Structural features of the avian influenza virus hemagglutinin that influence virulence

Structural features of the avian in¯uenza virus

hemagglutinin that in¯uence virulence

Michael L. Perdue*, David L. Suarez

S.E. Poultry Research Laboratory, 934 College Station Road, Athens, GA 30605, USA

Abstract

Analysis of the structure of the avian in¯uenza (AI) virus hemagglutinin (HA) gene and protein

has yielded a wealth of information on the virulence mechanisms of in¯uenza viruses. The AI

hemagglutinin appears to be unique in its capacity to accept basic amino acids at its proteolytic

cleavage site (PCS). The association of multiple basic (MB) amino acids, HA cleavage, tissue

spread and virulence by AI strains ®rst proposed in the late 1970s and early 1980s [Klenk, H.D.,

Rott, R., Orlich, M., 1977. J. Gen. Virol. 36, 151±161; Bosch, F.X., Garten, W., Klenk, H.D., Rott,

R., 1981. Virology 113, 725±735] has held fast for two decades now. While other structural

characteristics and other genes can certainly in¯uence virulence, the presence of MB amino acids at

the PCS has provided a hallmark structural feature which justi®es continuing sequence analysis of

emerging ®eld isolates of AI strains. In addition to this structural feature, the distal tip of the HA is

prone to appearance and disappearance of glycosylation sites, some of which have been associated

with virulence.

The recent outbreaks of highly pathogenic AI in Mexico, Australia, Pakistan, Hong Kong and in

the ongoing outbreak of moderately pathogenic H7 avian in¯uenza in the northeast US have all

provided new and useful information regarding the role of HA RNA and protein structure in both

virulence and host adaptation. We have previously noted that stable RNA secondary structure near

the PCS is related to the acquisition of virulence and have proposed that the secondary structure

may promote the insertion of basic amino acids. In this report we evaluate the phylogenetic

relationships for three recent isolates of highly pathogenic avian in¯uenza viruses and the possible

virulence factors associated with their primary and secondary structure. # 2000 Elsevier Science

B.V. All rights reserved.

Keywords: Avian in¯uenza; Hemagglutinin; Virulence; Glycosylation; Basic amino acids

Veterinary Microbiology 74 (2000) 77±86

* Corresponding author. Tel.: �1-706-546-3435; fax: �1-706-546-3161.

E-mail address: [email protected] (M.L. Perdue)

0378-1135/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.

PII: S 0 3 7 8 - 1 1 3 5 ( 0 0 ) 0 0 1 6 8 - 1

1. Introduction

Avian in¯uenza viruses (type A orthomyxoviruses) are distributed worldwide, and the

principal hosts are migrating waterfowl and shorebirds (Webster et al., 1992). Most of

these viruses appear to be adapted to their host populations in a state best described as

maintenance evolution. This state rarely results in disease in the wild bird host, but can

result in large-scale fecal distribution of virus into the environment (Ito et al., 1995;

Stallknecht, 1998). It is hypothesized that because of widespread distribution, the viruses

leave this state of `equilibrium' from time to time to enter other vertebrate populations.

The factors that promote this escape are not, however, clear. The overwhelming evidence

suggests that AI viruses were the precursors to in¯uenza virus populations that are now

adapted to and circulate in horses, swine and humans (Webster et al., 1993, 1995). Thus,

the potential for introduction of new populations of AI viruses into mammalian hosts exists

and the outcomes of such introductions are unpredictable. A commonly occurring scenario is

the entrance of these viruses into commercial poultry operations. Of the 15 hemagglutinin

and nine neuraminidase subtypes of AI, most have infected commercial poultry. Since the

viruses make these jumps within the same order (Aves) it is assumed, though not directly

demonstrated, that this transition is simpler than that into the mammalian population.

However, the most disturbing aspect of the movement of AIV from wild waterfowl to

commercial poultry has been the subsequent development of virulence in the new hosts.

Attempts to reproduce these virulence shifts in the laboratory have been only partially

successful (Horimoto et al., 1995; Perdue et al., 1996; Swayne et al., 1998), suggesting that

multiple unknown factors may contribute to virulence. As such, it is important that virus

evolution in the ®eld continues to be monitored. The information provided by following the

molecular epidemiology of the in¯uenza A viruses has been invaluable in understanding the

disease caused by in¯uenza viruses both in avian and mammalian hosts.

