results were revealed when the sub-

types of all 563 full-length genome D1 strains weredetermined and compared to the 21 strains from thepresent study. This analysis showed that ayw2 has aclose association with HBV D1 in all geographicalregions and D1 ayw2 is the most frequent subtype inthe Middle Eastern countries. Also, Iran, Syria, andTurkey have almost the same epidemiological profileof these subtypes with subgenotype D1. Althoughsubtypes adw2 and ayw4 are rare subtypes forgenotype D, in the current analysis a surprisinglyhigh 15 (2.85%) and 7 (1.25%) out of 563 HBV D1strains that were isolated from different countriesrepresented these subtypes, respectively.This detailed analysis revealed that except Poland,

none of the European countries examined displayed

J. Med. Virol. DOI 10.1002/jmv

Evolutionary Analysis of HBV Strains Circulating in Northern Coast of Persian Gulf 9

D1 ayw4. The subtype ayw4 in Europe is associatedwith subgenotype D4 [Pourkarim et al., 2010b] whilethis subtype is observed with subgenotype D1 in theMiddle Eastern countries like Iran and Syria(Table II). These epidemiological patterns suggestthat HBV subgenotype and subtype distributionshave been impacted by human migration across thedifferent continents. It also emphasizes that histori-cally, human mobility, and immigration had a largeimpact on the dispersal of this infectious agent. Thishypothesis is supported by some recent investiga-tions, which provided several lines of evidences forHBV co-divergence with modern humans that re-solves the spatial complexity of genotype D [Para-skevis et al., 2013].In this survey, eleven mutations at the MHR

region were detected, with sP120S being the mostmedically important variation (Fig. 3). It is wellknown that strains with this alteration at the S genecan escape vaccination and detection by diagnosticassays [Carman, 1997; Echevarria and Avellon,2006]. This variant was previously identified as themost common MHR mutation in Iranian strainsreported by different investigators from Iran [Garmiriet al., 2011; Mohebbi et al., 2012; Moradiet al., 2012], Serbia [Lazarevic et al., 2010] and inHBV carriers from Morocco [Kitab et al., 2011]. Al-though in the present study the number of substitu-tions in the “a determinant” motif were not amenableto statistical analysis, the number of mutations atthe first loop of the “a determinant” motif was twotimes higher than those in the second loop of thismotif. It has been shown that mutation frequenciesin the first loop of the “a determinant” motif arehigher during natural history of infection. Afteractive or passive immunization, more mutations arisein the second loop of the “a determinant” motif[Ogura et al., 1999; Chang, 2010]. Although stopcodons in the S antigen of two strains were detected,the stop codon at sW172� is considered to be associat-ed with rtA181T during lamivudine, entecavir, andadefovir therapy, which arises in a primary drug-resistance situation [Mahabadi et al., 2013]. Thetruncated S protein that results from this stop codonmay have a transactivating effect on several proteinsin host cells [Lai and Yeh, 2008; Yeh, 2010]. Thereare discrepancies in the surveys regarding the antivi-ral resistant variants among treatment-naı̈ve IranianHBV carriers in different studies [Amini-Bavil-Olyaeeet al., 2008; Mahabadi et al., 2013]. Although twostrains carried a stop codon at HBsAg, HBsAg wasstill detectable in those two cases.In this study, one strain showed a deletion up-

stream of the HBsAg ORF. A deletion at this regionis considered to be a marker for advanced liverdisease [Shen et al., 2009]. In line with presentfindings, deletions at the pre-S region in strainsisolated from Iranian blood donors and patients inthe end stage liver disease have previously beenreported [Garmiri et al., 2011; Mohebbi et al., 2012].

Compared to other genotypes, HBV genotype Ddemonstrates a higher rate of BCP and precoremutations [Lindh et al., 1997]. This feature may berelated to the higher evolutionary rate of HBVgenotype D [Paraskevis et al., 2013]. A high rate ofHBV precore mutations [G1896A and G1899A (47.6%and 14.3%)] and BCP mutations [A1762TþG1764A/T,and G1764A/TþC1766G/T (57.1% and 28.6%)] inblood donors was detected. These mutations in theprecore concur with a previous report from Iranianblood donors [Garmiri et al., 2011] and is compatiblewith results from asymptomatic carriers existingin other geographical regions of Iran [Poustchiet al., 2008; Veazjalali et al., 2009; Mohebbi et al.,2012]. Surprisingly, the BCP mutational rate issignificantly higher than previous reports from blooddonors or asymptomatic carriers in Iran. It is shownthat BCP and precore mutations may have a strongeffect on clinical outcome [Chu et al., 2002; Poustchiet al., 2008; Kitab et al., 2012]. Statistical analysisdemonstrated a significant relationship between pre-core mutations and HBV HBe antigen secretion(P< 0.05). In detail, all precore mutant strains (18/21; 86%) expressed no HBeAg protein and for allpatients harboring precore mutations, anti-HBeAgwere positive. This result corroborates previous re-ports of precore mutations affecting HbeAg secretionin asymptomatic carriers infected with HBV genotypeD [Garmiri et al., 2011; Mohebbi et al., 2012]. Also,the present study supports previous findings thatdemonstrated that strains carrying pc1764T,pc1766G mutations carried pc1757A also [Sendiet al., 2005]. Although, 1752C and 1753V have beenintroduced as viral mutations associated with HCC[Elkady et al., 2008], all such strains in this studyare asymptomatic HBV carriers.Mutations at hotspot amino acids of the X protein

overlapped with the core region known to be associat-ed with the transactivating function [Tanakaet al., 2006]. Only five strains (24%) showed the wildtype pattern. The rest of the strains carried at leastone or more mutations at the X protein, which mayhave a significant role in hepatocarcinogenesis [Kimet al., 2007; Pollicino et al., 2007]. High non-synony-mous mutational rate plus novel variations of hotspotamino acids of the X protein that were detected instrains circulating in this geographical region areremarkable findings.In conclusion, this study demonstrates that pa-

tients living in the northern coast of the PersianGulf, like other Iranian HBV carriers, have beeninfected mainly with HBV subgenotype D1, subtypeayw2. Specific mutations related to advanced liverdisease were found more frequently in these strainscompared to other strains isolated from asymptomaticcarriers in other regions of Iran. In addition, acomplete phylogenetic analysis of subgenotype D1strains suggests this country is the most probableplace for the common ancestor of HBV D1. Also,subgenotype D1 has a worldwide distribution and the

J. Med. Virol. DOI 10.1002/jmv

10 Pourkarim et al.

association of subtype ayw2 with subgenotype D1 inall geographical regions was confirmed, althoughother subtypes like D1 adw2, ayw1, ayw3, and ayw4have been well-integrated into this epidemiologicalprofile.

ACKNOWLEDGMENTS

Mahmoud Reza Pourkarim is supported by apostdoctoral grant from the “Fonds voor Wetenschap-pelijk Onderzoek (FWO) Vlaanderen.” The authorsthank Rina Amatya for a critical review of themanuscript.

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