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Short Communication |
Influenza Division, Coordinating Centers for Disease Control and Prevention, Atlanta, GA, USA
Correspondence
Natalie J. McDonald
Natalie.McDonald{at}cdc.hhs.gov
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ABSTRACT |
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Published online ahead of print on 6 September 2007 as DOI 10.1099/vir.0.83184-0.
Supplementary material is available with the online version of this paper.
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Coordinating Centers for Disease Control and Prevention.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). The high incidence of influenza cases is attributable to the ability of the influenza virus to escape immunity induced by prior infection or vaccination. This escape is potentiated by the accumulation of mutations in the surface glycoproteins haemagglutinin (HA), and to a lesser extent neuraminidase (NA), which confer antigenic change to the virus. This phenomenon, known as antigenic drift, necessitates annual vaccine updates to confer protection against the currently circulating strains.
Mutations in the HA molecule are considered to contribute almost entirely to the antigenic drift observed among influenza A viruses (Wilson & Cox, 1990). Those sites at the distal tip of the H1 HA molecule and on the side of the globular head near the receptor-binding pocket appear to be the main targets of the human immune response (Cox & Brokstad, 1999; Raymond et al., 1986; Sato et al., 2004). Influenza A viruses bind to sialic acids on the surface of target cells via a depression in the distal surface of the globular head of the HA molecule (Weis et al., 1988; Wilson et al., 1981). Several residues within this depression are highly conserved across the HA subtypes, including residues 98, 134, 138, 153 and 183 (H3 numbering) (Nobusawa et al., 1991). Amino acid substitutions within the receptor-binding pocket or the ‘second shell’ residues, including 190, 225 and 158, may alter the specificity toward certain types of galactosidic linkages, namely 
2–6Gal or 2–3Gal linkages (Aytay & Schulze, 1991; Matrosovich et al., 2000). Because of its location on the HA three-dimensional structure, mutations in the receptor-binding pocket or second shell residues can alter the antigenicity of a virus in addition to, or instead of, modifying receptor specificity or affinity (Daniels et al., 1984).
The HA1 domains of the HA genes from a subset of influenza A/H1N1 viruses collected worldwide from 1977 to 1999 were sequenced and analysed as described previously (Shaw et al., 2002; Xu et al., 2004). These isolates were selected to reflect the genetic and geographical distribution spectrum of influenza A/H1N1 viruses. Accession numbers for new and previously published influenza virus genes and amino acids alignments used in this study can be found in Supplementary Table S1 and Supplementary Fig. S1, respectively, available in JGV Online. The obtained sequences were assembled, aligned and edited using the DNASTAR (Madison, WI) and BIOEDIT version 5.0.6 (North Carolina State University) software. Phylogenetic trees were generated with the use of the MEGA version 3.1 software (Kumar et al., 2004).
Sequence alignments and phylogenetic analysis of influenza H1N1 strains isolated before 1994 revealed two clades, the A/Bayern/7/95-like clade and the A/Hebei/52/94-like clade, which although genetically distinguishable were antigenically indistinguishable. In 1995, virus isolates emerged in China that had a deletion of aa 134 (Fig. 1). While a comparable deletion had been previously observed in strains from 1935 (A/Alaska/35) and from an immunocompromised child (Rocha et al., 1991), the Chinese deletion lineage became the predecessor of the current human H1 viruses (Nakajima et al., 2000). Based on the phylogenetic tree, three strains were chosen for reverse genetic experiments to examine the genetic basis of the antigenic differences that arose during this period; A/Hebei/52/94 was the Chinese virus precursor to the deletion mutant, A/Beijing/262/95, and A/Shenzhen/227/95 from the A/Bayern/7/95-like lineage, which did not give rise to a deletion and eventually became extinct.
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). Lysine was inserted at position 134 in the A/Beijing/262/95 HA gene, or deleted in the A/Hebei/52/94 and A/Shenzhen/227/95 HA genes using the QuikChange Site-Directed Mutagenesis kit (Stratagene), and the resulting wild-type and mutant plasmids were sequenced both to confirm the plasmid identities and the absence of additional changes.
Infectious recombinant influenza A viruses were rescued by cotransfecting 293T cells with six A/Puerto Rico/8/34 (PR8) vRNA and protein expression plasmids (pCI-PR8-PB1, pCI-PR8-PB2, pCI-PR8-PA, pCI-PR8-NP, pCI-PR8-M and pCI-PR8-NS) (Subbarao et al., 2003) and two plasmids bearing the wild-type NA and either a wild-type or modified HA gene using TransIT-LT1 (Mirrus) as described previously (Hoffmann et al., 2000). Nine to 11-day-old embryonated chicken eggs were infected with 0.2 ml from each transfection, and recombinant virus and vRNA was isolated following incubation for 72 h at 35 °C. An HA1 amplicon was generated by RT-PCR and subsequently sequenced to ensure virus identity. Aliquots of recombinant viral stocks in allantoic fluid were maintained at –80 °C.
