A Bayesian approach to incorporate structural data into the mapping of genotype to antigenic phenotype of influenza A(H3N2) viruses.

Surface antigens of pathogens are commonly targeted by vaccine-elicited antibodies but antigenic variability, notably in RNA viruses such as influenza, HIV and SARS-CoV-2, pose challenges for control by vaccination. For example, influenza A(H3N2) entered the human population in 1968 causing a pandemic and has since been monitored, along with other seasonal influenza viruses, for the emergence of antigenic drift variants through intensive global surveillance and laboratory characterisation. Statistical models of the relationship between genetic differences among viruses and their antigenic similarity provide useful information to inform vaccine development, though accurate identification of causative mutations is complicated by highly correlated genetic signals that arise due to the evolutionary process. Here, using a sparse hierarchical Bayesian analogue of an experimentally validated model for integrating genetic and antigenic data, we identify the genetic changes in influenza A(H3N2) virus that underpin antigenic drift. We show that incorporating protein structural data into variable selection helps resolve ambiguities arising due to correlated signals, with the proportion of variables representing haemagglutinin positions decisively included, or excluded, increased from 59.8% to 72.4%. The accuracy of variable selection judged by proximity to experimentally determined antigenic sites was improved simultaneously. Structure-guided variable selection thus improves confidence in the identification of genetic explanations of antigenic variation and we also show that prioritising the identification of causative mutations is not detrimental to the predictive capability of the analysis. Indeed, incorporating structural information into variable selection resulted in a model that could more accurately predict antigenic assay titres for phenotypically-uncharacterised virus from genetic sequence. Combined, these analyses have the potential to inform choices of reference viruses, the targeting of laboratory assays, and predictions of the evolutionary success of different genotypes, and can therefore be used to inform vaccine selection processes.

Temporal and Gene Reassortment Analysis of Influenza C Virus Outbreaks in Hong Kong, SAR, China.

From 2014 to week 07/2020 the Centre for Health Protection in Hong Kong conducted screening for influenza C virus (ICV). A retrospective analysis of ICV detections to week 26/2019 revealed persistent low-level circulation with outbreaks occurring biennially in the winters of 2015 to 2016 and 2017 to 2018 (R. S. Daniels et al., J Virol 94:e01051-20, 2020, https://doi.org/10.1128/JVI.01051-20). Here, we report on an outbreak occurring in 2019 to 2020, reinforcing the observation of biennial seasonality in Hong Kong. All three outbreaks occurred in similar time frames, were subsequently dwarfed by seasonal epidemics of influenza types A and B, and were caused by similar proportions of C/Kanagawa/1/76 (K)-lineage and C/São Paulo/378/82 S1- and S2-sublineage viruses. Ongoing genetic drift was observed in all genes, with some evidence of amino acid substitution in the hemagglutinin-esterase-fusion (HEF) glycoprotein possibly associated with antigenic drift. A total of 61 ICV genomes covering the three outbreaks were analyzed for reassortment, and 9 different reassortant constellations were identified, 1 K-lineage, 4 S1-sublineage, and 4 S2-sublineage, with 6 of these being identified first in the 2019-1920 outbreak (2 S2-lineage and 4 S1-lineage). The roles that virus interference/enhancement, ICV persistent infection, genome evolution, and reassortment might play in the observed seasonality of ICV in Hong Kong are discussed. IMPORTANCE Influenza C virus (ICV) infection of humans is common, with the great majority of people being infected during childhood, though reinfection can occur throughout life. While infection normally results in "cold-like" symptoms, severe disease cases have been reported in recent years. However, knowledge of ICV is limited due to poor systematic surveillance and an inability to propagate the virus in large amounts in the laboratory. Following recent systematic surveillance in Hong Kong SAR, China, and direct ICV gene sequencing from clinical specimens, a 2-year cycle of disease outbreaks (epidemics) has been identified, with gene mixing playing a significant role in ICV evolution. Studies like those reported here are key to developing an understanding of the impact of influenza C virus infection in humans, notably where comorbidities exist and severe respiratory disease can develop.

Correction: Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans.

[This corrects the article DOI: 10.1371/journal.ppat.1009330.].

Correction: Breadth and function of antibody response to acute SARS-CoV-2 infection in humans.

[This corrects the article DOI: 10.1371/journal.ppat.1009352.].

Breadth and function of antibody response to acute SARS-CoV-2 infection in humans.

Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 3.5% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-receptor-binding domain (RBD), three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two recovered patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. Finally, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.

Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans.

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

Reduced sialidase activity of influenza A(H3N2) neuraminidase associated with positively charged amino acid substitutions.

