Antigen-Antibody docking reveals the molecular basis for cross-reactivity of the 1918 and 2009 Influenza A/H1N1 pandemic viruses

To understand the reported cross-reactivity of the 2009 H1N1 and the 1918 H1N1 pandemic viruses we docked the crystal structure of 2D1, an antibody derived from a survivor of the 1918 pandemic, to the structures of hemaglutinin (HA) of the 2009 strain and seasonal H1 vaccine strains. Our studies revealed that 2D1 binds to the 2009 HA at antigenic site 'Sa', with stabilizing contacts, similar to that in an available co-crystal structure of 2D1-1918 HA. However, 2D1 failed to bind to the known antigenic sites in the HAs of seasonal strains. Our study thus reveals the molecular basis for pre-existing immunity in elderly people to the 2009 pandemic virus.     

Protection of Mice against Lethal Challenge with 2009 H1N1 Influenza A Virus by 1918-Like and Classical Swine H1N1 Based Vaccines

The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918–1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses.     

Antibody Recognition of the Pandemic H1N1 Influenza Virus Hemagglutinin Receptor Binding Site

Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines.     

Genetic Requirement for Hemagglutinin Glycosylation and Its Implications for Influenza A H1N1 Virus Evolution

Influenza A virus has evolved and thrived in human populations. Since the 1918 influenza A pandemic, human H1N1 viruses had acquired additional N-linked glycosylation (NLG) sites within the globular head region of hemagglutinin (HA) until the NLG-free HA head pattern of the 1918 H1N1 virus was renewed with the swine-derived 2009 pandemic H1N1 virus. Moreover, the HA of the 2009 H1N1 virus appeared to be antigenically related to that of the 1918 H1N1 virus. Hence, it is possible that descendants of the 2009 H1N1 virus might recapitulate the acquisition of HA head glycosylation sites through their evolutionary drift as a means to evade preexisting immunity. We evaluate here the evolution signature of glycosylations found in the globular head region of H1 HA in order to determine their impact in the virulence and transmission of H1N1 viruses. We identified a polymorphism at HA residue 147 associated with the acquisition of glycosylation at residues 144 and 172. By in vitro and in vivo analyses using mutant viruses, we also found that the polymorphism at HA residue 147 compensated for the loss of replication, virulence, and transmissibility associated with the presence of the N-linked glycans. Our findings suggest that the polymorphism in H1 HA at position 147 modulates viral fitness by buffering the constraints caused by N-linked glycans and provide insights into the evolution dynamics of influenza viruses with implications in vaccine immunogenicity.     

Comparative analysis of hemagglutinin of 2009 H1N1 influenza A pandemic indicates its evolution to 1918 H1N1 pandemic

To gain insight into the possible origin of the hemagglutinin of 2009 outbreak, we performed its comparative analysis with hemagglutinin of influenza viral strains from 2005 to 2008 and the past pandemics of 1977, 1968, 1957 and 1918. This insilico analysis showed a maximum sequence similarity between 2009 and 1918 pandemics. Primary structure analysis, antigenic and glycosylation site analyses revealed that this protein has evolved from 1918 pandemic. Phylogenetic analysis of HA amino acid sequence of 2009 influenza A(H1N1) viruses indicated that this virus possesses a distinctive evolutionary trait with 1918 influenza A virus. Although the disordered sequences are different among all the isolates, the disordered positions and sequences between 2009 and 1918 isolates show a greater similarity. Thus these analyses contribute to the evidence of the evolution of 2009 pandemic from 1918 influenza pandemic. This is the first computational evolutionary analysis of HA protein of 2009 H1N1 pandemic.     