The HA protein appears to be the most important protein for virulence of AI viruses. The

protein contains the receptor binding site, at least four variable antigenic sites that help evade

immune systems and it speci®es the proteolytic cleavage site necessary for processing the HA

and releasing the amino acids required for fusion. The major changes in the AI hemagglutinin

protein that accompany acquisition of virulence in the ®eld have been well documented and

include changes in the glycosylation patterns of the HA1 and in the structure of the proteolytic

cleavage site. The changes in glycosylation patterns are less consistent in virulence

development than are the acquisition of basic amino acids at the PCS. Our lab established the

addition of basic amino acids by duplicated insertions at the PCS as the most consistent

change associated with virulence. We have further shown that predicted stable secondary

structure near the PCS is likewise associated with the insertion event (Perdue et al., 1997). In

this report, we examine the data from recent outbreaks of lethal AI to assess the participation

of these proposed virulence factors in the appearance of virulence in the ®eld.

2. Materials and methods

Nucleotide sequence analysis and computer-assisted phylogenetic analysis of HA gene

sequences have been the most widely used tools for following virulence patterns among

78 M.L. Perdue, D.L. Suarez / Veterinary Microbiology 74 (2000) 77±86

®eld isolates of AI strains. Nucleotide sequences were obtained using an ABI 373

automated sequencer as previously described (Garcia et al., 1996, 1997). The library of

complete HA sequences has grown rapidly in recent years. Knowing the entire sequence

of the HA has provided data for computerized analysis of the secondary structure of the

HA RNA as well. The algorithms of (Zucker, 1989; Zucker et al., 1991) available through

GCG software (Madison, WI) were used to fold predicted secondary structures of AI

hemagglutinin RNA. Phylogenetic analyses were performed using PAUP (Suarez et al.,

1998, 1999). We are grateful to Dr. Alan Gould and Paul Selleck of CSIRO for providing

the Australian AI isolate. We are grateful to Drs. Dennis Alexander and Jill Banks of the

Central Veterinary Laboratory, Weybridge, England for providing the Italian H5 AI

isolate and for providing full length sequences of Pakistan H7 isolates for analysis.

3. Results

Two recent publications have reviewed in some detail the roles of hemagglutinin

glycosylation sites (Matrosovich et al., 1999) and the multibasic cleavage site

(Steinhauer, 1999) in the virulence of ®eld strains of AI. Glycosylation sites appear to

affect virulence in one of two ways. The ®rst is at the level of binding to the host cell

receptor. For example, the presence of carbohydrate near the receptor of an AI virus A/

Ck/Victoria/76 (H7N3) strain was ®rst directly associated with virulence in our prior

laboratory studies (Perdue et al., 1995). If as little as 1% of the population contained the

CHO binding site, virulence was measurably increased. With the Hong Kong origin

viruses, the presence of the carbohydrate at Position 156 near the receptor (Suarez et al.,

1998) yielded a less dramatic virulence effect. Studies in both chickens and mice have

shown no statistical difference in virulence potential when the CHO site is present (data

not shown). Matrosovich et al. (1999) proposed that receptor glycosylation sites enhanced

virulence potential and were closely associated with adaptation in chickens.

Fig. 1 demonstrates the phylogenetic relationships among recent highly pathogenic

isolates of avian in¯uenza viruses of the H5 and H7 subtypes. The newest isolates from

Italy and Hong Kong (Suarez et al., 1998) fall within the Eurasian lineage that has been

known for some time and appear to have evolved as a result of continued circulation of

these viruses. The Australia isolate is closely related to the other isolates that have

circulated and appeared sporadically since 1976. The Pakistan isolates are interesting in

that their closest characterized relative based on HA sequence is an ostrich virus

originally collected in South Africa (data not shown). While unquestionably of the

Eurasian lineage, its relationship to the most recent H7 isolates from Europe is not clear.

Table 1 summarizes both new and previously published data on the recent isolates of

avian in¯uenza where glycosylation, insertions of basic amino acids at the HA cleavage

site, and stable secondary structure in the RNA have clearly been linked to virulence. Two

of the newest isolates of AI have no additional glycosylation sites near the receptor, even

though they came from chickens, so the in¯uence of glycosylation near the receptor is

sometimes related to the virulent phenotype but not always clearly.