HA and HA inhibition (HAI) assays were performed using standard techniques as described previously (Kendal et al., 1982), using turkey red blood cells (University of Georgia, Athens, GA). Seronegative and post-infection ferret antisera raised against the reference H1N1 viruses were provided by our colleagues in the Diagnostic and Strain Surveillance Branch of the Influenza Division. Briefly, serologically naïve ferrets were inoculated intranasally with 1 ml diluted virus. Serum was collected via cardiac puncture 14 days post-immunization according to Institutional Animal Care and Use Committee regulations (National Research Council, 2002).
Nearly all the recombinant viruses grew well in eggs, reaching HA titres of 1024. The two exceptions were the A/Hebei/52/94 strain bearing a deletion of K134 (Heb/
K134), which grew to a titre of 64, and the A/Beijing/262/95 strain bearing an insertion of K134 (Bei/insK134), which could only be rescued when the NA gene of A/Beijing/262/95 was replaced with that of A/Hebei/52/94. These two NA polypeptides differ by only 1 aa; A/Hebei/52/94 bears an alanine at position 13, while A/Beijing/262/95 bears a valine [Supplementary Fig. S1(b) available in JGV Online]. The reassortant wild-type (Bei/WT Heb/NA) and mutant (Bei/insK134 Heb/NA) viruses were able to grow to a titre of 1024. In contrast, both the reassortant viruses bearing either the wild-type or K134 mutant HA of A/Hebei/52/94 and A/Beijing/262/95 NA were able to be rescued although at significantly lower titres (256 and 64, respectively). While the role of residue 13 of the NA glycoprotein in the replication of these recombinant viruses is unclear, the functional interaction between the activities of the HA and NA glycoproteins of influenza is well documented (Baigent & McCauley, 2001; Baum & Paulson, 1991; Kaverin et al., 2000; Lu et al., 2005; Wagner et al., 2000).
The effects of K134 insertion or deletion on the three-dimensional structure of the HA trimer were investigated by producing molecular graphic images based on the PR8 and A/swine/Iowa/30 HA trimers using the Chimera package (Computer Graphics Laboratory, University of California, San Francisco, USA) (Gamblin et al., 2004; Huang et al., 1996; Sanner et al., 1996). The models predicted that residue 134 lies along the edge of the receptor-binding pocket, potentially reducing the size of the pocket (Fig. 2). Addition of K134 did not appear to cause gross changes in the three-dimensional structure of HA, but was predicted to introduce a strong-positive charge on the right side of the receptor-binding pocket as well as a slightly reduced negative charge at the bottom of the pocket (Supplementary Fig. S2 available in JGV Online). The predicted change in charge distribution did not impact HA periodate sensitivity of the recombinant A/Beijing/262/95 viruses, while the deletion of K134 in A/Hebei/52/94 HA did result in a modest increase in sensitivity to periodate of the recombinant viruses (data not shown), suggesting that residue 134 might alter the specificity or the avidity of receptor binding. Additional amino acid differences in the receptor-binding pocket of A/Hebei/52/94 and A/Beijing/262/95 HAs, including residues 190, 225 and 226 (H3 numbering) known to impact receptor specificity, may play a role in magnifying the effect of deleting residue 134 in A/Hebei/52/94 HA (Supplementary Fig. S1 available in JGV Online) (Matrosovich et al., 1997, 2000). Interestingly, Kaverin et al. (2000) reported that virus aggregation, resulting from incompatibilities between the HA and NA glycoprotein activities, could be overcome by mutation in the vicinity of the receptor pocket of HA by increasing the local negative charge. This increased negative charge was presumed to decrease the affinity of HA for sialic acid. The failure to rescue Bei/insK134 with its homologous NA may be due to the increased positive charge introduced around the pocket by K134, which may result in stronger affinity for the sialic acid, and consequently require compensatory changes in NA activity to allow viral entry and release. The role of residue 13 in NA activity is the subject of continuing investigation.