N-Linked Glycosylation of the Hemagglutinin Protein Influences Virulence and Antigenicity of the 1918 Pandemic and Seasonal H1N1 Influenza A Viruses

The hemagglutinin (HA) protein is a major virulence determinant for the 1918 pandemic influenza virus; however, it encodes no known virulence-associated determinants. In comparison to seasonal influenza viruses of lesser virulence, the 1918 H1N1 virus has fewer glycosylation sequons on the HA globular head region. Using site-directed mutagenesis, we found that a 1918 HA recombinant virus, of high virulence, could be significantly attenuated in mice by adding two additional glycosylation sites (asparagine [Asn] 71 and Asn 286) on the side of the HA head. The 1918 HA recombinant virus was further attenuated by introducing two additional glycosylation sites on the top of the HA head at Asn 142 and Asn 172. In a reciprocal experimental approach, deletion of HA glycosylation sites (Asn 142 and Asn 177, but not Asn 71 and Asn 104) from a seasonal influenza H1N1 virus, A/Solomon Islands/2006 (SI/06), led to increased virulence in mice. The addition of glycosylation sites to 1918 HA and removal of glycosylation sites from SI/06 HA imposed constraints on the theoretical structure surrounding the glycan receptor binding sites, which in turn led to distinct glycan receptor binding properties. The modification of glycosylation sites for the 1918 and SI/06 viruses also caused changes in viral antigenicity based on cross-reactive hemagglutinin inhibition antibody titers with antisera from mice infected with wild-type or glycan mutant viruses. These results demonstrate that glycosylation patterns of the 1918 and seasonal H1N1 viruses directly contribute to differences in virulence and are partially responsible for their distinct antigenicity.     

Minor changes in the hemagglutinin of influenza A(H1N1)2009 virus alter its antigenic properties

Background

The influenza A(H1N1)2009 virus has been the dominant type of influenza A virus in Finland during the 2009–2010 and 2010–2011 epidemic seasons. We analyzed the antigenic characteristics of several influenza A(H1N1)2009 viruses isolated during the two influenza seasons by analyzing the amino acid sequences of the hemagglutinin (HA), modeling the amino acid changes in the HA structure and measuring antibody responses induced by natural infection or influenza vaccination.

Methods/Results

Based on the HA sequences of influenza A(H1N1)2009 viruses we selected 13 different strains for antigenic characterization. The analysis included the vaccine virus, A/California/07/2009 and multiple California-like isolates from 2009–2010 and 2010–2011 epidemic seasons. These viruses had two to five amino acid changes in their HA1 molecule. The mutation(s) were located in antigenic sites Sa, Ca1, Ca2 and Cb region. Analysis of the antibody levels by hemagglutination inhibition test (HI) indicated that vaccinated individuals and people who had experienced a natural influenza A(H1N1)2009 virus infection showed good immune responses against the vaccine virus and most of the wild-type viruses. However, one to two amino acid changes in the antigenic site Sa dramatically affected the ability of antibodies to recognize these viruses. In contrast, the tested viruses were indistinguishable in regard to antibody recognition by the sera from elderly individuals who had been exposed to the Spanish influenza or its descendant viruses during the early 20^th century.

Conclusions

According to our results, one to two amino acid changes (N125D and/or N156K) in the major antigenic sites of the hemagglutinin of influenza A(H1N1)2009 virus may lead to significant reduction in the ability of patient and vaccine sera to recognize A(H1N1)2009 viruses.     

Influenza Human Monoclonal Antibody 1F1 Interacts with Three Major Antigenic Sites and Residues Mediating Human Receptor Specificity in H1N1 Viruses