The hypothesis outlined in Perdue et al. (1997) proposes that non-pathogenic strains

can give rise to highly pathogenic strains by insertion mutations. The proposed model

M.L. Perdue, D.L. Suarez / Veterinary Microbiology 74 (2000) 77±86 79

should meet three conditions: (1) insertions are made up of purines; (2) insertions are

tandem duplications in multiples of three nucleotides (with duplications of six and nine

nucleotides being the most convincing); (3) in the positive sense RNA template a

region(s) of relatively more stable predicted secondary structure will be encountered

immediately 50 to the PCS.

Unfortunately in most cases, a non-pathogenic precursor has not been identi®ed for

lethal outbreaks; a non-pathogenic precursor is generally the exception rather than the

Fig. 1. Phylogenetic pro®les of the hemagglutinin gene of H5 and H7 isolates. The hemagglutinin sequences

were aligned and analyzed using the PAUP program. Isolates analyzed for the data in Table 1 are shown in bold.

(A) H5 subtype (B) H7 subtype.


rule. Thus, it is dif®cult to evaluate the stable secondary structure hypothesis for each

outbreak as the level of replication and mutation following the virulence shift may not be

known. If a single purine transition is allowed in the proposed insertion region, thus far all

but two highly pathogenic isolates with insertions have completely followed the

conditions: A/Tern/S.A./59 and the early FPV isolates from Germany such as A/Fowl/

Rostock/34. The South African isolate is the only know highly pathogenic wild bird

isolate and the passage history of the old German isolates we have analyzed is unknown,

making them really unsuitable for the analysis. The HA sequence of ®ve recent highly

Fig. 1 (Continued ).


pathogenic isolates have been evaluated for this report and have all satis®ed the

conditions in the hypothesis (Fig. 2A, B; Table 1).

Fig. 2 illustrates folding patterns using the Zucker mfold algorithm in the GCG gene

analysis package. Nucleotides 200-1599 from the positive sensed HA RNA from isolates

of the H5 (A/Ck/Hong Kong/220/97 and H7 (A/Ck/New South Wales/1688/97) subtypes

from the two most recent highly pathogenic outbreaks of AI were folded as was the A/Ck/

New York/8030/96. The patterns for the HP isolates clearly show an increased predicted

stability in the RNA structure 50 to the PCS as the hypothesis predicts. Overlapping

foldings using other stretches of 1400 nucleotides (the most the Zucker algorithm can

currently accommodate) showed that the regions in the HP strains remained stable (data

not shown). As seen in analyses of the HA genes of other strains, other regions of stable

secondary structure are encountered as well, but regions of high purine content were not

associated with these predicted structures. Also no other duplications or amino acid

insertions at other sites in the HA of the ®ve isolates in Table 1 were found (data not

shown).

Evaluation of H5 and H7 sequences from virus isolates that have not been associated

with the transition to the HP phenotype yields more variable results. Many of the folding

patterns appear similar to that shown in Fig. 1C. This ®gure illustrates the folding of the

non-pathogenic H7 isolate from New York. Other H5 and H7 non-pathogenic strains

occasionally do exhibit regions of stable secondary structure near the PCS but not as

dramatically as seen in the pre-HP and HP isolates (data not shown).

4. Discussion

The expression of virulence by in¯uenza viruses is a complicated process, and attempts

to force in¯uenza viruses into molecular or biological dogma are generally doomed to

failure. Analyses of naturally occurring isolates, however, have indicated an apparent

virulence-related preference for certain structural features associated with the

hemagglutinin. The most dif®cult of these to measure is the proposed relationship

Table 1

Structural features of the HA gene from recent avian in¯uenza isolates associated with lethal outbreaks

worldwidea

Isolate/outbreak and subtype Tandem

duplication/

insertion

Additional

Receptor

CHO sites

Increasing stable

RNA secondary

structure 50 to PCS

A/Ck/NSW/1688/97 (H7N4) Yesb No Yes

A/Ck/HK/220/97 (H5N1) Yes Yes Yes

A/Ck/Italy/1485-330/97 (H5N2) Yes Yes Yes

A/Ck/Pakistan/447/94 (H7N3) Yes No Yes

A/Ck/Queretaro/7653-20/95 (H5N2) Yes Yes Yes

aCk�chicken; H�Hemagglutinin; N�Neuraminidase; NSW�New South Wales, Australia; HK�Hong

Kong, China; CHO�carbohydrate; PCS�Proteolytic cleavage site.bIn this case the tandem duplication was five of six nucleotides: 1005GAGAAG.GAGAAA1016.