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and Supplementary Table S2). Furthermore, the A/Beijing/262/95 reassortant bearing its homologous wild-type HA and the wild-type NA of A/Hebei/52/94 did not have an altered antigenic profile compared to the reference antigen. However, insertion of K at position 134 in the A/Beijing/262/95 HA gene resulted in the loss of titre to the homologous antiserum, and a significant gain of reactivity to both the A/Hebei/52/94 and A/Shenzhen/227/95 antisera. Likewise, deletion of K134 in either the A/Hebei/52/94 or the A/Shenzhen/227/95 HA (Shen/K134) genes resulted in a virus with antigenic properties characteristic of A/Beijing/262/95. One additional substitution, at residue 166 of the HA1 domain is shared between the A/Hebei/52/94 and A/Shenzhen/227/95 genetic groups versus the A/Beijing/262/95 genetic group (Supplementary Fig. S1 available in JGV Online). This residue lies immediately C-terminal to a potential N-glycosylation site in antibody combining site D on the opposite surface of the HA monomer with respect to the receptor-binding pocket, and lies several residues away from the adjacent pocket of the assembled trimer (Caton et al., 1982). Amino acid substitution at residue 166 did not alter the antigenic properties compared to the wild-type or K134 mutant viruses (data not shown). However, it has been reported that structural conformation of the consensus N–X–T/S can impact the usage of the site (Bause et al., 1982; Bause, 1983). The substitution of N for K C-terminal to this site in A/Beijing/262/95 may alter the local conformation, resulting in differential usage of the site or altered conformation of the carbohydrate. In H3N2 influenza A, glycosylation near the receptor-binding pocket can alter the dependence on NA activity in viral growth (Baigent & McCauley, 2001). At present, it is unknown whether A/Beijing/262/95 HA is differentially glycosylated compared with that of A/Hebei/52/94 or if glycosylation at this site may also affect NA activity requirements in coordination with the presence or absence of K134.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Baigent, S. J. & McCauley, J. W. (2001). Glycosylation of haemagglutinin and stalk-length of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture. Virus Res 79, 177–185.[CrossRef][Medline]
Baum, L. G. & Paulson, J. C. (1991). The N2 neuraminidase of human influenza virus has acquired a substrate specificity complementary to the hemagglutinin receptor specificity. Virology 180, 10–15.[CrossRef][Medline]
Bause, E. (1983). Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem J 209, 331–336.[Medline]
Bause, E., Hettkamp, H. & Legler, G. (1982). Conformational aspects of N-glycosylation of proteins. Studies with linear and cyclic peptides as probes. Biochem J 203, 761–768.[Medline]
Caton, A. J., Brownlee, G. G., Yewdell, J. W. & Gerhard, W. (1982). The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell 31, 417–427.[CrossRef][Medline]
Cox, R. J. & Brokstad, K. A. (1999). The postvaccination antibody response to influenza virus proteins. APMIS 107, 289–296.[Medline]
Daniels, R. S., Douglas, A. R., Skehel, J. J., Wiley, D. C., Naeve, C. W., Webster, R. G., Rogers, G. N. & Paulson, J. C. (1984). Antigenic analyses of influenza virus haemagglutinins with different receptor-binding specificities. Virology 138, 174–177.[CrossRef][Medline]
Gamblin, S. J., Haire, L. F., Russell, R. J., Stevens, D. J., Xiao, B., Ha, Y., Vasisht, N., Steinhauer, D. A., Daniels, R. S. & other authors (2004). The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science 303, 1838–1842.
Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G. & Webster, R. G. (2000). A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A 97, 6108–6113.
Huang, C. C., Couch, G. S., Pettersen, E. F. & Ferrin, T. E. (1996). Chimera: an extensible molecular modeling application constructed using standard components. In Pacific Symposium on Biocomputing ‘96, p. 724. Edited by L. Hunter & T. E. Klein. Singapore: World Scientific Publishing.
Kaverin, N. V., Matrosovich, M. N., Gambaryan, A. S., Rudneva, I. A., Shilov, A. A., Varich, N. L., Makarova, N. V., Kropotkina, E. A. & Sinitsin, B. V. (2000). Intergenic HA-NA interactions in influenza A virus: postreassortment substitutions of charged amino acid in the hemagglutinin of different subtypes. Virus Res 66, 123–129.[CrossRef][Medline]
Kendal, A. P., Pereira, M. S. & Skehel, J. J. (1982). Hemagglutination inhibition. In Concepts and Procedures for Laboratory-Based Influenza Surveillance, pp. B17–B35. Edited by A. P. Kendal, M. S. Pereira & J. J. Skehel. Atlanta, GA: Centers for Disease Control and Prevention.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Lu, B., Zhou, H., Ye, D., Kemble, G. & Jin, H. (2005). Improvement of influenza A/Fujian/411/02 (H3N2) virus growth in embryonated chicken eggs by balancing the hemagglutinin and neuraminidase activities, using reverse genetics. J Virol 79, 6763–6771.