Most monoclonal antibodies (mAbs) to the influenza A virus hemagglutinin (HA) head domain exhibit very limited breadth of inhibitory activity due to antigenic drift in field strains. However, mAb 1F1, isolated from a 1918 influenza pandemic survivor, inhibits select human H1 viruses (1918, 1943, 1947, and 1977 isolates). The crystal structure of 1F1 in complex with the 1918 HA shows that 1F1 contacts residues that are classically defined as belonging to three distinct antigenic sites, Sa, Sb and Ca₂. The 1F1 heavy chain also reaches into the receptor binding site (RBS) and interacts with residues that contact sialoglycan receptors and determine HA receptor specificity. The 1F1 epitope is remarkably similar to the previously described murine HC63 H3 epitope, despite significant sequence differences between H1 and H3 HAs. Both antibodies potently inhibit receptor binding, but only HC63 can block the pH-induced conformational changes in HA that drive membrane fusion. Contacts within the RBS suggested that 1F1 may be sensitive to changes that alter HA receptor binding activity. Affinity assays confirmed that sequence changes that switch the HA to avian receptor specificity affect binding of 1F1 and a mAb possessing a closely related heavy chain, 1I20. To characterize 1F1 cross-reactivity, additional escape mutant selection and site-directed mutagenesis were performed. Residues 190 and 227 in the 1F1 epitope were found to be critical for 1F1 reactivity towards 1918, 1943 and 1977 HAs, as well as for 1I20 reactivity towards the 1918 HA. Therefore, 1F1 heavy-chain interactions with conserved RBS residues likely contribute to its ability to inhibit divergent HAs.     

A broadly neutralizing human monoclonal antibody that recognizes a conserved, novel epitope on the globular head of the influenza H1N1 virus hemagglutinin

The conserved influenza virus hemagglutinin (HA) stem domain elicits cross-reactive antibodies, but epitopes in the globular head typically elicit strain-specific responses because of the hypervariability of this region. We isolated human monoclonal antibody 5J8, which neutralized a broad spectrum of 20th century H1N1 viruses and the 2009 pandemic H1N1 virus. Fine mapping of the interaction unexpectedly revealed a novel epitope between the receptor-binding pocket and the Ca₂ antigenic site on HA. This antibody exposes a new mechanism underlying broad immunity against H1N1 influenza viruses and identifies a conserved epitope that might be incorporated into engineered H1 virus vaccines.     

Cooperation between the Hemagglutinin of Avian Viruses and the Matrix Protein of Human Influenza A Viruses

To analyze the compatibility of avian influenza A virus hemagglutinins (HAs) and human influenza A virus matrix (M) proteins M1 and M2, we doubly infected Madin-Darby canine kidney cells with amantadine (1-aminoadamantane hydrochloride)-resistant human viruses and amantadine-sensitive avian strains. By using antisera against the human virus HAs and amantadine, we selected reassortants containing the human virus M gene and the avian virus HA gene. In our system, high virus yields and large, well-defined plaques indicated that the avian HAs and the human M gene products could cooperate effectively; low virus yields and small, turbid plaques indicated that cooperation was poor. The M gene products are among the primary components that determine the species specificities of influenza A viruses. Therefore, our system also indicated whether the avian HA genes effectively reassorted into the genome and replaced the HA gene of the prevailing human influenza A viruses. Most of the avian HAs that we tested efficiently cooperated with the M gene products of the early human A/PR/8/34 (H1N1) virus; however, the avian HAs did not effectively cooperate with the most recently isolated human virus that we tested, A/Nanchang/933/95 (H3N2). Cooperation between the avian HAs and the M proteins of the human A/Singapore/57 (H2N2) virus was moderate. These results suggest that the currently prevailing human influenza A viruses might have lost their ability to undergo antigenic shift and therefore are unable to form new pandemic viruses that contain an avian HA, a finding that is of great interest for pandemic planning.     