Neuraminidase (NA) inhibitors (NAI), oseltamivir and zanamivir, are the main antiviral medications for influenza and monitoring of susceptibility to these antivirals is routinely done by determining 50 % inhibitory concentrations (IC50) with MUNANA substrate. During 2010-2019, levels of A(H3N2) viruses presenting reduced NAI inhibition (RI) were low (~0.75 %) but varied year-on-year. The highest proportions of viruses showing RI were observed during the 2013-2014, 2016-2017 and 2017-2018 Northern Hemisphere seasons. The majority of RI viruses were found to contain positively charged NA amino acid substitutions of N329K, K/S329R, S331R or S334R, being notably higher during the 2016-2017 season. Sialidase activity kinetics were determined for viruses of RI phenotype and contemporary wild-type (WT) viruses showing close genetic relatedness and displaying normal inhibition (NI). RI phenotypes resulted from reduced sialidase activity compared to relevant WT viruses. Those containing S329R or N329K or S331R showed markedly higher Km for the substrate and Ki values for NAIs, while those with S334R showed smaller effects. Substitutions at N329 and S331 disrupt a glycosylation sequon (NDS), confirmed to be utilised by mass spectrometry. However, gain of positive charge at all three positions was the major factor influencing the kinetic effects, not loss of glycosylation. Because of the altered enzyme characteristics NAs carrying these substitutions cannot be assessed reliably for susceptibility to NAIs using standard MUNANA-based assays due to reductions in the affinity of the enzyme for its substrate and the concentration of the substrate usually used.

Broadly Inhibiting Antineuraminidase Monoclonal Antibodies Induced by Trivalent Influenza Vaccine and H7N9 Infection in Humans.

The majority of antibodies induced by influenza neuraminidase (NA), like those against hemagglutinin (HA), are relatively specific to viruses isolated within a limited time window, as seen in serological studies and the analysis of many murine monoclonal antibodies (MAbs). We report three broadly reactive human MAbs targeting N1 NA. Two were isolated from a young adult vaccinated with trivalent influenza vaccine (TIV), which inhibited N1 NA from viruses isolated from humans over a period of a hundred years. The third antibody, isolated from a child with acute mild H7N9 infection, inhibited both group 1 N1 and group 2 N9 NAs. In addition, the antibodies cross-inhibited the N1 NAs of highly pathogenic avian H5N1 influenza viruses. These antibodies are protective in prophylaxis against seasonal H1N1 viruses in mice. This study demonstrates that human antibodies to N1 NA with exceptional cross-reactivity can be recalled by vaccination and highlights the importance of standardizing the NA antigen in seasonal vaccines to offer optimal protection.IMPORTANCE Antibodies to the influenza virus NA can provide protection against influenza disease. Analysis of human antibodies to NA lags behind that of antibodies to HA. We show that human monoclonal antibodies against NA induced by vaccination and infection can be very broadly reactive, with the ability to inhibit a wide spectrum of N1 NAs on viruses isolated between 1918 and 2018. This suggests that antibodies to NA may be a useful therapy and that the efficacy of influenza vaccines could be enhanced by ensuring the appropriate content of NA antigen.

Molecular Characterization of Influenza C Viruses from Outbreaks in Hong Kong SAR, China.

In 2014, the Centre for Health Protection in Hong Kong introduced screening for influenza C virus (ICV) as part of its routine surveillance for infectious agents in specimens collected from patients presenting with symptoms of respiratory viral infection, including influenza-like illness (ILI). A retrospective analysis of ICV detections up to week 26 of 2019 revealed persistent low-level circulation, with two outbreaks having occurred in the winters of 2015 to 2016 and 2017 to 2018. These outbreaks occurred at the same time as, and were dwarfed by, seasonal epidemics of influenza types A and B. Gene sequencing studies on stored ICV-positive clinical specimens from the two outbreaks have shown that the hemagglutinin-esterase (HE) genes of the viruses fall into two of the six recognized genetic lineages (represented by C/Kanagawa/1/76 and C/São Paulo/378/82), with there being significant genetic drift compared to earlier circulating viruses within both lineages. The location of a number of encoded amino acid substitutions in hemagglutinin-esterase fusion (HEF) glycoproteins suggests that antigenic drift may also have occurred. Observations of ICV outbreaks in other countries, with some of the infections being associated with severe disease, indicates that ICV infection has the potential to have significant clinical and health care impacts in humans.IMPORTANCE Influenza C virus infection of humans is common, and reinfection can occur throughout life. While symptoms are generally mild, severe disease cases have been reported, but knowledge of the virus is limited, as little systematic surveillance for influenza C virus is conducted and the virus cannot be studied by classical virologic methods because it cannot be readily isolated in laboratories. A combination of systematic surveillance in Hong Kong SAR, China, and new gene sequencing methods has been used in this study to assess influenza C virus evolution and provides evidence for a 2-year cycle of disease outbreaks. The results of studies like that reported here are key to developing an understanding of the impact of influenza C virus infection in humans and how virus evolution might be associated with epidemics.