Maines, T. R., Chen, L. M., Matsuoka, Y., Chen, H., Rowe, T., Ortin, J., Falcon, A., Nguyen, T. H., Mai, L. Q. & other authors (2006). Lack of transmission of H5N1 avian-human reassortant influenza viruses in a ferret model. Proc Natl Acad Sci U S A 103, 12121–12126.
Matrosovich, M. N., Gambaryan, A. S., Teneberg, S., Piskarev, V. E., Yamnikova, S. S., Lvov, D. K., Robertson, J. S. & Karlsson, K. A. (1997). Avian influenza A viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology 233, 224–234.[CrossRef][Medline]
Matrosovich, M., Tuzikov, A., Bovin, N., Gambaryan, A., Klimov, A., Castrucci, M. R., Donatelli, I. & Kawaoka, Y. (2000). Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J Virol 74, 8502–8512.
Nakajima, S., Nishikawa, F. & Nakajima, K. (2000). Comparison of the evolution of recent and late phase of old influenza A (H1N1) viruses. Microbiol Immunol 44, 841–847.[Medline]
National Research Council (2002). Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academy Press.
Nobusawa, E., Aoyama, T., Kato, H., Suzuki, Y., Tateno, Y. & Nakajima, K. (1991). Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses. Virology 182, 475–485.[CrossRef][Medline]
Raymond, F. L., Caton, A. J., Cox, N. J., Kendal, A. P. & Brownlee, G. G. (1986). The antigenicity and evolution of influenza H1 haemagglutinin, from 1950–1957 and 1977–1983: two pathways from one gene. Virology 148, 275–287.[CrossRef][Medline]
Rocha, E., Cox, N. J., Black, R. A., Harmon, M. W., Harrison, C. J. & Kendal, A. P. (1991). Antigenic and genetic variation in influenza A (H1N1) virus isolates recovered from a persistently infected immunodeficient child. J Virol 65, 2340–2350.
Sanner, M. F., Olson, A. J. & Spehner, J. C. (1996). Reduced surface: an efficient way to compute molecular surfaces. Biopolymers 38, 305–320.[CrossRef][Medline]
Sato, K., Morishita, T., Nobusawa, E., Tonegawa, K., Sakae, K., Nakajima, S. & Nakajima, K. (2004). Amino-acid change on the antigenic region B1 of H3 haemagglutinin may be a trigger for the emergence of drift strain of influenza A virus. Epidemiol Infect 132, 399–406.[CrossRef][Medline]
Shaw, M. W., Xu, X., Li, Y., Normand, S., Ueki, R. T., Kunimoto, G. Y., Hall, H., Klimov, A., Cox, N. J. & Subbarao, K. (2002). Reappearance and global spread of variants of influenza B/Victoria/2/87 lineage viruses in the 2000–2001 and 2001–2002 seasons. Virology 303, 1–8.[CrossRef][Medline]
Stohr, K. (2002). Influenza–WHO cares. Lancet Infect Dis 2, 517[CrossRef][Medline]
Subbarao, K., Chen, H., Swayne, D., Mingay, L., Fodor, E., Brownlee, G., Xu, X., Lu, X., Katz, J. & other authors (2003). Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics. Virology 305, 192–200.[CrossRef][Medline]
Wagner, R., Wolff, T., Herwig, A., Pleschka, S. & Klenk, H. D. (2000). Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics. J Virol 74, 6316–6323.
Weis, W., Brown, J. H., Cusack, S., Paulson, J. C., Skehel, J. J. & Wiley, D. C. (1988). Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature 333, 426–431.[CrossRef][Medline]
Wilson, I. A. & Cox, N. J. (1990). Structural basis of immune recognition of influenza virus hemagglutinin. Annu Rev Immunol 8, 737–771.[CrossRef][Medline]
Wilson, I. A., Skehel, J. J. & Wiley, D. C. (1981). Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature 289, 366–373.[CrossRef][Medline]
Xu, X., Lindstrom, S. E., Shaw, M. W., Smith, C. B., Hall, H. E., Mungall, B. A., Subbarao, K., Cox, N. J. & Klimov, A. (2004). Reassortment and evolution of current human influenza A and B viruses. Virus Res 103, 55–60.[CrossRef][Medline]
Received 24 May 2007;
accepted 21 August 2007.
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