Fig. 2. Secondary structure stability pro®les for two recent highly pathogenic avian in¯uenza isolates (A) and

(B) and a non-pathogenic isolate from 1996 (C). The Zucker algorithm in the GCG software package was

employed to perform analysis of base pairing probabilities for nucleotide positions 200-1599 of the RNA

sequences of (A) A/chicken/Hong Kong/220/97; (B) A/chicken/New South Wales/1688/97 and (C) A/chicken/

NY/1996. The Australia isolate was kindly provided by Dr. Alan Gould and Paul Selleck of CSIRO, Geelong,

Australia. The arrow denotes the position of the proteolytic cleavage site.


between increased stable secondary structure near the PCS and the potential for

duplication/insertion events at that site. The use of predictive computer programs to

evaluate secondary structure is more believable when supported by physical analysis of

the RNA using speci®c treatments to measure hairpins or other base paired structures.

While this is an ongoing quest in our laboratory, it has proved dif®cult to achieve. This

may be due in part to the variability in the RNA sequence and the lack of any constant

structural regions required for replication outside the ends of the in¯uenza RNAs. So we

are left with computer-predicted structures that differ dramatically among the various

strains (see Fig. 2; Perdue et al., 1997). In many cases, as clearly shown in Fig. 1A,

regions of apparent stable secondary structures are found at sites other than the PCS. Thus

far, these other sites have not shown insertions or regions of high purine content, as seen

at the PCS (data not shown). In fact, the occurrence of insertions of amino acids in the

HA protein of in¯uenza A viruses in general is a rare occurrence outside of the PCS.

It is possible that insertions occur primarily at the cleavage site because of the

preponderance of purines there (Perdue et al., 1997). It may be further due to the fact that

the site is capable of physically accepting a number of amino acids without disrupting the

ability to cleave the HA (Khatchikian et al., 1989). The occasional presence of regions of

such stable structure in H5 and H7 and other strains not associated with HP outbreaks

indicates that the presence may not be suf®cient to result in a virulence shift. It is likely

that, as in expression of other phenotypes by in¯uenza A viruses, optimal gene

constellations must be achieved as a pre-requisite to the insertion event. It is entirely

possible, for example, that a polymerase complex capable of generating such an insertion

may be required in addition to the presence of the stable secondary structure.

Fig. 2 (Continued ).


There is little indication from analysis of these most recent isolates that additional

glycosylation sites near the receptor are strictly related to virulence in the ®eld. Certainly

there is an association, but not nearly to the degree of the association seen with the

presence of the multiple basic amino acids at the cleavage site. Additionally, we have

identi®ed recent non-pathogenic strains in our lab with multiple potential carbohydrate

addition sites on the globular tip (data not shown). It has been suggested that these sites

may represent adaptation to the chicken (Garcia et al., 1997; Matrosovich et al., 1999)

and precede development of virulence in the ®eld. Other major alterations in the HA

structure have been described for ®eld isolates of AI as have the rates of mutations in

these viruses (Suarez et al., 1999). It is clear that AI viruses evolve at high levels in

poultry. It will be important to continue sequencing the entire HA genes from ®eld

isolates to follow this process, particularly in regions where subtypes H7 and H5 strains

continue to circulate. The information gained should be invaluable in assessing potential

for shifting from a non-lethal to a lethal infection in the ®eld as well as potential for

crossing taxonomic barriers.

Acknowledgements

This work was supported entirely by USDA funds provided through the Agricultural

Research Service to CRIS research project 6612-32000-022. The authors thank Patsy

Decker and Suzanne DeBlois for their expert technical assistance.

References

Garcia, M., Crawford, J., Latimer, J.W., Rivera-Cruz, E., Perdue, M., 1996. Heterogeneity in the hemagglutinin

gene and emergence of the highly pathogenic phenotype among recent H5N2 avian in¯uenza viruses from

Mexico. J. Gen. Virol. 77, 1493±1504.