A single base-pair change in 2009 H1N1 hemagglutinin increases human receptor affinity and leads to efficient airborne viral transmission in ferrets

The 2009 H1N1 influenza A virus continues to circulate among the human population as the predominant H1N1 subtype. Epidemiological studies and airborne transmission studies using the ferret model have shown that the transmission efficiency of 2009 H1N1 viruses is lower than that of previous seasonal strains and the 1918 pandemic H1N1 strain. We recently correlated this reduced transmission efficiency to the lower binding affinity of the 2009 H1N1 hemagglutinin (HA) to α2→6 sialylated glycan receptors (human receptors). Here we report that a single point mutation (Ile219→Lys; a base pair change) in the glycan receptor-binding site (RBS) of a representative 2009 H1N1 influenza A virus, A/California/04/09 or CA04/09, quantitatively increases its human receptor-binding affinity. The increased human receptor-affinity is in the same range as that of the HA from highly transmissible seasonal and 1918 pandemic H1N1 viruses. Moreover, a 2009 H1N1 virus carrying this mutation in the RBS (generated using reverse genetics) transmits efficiently in ferrets by respiratory droplets thereby reestablishing our previously observed correlation between human receptor-binding affinity and transmission efficiency. These findings are significant in the context of monitoring the evolution of the currently circulating 2009 H1N1 viruses.     

Naturally Occurring Human Monoclonal Antibodies Neutralize both 1918 and 2009 Pandemic Influenza A (H1N1) Viruses

The 2009 pandemic influenza A (H1N1) virus exhibits hemagglutinin protein sequence homology with the 1918 pandemic influenza virus. We found that human monoclonal antibodies recognized the Sa antigenic site on the head domains of both 1918 and 2009 hemagglutinins, a site that is hypervariable due to immune selection. These antibodies exhibited high potency against the 2009 virus in vitro, and one exerted a marked therapeutic effect in vivo.In March and April of 2009, an outbreak of a swine-origin novel H1N1 influenza A virus began in Mexico (2). As of 29 November 2009, worldwide, more than 207 countries and overseas territories or communities have reported laboratory-confirmed cases of 2009 A (H1N1), including at least 8,768 deaths. (http://www.who.int/csr/don/2009_12_04/en/index.html). The hemagglutinin (HA) gene of the 2009 A (H1N1) strain has been present in classical swine H1N1 viruses that have circulated in pigs at least since the discovery by Shope in the 1930s (Table 10, 11, 16). In contrast, the HA of human H1N1 influenza viruses circulating from 1918 to 1957 and from 1977 to present drifted progressively away from the 1918 virus HA (Table ​(Table1d)1d) (8, 14). Elderly subjects born prior to 1918 were found to have serum neutralizing antibody titers to the A/California/04/2009 (CA04) virus (5, 6).

TABLE 1.

Alignment of the amino acids in site Sa of the HA of representative swine or human influenza viruses from the 20th century^a

Evolutionary pathways of the pandemic influenza A (H1N1) 2009 in the UK

The emergence of the influenza (H1N1) 2009 virus provided a unique opportunity to study the evolution of a pandemic virus following its introduction into the human population. Virological and clinical surveillance in the UK were comprehensive during the first and second waves of the pandemic in 2009, with extensive laboratory confirmation of infection allowing a detailed sampling of representative circulating viruses. We sequenced the complete coding region of the haemagglutinin (HA) segment of 685 H1N1 pandemic viruses selected without bias during two waves of pandemic in the UK (April-December 2009). Phylogenetic analysis showed that although temporal accumulation of amino acid changes was observed in the HA sequences, the overall diversity was less than that typically seen for seasonal influenza A H1N1 or H3N2. There was co-circulation of multiple variants as characterised by signature amino acid changes in the HA. A specific substitution (S203T) became predominant both in UK and global isolates. No antigenic drift occurred during 2009 as viruses with greater than four-fold reduction in their haemagglutination inhibition (HI) titre ("low reactors") were detected in a low proportion (3%) and occurred sporadically. Although some limited antigenic divergence in viruses with four-fold reduction in HI titre might be related to the presence of 203T, additional studies are needed to test this hypothesis.     

Neuraminidase and Hemagglutinin Matching Patterns of a Highly Pathogenic Avian and Two Pandemic H1N1 Influenza A Viruses

Background

Influenza A virus displays strong reassortment characteristics, which enable it to achieve adaptation in human infection. Surveying the reassortment and virulence of novel viruses is important in the prevention and control of an influenza pandemic. Meanwhile, studying the mechanism of reassortment may accelerate the development of anti-influenza strategies.