Pandemic potential of highly pathogenic avian influenza clade 2.3.4.4 A(H5) viruses.

The panzootic caused by A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) A(H5) viruses has occurred in multiple waves since 1996. From 2013 onwards, clade 2.3.4.4 viruses of subtypes A(H5N2), A(H5N6), and A(H5N8) emerged to cause panzootic waves of unprecedented magnitude among avian species accompanied by severe losses to the poultry industry around the world. Clade 2.3.4.4 A(H5) viruses have expanded in distinct geographical and evolutionary pathways likely via long distance migratory bird dispersal onto several continents and by poultry trade among neighboring countries. Coupled with regional circulation, the viruses have evolved further by reassorting with local viruses. As of February 2019, there have been 23 cases of humans infected with clade 2.3.4.4 H5N6 viruses, 16 (70%) of which had fatal outcomes. To date, no HPAI A(H5) virus has caused sustainable human-to-human transmission. However, due to the lack of population immunity in humans and ongoing evolution of the virus, there is a continuing risk that clade 2.3.4.4 A(H5) viruses could cause an influenza pandemic if the ability to transmit efficiently among humans was gained. Therefore, multisectoral collaborations among the animal, environmental, and public health sectors are essential to conduct risk assessments and develop countermeasures to prevent disease and to control spread. In this article, we describe an assessment of the likelihood of clade 2.3.4.4 A(H5) viruses gaining human-to-human transmissibility and impact on human health should such human-to-human transmission occur. This structured analysis assessed properties of the virus, attributes of the human population, and ecology and epidemiology of these viruses in animal hosts.

Global circulation patterns of seasonal influenza viruses vary with antigenic drift.

Understanding the spatiotemporal patterns of emergence and circulation of new human seasonal influenza virus variants is a key scientific and public health challenge. The global circulation patterns of influenza A/H3N2 viruses are well characterized, but the patterns of A/H1N1 and B viruses have remained largely unexplored. Here we show that the global circulation patterns of A/H1N1 (up to 2009), B/Victoria, and B/Yamagata viruses differ substantially from those of A/H3N2 viruses, on the basis of analyses of 9,604 haemagglutinin sequences of human seasonal influenza viruses from 2000 to 2012. Whereas genetic variants of A/H3N2 viruses did not persist locally between epidemics and were reseeded from East and Southeast Asia, genetic variants of A/H1N1 and B viruses persisted across several seasons and exhibited complex global dynamics with East and Southeast Asia playing a limited role in disseminating new variants. The less frequent global movement of influenza A/H1N1 and B viruses coincided with slower rates of antigenic evolution, lower ages of infection, and smaller, less frequent epidemics compared to A/H3N2 viruses. Detailed epidemic models support differences in age of infection, combined with the less frequent travel of children, as probable drivers of the differences in the patterns of global circulation, suggesting a complex interaction between virus evolution, epidemiology, and human behaviour.

Receptor binding by H10 influenza viruses.

H10N8 follows H7N9 and H5N1 as the latest in a line of avian influenza viruses that cause serious disease in humans and have become a threat to public health. Since December 2013, three human cases of H10N8 infection have been reported, two of whom are known to have died. To gather evidence relating to the epidemic potential of H10 we have determined the structure of the haemagglutinin of a previously isolated avian H10 virus and we present here results relating especially to its receptor-binding properties, as these are likely to be major determinants of virus transmissibility. Our results show, first, that the H10 virus possesses high avidity for human receptors and second, from the crystal structure of the complex formed by avian H10 haemagglutinin with human receptor, it is clear that the conformation of the bound receptor has characteristics of both the 1918 H1N1 pandemic virus and the human H7 viruses isolated from patients in 2013 (ref. 3). We conclude that avian H10N8 virus has sufficient avidity for human receptors to account for its infection of humans but that its preference for avian receptors should make avian-receptor-rich human airway mucins an effective block to widespread infection. In terms of surveillance, particular attention will be paid to the detection of mutations in the receptor-binding site of the H10 haemagglutinin that decrease its avidity for avian receptor, and could enable it to be more readily transmitted between humans.

Detection of Influenza C Virus Infection among Hospitalized Patients, Cameroon.