Garcia, M., Suarez, D.L., Crawford, J.M., Latimer, J.W., Slemons, R.D., Swayne, D.E., Perdue, M.L., 1997.

Evolution of H5 subtype avian in¯uenza A viruses in North America. Virus Res. 51, 115±124.

Horimoto, T., Rivera, E., Pearson, J., Senne, D., Krauss, S., Kawaoka, Y., Webster, R.G., 1995. Origin and

molecular changes associated with emergence of a highly pathogenic H5N2 in¯uenza virus in Mexico.

Virology 213, 223±230.

Ito, T., Okazaki, K., Kawaoka, Y., Takada, A., Webster, R.G., Kida, H., 1995. Perpetuation of in¯uenza A viruses

in Alaskan waterfowl reservoirs. Arch. Virol. 140, 1163±1172.

Khatchikian, E., Orlich, M., Rott, R., 1989. Increased viral pathogenicity after insertion of a 28S ribosomal RNA

sequence into the hemagglutinin gene of an avian in¯uenza virus. Nature 340, 156±157.

Matrosovich, M., Zhou, N., Kawaoka, Y., Webster, R., 1999. The surface glycoproteins of H5 in¯uenza viruses

isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J. Virol. 73, 1146±1155.

Perdue, M.L., Latimer, J.W., Crawford, J.M., 1995. A novel carbohydrate addition site on the hemagglutinin

protein of a highly pathogenic H7 subtype avian in¯uenza virus. Virology 213, 276±281.

Perdue, M.L., Garcia, M., Senne, D., Fraire, M., 1997. Virulence-associated sequence duplication at the

hemagglutinin cleavage site of avian in¯uenza viruses. Virus Res. 49, 173±186.

Perdue, M.L., Garcia, M., Beck, J., Brugh, M., Swayne, D.E., 1996. An Arg-Lys insertion at the hemagglutinin

cleavage site of an H5N2 avian in¯uenza isolate. Virus Genes 12, 77±84.

Stallknecht, D., 1998. Ecology and epidemiology of avian in¯uenza viruses in wild bird populations: waterfowl,

shorebirds, pelicans, and cormorants. In: Proceedings of the Fourth International Symposium on Avian

In¯uenza. Athens, GA. US Animal Health Association, pp. 61±69.


Steinhauer, D.A., 1999. Role of hemagglutinin cleavage for the pathogenicity of in¯uenza virus. Virology 258,

1±20.

Suarez, D.L., Perdue, M.L., Cox, N., Rowe, T., Bender, C., Huang, J., Swayne, D.E., 1998. Comparisons of

highly virulent H5N1 in¯uenza viruses isolated from humans and chickens from Hong Kong. J. Virol. 72,

6678±6688.

Suarez, D., Garcia, M., Latimer, J., Senne, .D., Perdue, M.L., 1999. Phylogenetic analysis of H7 avian in¯uenza

viruses isolated from the live bird markets of the Northeast United States. J. Virol. 73, 3567±3573.

Swayne, D., Beck, J., Garcia, M., Perdue, M., Brugh, M., 1998. Pathogenicity shifts in experimental avian

in¯uenza virus infections in chickens. In: Proceedings of the Fourth International Symposium on Avian

In¯uenza. Athens GA. US Animal Health Association, pp. 171±181.

Webster, R.G., Sharp, G.B., Claas, E.C., 1995. Interspecies transmission of in¯uenza viruses. Am. J. Resp. Crit.

Care Med. 152, S25±30.

Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., Kawaoka, Y., 1992. Evolution and ecology of

in¯uenza A viruses. Microbiol. Rev. 56, 152±179.

Webster, R.G., Wright, S.M., Castrucci, M.R., Bean, W.J., Kawaoka, Y., 1993. In¯uenza Ð a model of an

emerging virus disease. Intervirology 35, 16±25.

Zucker, M., 1989. On ®nding all suboptimal foldings of an RNA molecule. Science 244, 48±52.

Zucker, M., Jaeger, J., Turner, D., 1991. A comparison of optimal and suboptimal RNA secondary structure

predicted by free energy minimization with structures determined by phylogenetic comparison. Nucleic

Acids Res. 19, 2707±2714.


Structural features of the avian influenza virus hemagglutinin that influence virulence

Documents

Transcript of Structural features of the avian influenza virus hemagglutinin that influence virulence