Methodology/Principal Findings

The hemagglutinin (HA) and neuraminidase (NA) matching patterns of two pandemic H1N1 viruses (the 1918 and current 2009 strains) and a highly pathogenic avian influenza A virus (H5N1) were studied using a pseudotyped particle (pp) system. Our data showed that four of the six chimeric HA/NA combinations could produce infectious pps, and that some of the chimeric pps had greater infectivity than did their ancestors, raising the possibility of reassortment among these viruses. The NA of H5N1 (A/Anhui/1/2005) could hardly reassort with the HAs of the two H1N1 viruses. Many biological characteristics of HA and NA, including infectivity, hemagglutinating ability, and NA activity, are dependent on their matching pattern.

Conclusions/Significance

Our data suggest the existence of an interaction between HA and NA, and the HA NA matching pattern is critical for valid viral reassortment.     

Hemagglutinin and neuraminidase matching patterns of two influenza A virus strains related to the 1918 and 2009 global pandemics

The current pandemic influenza A (H1N1) virus has revealed a complicated reassortment of various influenza A viruses. The biological study of these viruses, especially of the viral envelope proteins hemagglutinin (HA) and neuraminidase (NA), is urgently needed for the control and prevention of H1N1 viruses. We have generated H1N1-2009 and H1N1-1918 pseudotyped particles (pp) with high infectivity. Combinations of HA1918 + NA2009 and HA2009 + NA1918 also formed infectious H1N1pps, among which the HA2009 + NA1918 combination resulted in the most highly infectious pp. Our study demonstrated that some reassortments of H1N1 viruses may hold the potential to produce higher infectivity than do their ancestors.     

Genetic diversity of the 2009 pandemic influenza A(H1N1) viruses in Finland

Background

In Finland, the first infections caused by the 2009 pandemic influenza A(H1N1) virus were identified on May 10. During the next three months almost all infections were found from patients who had recently traveled abroad. In September 2009 the pandemic virus started to spread in the general population, leading to localized outbreaks and peak epidemic activity was reached during weeks 43–48.

Methods/Results

The nucleotide sequences of the hemagglutinin (HA) and neuraminidase (NA) genes from viruses collected from 138 patients were determined. The analyzed viruses represented mild and severe infections and different geographic regions and time periods. Based on HA and NA gene sequences, the Finnish pandemic viruses clustered in four groups. Finnish epidemic viruses and A/California/07/2009 vaccine virus strain varied from 2–8 and 0–5 amino acids in HA and NA molecules, respectively, giving a respective maximal evolution speed of 1.4% and 1.1%. Most amino acid changes in HA and NA molecules accumulated on the surface of the molecule and were partly located in antigenic sites. Three severe infections were detected with a mutation at HA residue 222, in two viruses with a change D222G, and in one virus D222Y. Also viruses with change D222E were identified. All Finnish pandemic viruses were sensitive to oseltamivir having the amino acid histidine at residue 275 of the neuraminidase molecule.

Conclusions

The Finnish pandemic viruses were quite closely related to A/California/07/2009 vaccine virus. Neither in the HA nor in the NA were changes identified that may lead to the selection of a virus with increased epidemic potential or exceptionally high virulence. Continued laboratory-based surveillance of the 2009 pandemic influenza A(H1N1) is important in order to rapidly identify drug resistant viruses and/or virus variants with potential ability to cause severe forms of infection and an ability to circumvent vaccine-induced immunity.     