We report 3 cases of influenza C virus in children hospitalized with severe acute respiratory infection in Cameroon. Two of these case-patients had grave clinical manifestations, but all 3 recovered. The lack of specific antiviral drugs for influenza C virus highlights the need to identify and describe cases involving this virus.

Segment 2 from influenza A(H1N1) 2009 pandemic viruses confers temperature-sensitive haemagglutinin yield on candidate vaccine virus growth in eggs that can be epistatically complemented by PB2 701D.

Candidate vaccine viruses (CVVs) for seasonal influenza A virus are made by reassortment of the antigenic virus with an egg-adapted strain, typically A/Puerto Rico/8/34 (PR8). Many 2009 A(H1N1) pandemic (pdm09) high-growth reassortants (HGRs) selected this way contain pdm09 segment 2 in addition to the antigenic genes. To investigate this, we made CVV mimics by reverse genetics (RG) that were either 6 : 2 or 5 : 3 reassortants between PR8 and two pdm09 strains, A/California/7/2009 (Cal7) and A/England/195/2009, differing in the source of segment 2. The 5 : 3 viruses replicated better in MDCK-SIAT1 cells than the 6 : 2 viruses, but the 6 : 2 CVVs gave higher haemagglutinin (HA) antigen yields from eggs. This unexpected phenomenon reflected temperature sensitivity conferred by pdm09 segment 2, as the egg HA yields of the 5 : 3 viruses improved substantially when viruses were grown at 35 °C compared with 37.5 °C, whereas the 6 : 2 virus yields did not. However, the authentic 5 : 3 pdm09 HGRs, X-179A and X-181, were not markedly temperature sensitive despite their PB1 sequences being identical to that of Cal7, suggesting compensatory mutations elsewhere in the genome. Sequence comparisons of the PR8-derived backbone genes identified polymorphisms in PB2, NP, NS1 and NS2. Of these, PB2 N701D affected the temperature dependence of viral transcription and, furthermore, improved and drastically reduced the temperature sensitivity of the HA yield from the 5 : 3 CVV mimic. We conclude that the HA yield of pdm09 CVVs can be affected by an epistatic interaction between PR8 PB2 and pdm09 PB1, but that this can be minimized by ensuring that the backbones used for vaccine manufacture in eggs contain PB2 701D.

Receptor binding by an H7N9 influenza virus from humans.

Of the 132 people known to have been infected with H7N9 influenza viruses in China, 37 died, and many were severely ill. Infection seems to have involved contact with infected poultry. We have examined the receptor-binding properties of this H7N9 virus and compared them with those of an avian H7N3 virus. We find that the human H7 virus has significantly higher affinity for α-2,6-linked sialic acid analogues ('human receptor') than avian H7 while retaining the strong binding to α-2,3-linked sialic acid analogues ('avian receptor') characteristic of avian viruses. The human H7 virus does not, therefore, have the preference for human versus avian receptors characteristic of pandemic viruses. X-ray crystallography of the receptor-binding protein, haemagglutinin (HA), in complex with receptor analogues indicates that both human and avian receptors adopt different conformations when bound to human H7 HA than they do when bound to avian H7 HA. Human receptor bound to human H7 HA exits the binding site in a different direction to that seen in complexes formed by HAs from pandemic viruses and from an aerosol-transmissible H5 mutant. The human-receptor-binding properties of human H7 probably arise from the introduction of two bulky hydrophobic residues by the substitutions Gln226Leu and Gly186Val. The former is shared with the 1957 H2 and 1968 H3 pandemic viruses and with the aerosol-transmissible H5 mutant. We conclude that the human H7 virus has acquired some of the receptor-binding characteristics that are typical of pandemic viruses, but its retained preference for avian receptor may restrict its further evolution towards a virus that could transmit efficiently between humans, perhaps by binding to avian-receptor-rich mucins in the human respiratory tract rather than to cellular receptors.

Receptor binding by a ferret-transmissible H5 avian influenza virus.

Cell-surface-receptor binding by influenza viruses is a key determinant of their transmissibility, both from avian and animal species to humans as well as from human to human. Highly pathogenic avian H5N1 viruses that are a threat to public health have been observed to acquire affinity for human receptors, and transmissible-mutant-selection experiments have identified a virus that is transmissible in ferrets, the generally accepted experimental model for influenza in humans. Here, our quantitative biophysical measurements of the receptor-binding properties of haemagglutinin (HA) from the transmissible mutant indicate a small increase in affinity for human receptor and a marked decrease in affinity for avian receptor. From analysis of virus and HA binding data we have derived an algorithm that predicts virus avidity from the affinity of individual HA-receptor interactions. It reveals that the transmissible-mutant virus has a 200-fold preference for binding human over avian receptors. The crystal structure of the transmissible-mutant HA in complex with receptor analogues shows that it has acquired the ability to bind human receptor in the same folded-back conformation as seen for HA from the 1918, 1957 (ref. 4), 1968 (ref. 5) and 2009 (ref. 6) pandemic viruses. This binding mode is substantially different from that by which non-transmissible wild-type H5 virus HA binds human receptor. The structure of the complex also explains how the change in preference from avian to human receptors arises from the Gln226Leu substitution, which facilitates binding to human receptor but restricts binding to avian receptor. Both features probably contribute to the acquisition of transmissibility by this mutant virus.