A cross-neutralizing antibody between HIV-1 and influenza virus

Incessant antigenic evolution enables the persistence and spread of influenza virus in the human population. As the principal target of the immune response, the hemagglutinin (HA) surface antigen on influenza viruses continuously acquires and replaces N-linked glycosylation sites to shield immunogenic protein epitopes using host-derived glycans. Anti-glycan antibodies, such as 2G12, target the HIV-1 envelope protein (Env), which is even more extensively glycosylated and contains under-processed oligomannose-type clusters on its dense glycan shield. Here, we illustrate that 2G12 can also neutralize human seasonal influenza A H3N2 viruses that have evolved to present similar oligomannose-type clusters on their HAs from around 20 years after the 1968 pandemic. Using structural biology and mass spectrometric approaches, we find that two N-glycosylation sites close to the receptor binding site (RBS) on influenza hemagglutinin represent the oligomannose cluster recognized by 2G12. One of these glycan sites is highly conserved in all human H3N2 strains and the other emerged during virus evolution. These two N-glycosylation sites have also become crucial for fitness of recent H3N2 strains. These findings shed light on the evolution of the glycan shield on influenza virus and suggest 2G12-like antibodies can potentially act as broad neutralizers to target human enveloped viruses.     

Evolutionary analyses on the HA gene ofpandemic H1N1/09: early findings

The HA protein is responsible for influenza virus attachment and the subsequent fusion of viral and cellular membranes. Antigenic drift is driven by an accumulation of point mutations in the HA. And, the receptor-binding specificity of HA is responsible for the host range restriction of the virus. In April 2009, large outbreaks of novel H1N1 influenza in human population were reported from North America. The pandemic H1N1 virus originated from swine influenza virus. Evolutionary process of the pandemic virus after its introduction to human population remains to be clarified. We conducted phylogenetic analyses constructing a phylogenetic tree for and calculating site-by-site selective pressures in the HA gene. Phylogenetic tree showed that pandemic viruses were not clustered clearly by their geographical location or isolation time in the phylogenetic tree. The virus has been circulating the globe extensively with multiple introductions into most geographical areas. We found 3 sites positively selected in the HA gene for pandemic H1N1 virus. Among them, position 206 is located in an antigenic site. We did not find significant negative selection on any of the receptor binding sites. The virus has been evolving under unique selective pressure.     

Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals

Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3'SL-PAA and 6'SLN-PAA, which contained, respectively, 3'-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6'-sialyl(N-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6'SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q-->L, increased binding to 6'SLN-PAA, while among H1 swine viruses, the 190E-->D and 225G-->E mutations in the HA appeared important for the increased affinity of the viruses for 6'SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.     

Glycosylation on Hemagglutinin Affects the Virulence and Pathogenicity of Pandemic H1N1/2009 Influenza A Virus in Mice

The two glycosylation sites (Asn142 and Asn177) were observed in the HA of most human seasonal influenza A/H1N1 viruses, while none in pandemic H1N1/2009 influenza A (pH1N1) viruses. We investigated the effect of the two glycosylation sites on viral virulence and pathogenicity in mice using recombinant pH1N1. The H1N1/144 and H1N1/177 mutants which gained potential glycosylation sites Asn142 and Asn177 on HA respectively were generated from A/Mexico/4486/2009(H1N1) by site-directed mutagenesis and reverse genetics, the same as the H1N1/144+177 gained both glycosylation sites Asn142 and Asn177. The biological characteristics and antigenicity of the mutants were compared with wild-type pH1N1. The virulence and pathogenicity of recombinants were also detected in mice. Our results showed that HA antigenicity and viral affinity for receptor may change with introduction of the glycosylation sites. Compared with wild-type pH1N1, the mutant H1N1/177 displayed an equivalent virus titer in chicken embryos and mice, and increased virulence and pathogenicity in mice. The H1N1/144 displayed the highest virus titer in mice lung. However, the H1N1/144+177 displayed the most serious alveolar inflammation and pathogenicity in infected mice. The introduction of the glycosylation sites Asn144 and Asn177 resulted in the enhancement on virulence and pathogenicity of pH1N1 in mice, and was also associated with the change of HA antigenicity and the viral affinity for receptor.