Association of Increased Receptor-Binding Avidity of Influenza A(H9N2) Viruses with Escape from Antibody-Based Immunity and Enhanced Zoonotic Potential.

We characterized 55 influenza A(H9N2) viruses isolated in Pakistan during 2014-2016 and found that the hemagglutinin gene is of the G1 lineage and that internal genes have differentiated into a variety of novel genotypes. Some isolates had up to 4-fold reduction in hemagglutination inhibition titers compared with older viruses. Viruses with hemagglutinin A180T/V substitutions conveyed this antigenic diversity and also caused up to 3,500-fold greater binding to avian-like and >20-fold greater binding to human-like sialic acid receptor analogs. This enhanced binding avidity led to reduced virus replication in primary and continuous cell culture. We confirmed that altered receptor-binding avidity of H9N2 viruses, including enhanced binding to human-like receptors, results in antigenic variation in avian influenza viruses. Consequently, current vaccine formulations might not induce adequate protective immunity in poultry, and emergence of isolates with marked avidity for human-like receptors increases the zoonotic risk.

Characterization of neutralizing epitopes in antigenic site B of recently circulating influenza A(H3N2) viruses.

Influenza A(H3N2) viruses are associated with outbreaks worldwide and can cause disease with severe complications. The impact can be reduced by vaccination, which induces neutralizing antibodies that mainly target the haemagglutinin glycoprotein (HA). In this study we generated neutralizing mouse monoclonal antibodies (mAbs) against A/Victoria/361/2011 and identified their epitopes by generating and sequencing escape viruses. The epitopes are located in antigenic site B, which is near the receptor-binding site and is immunodominant in humans. Amino acid (aa) substitutions at positions 156, 158, 159, 189, 190 and 193 in antigenic site B led to reduced ability of mAbs to block receptor-binding. The majority of A(H3N2) viruses that have been circulating since 2014 are antigenically distinct from previous A(H3N2) viruses. The neutralization-sensitive epitopes in antigenic site B of currently circulating viruses were examined with these mAbs. We found that clade 3C.2a viruses, possessing an additional potential glycosylation site at HA1 position N158, were poorly recognized by some of the mAbs, but other residues, notably at position 159, also affected antibody binding. Through a mass spectrometric (MS) analysis of HA, the glycosylated sites of HA1 were established and we determined that residue 158 of HA1 was glycosylated and so modified a neutralization-sensitive epitope. Understanding and monitoring individual epitopes is likely to improve vaccine strain selection.

Role of Neuraminidase in Influenza A(H7N9) Virus Receptor Binding.

Influenza A(H7N9) viruses have caused a large number of zoonotic infections since their emergence in 2013. They remain a public health concern due to the repeated high levels of infection with these viruses and their perceived pandemic potential. A major factor that determines influenza A virus fitness and therefore transmissibility is the interaction of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) with the cell surface receptor sialic acid. Typically, the HA is responsible for binding to the sialic acid to allow virus internalization and the NA is a sialidase responsible for cleaving sialic acid to aid virus spread and release. N9 NA has previously been shown to have receptor binding properties mediated by a sialic acid binding site, termed the hemadsorption (Hb) site, which is discrete from the enzymatically active sialidase site. This study investigated the N9 NA from a zoonotic H7N9 virus strain in order to determine its possible role in virus receptor binding. We demonstrate that this N9 NA has an active Hb site which binds to sialic acid, which enhances overall virus binding to sialic acid receptor analogues. We also show that the N9 NA can also contribute to receptor binding due to unusual kinetic characteristics of the sialidase site which specifically enhance binding to human-like α2,6-linked sialic acid receptors.IMPORTANCE The interaction of influenza A virus glycoproteins with cell surface receptors is a major determinant of infectivity and therefore transmissibility. Understanding these interactions is important for understanding which factors are necessary to determine pandemic potential. Influenza A viruses generally mediate binding to cell surface sialic acid receptors via the hemagglutinin (HA) glycoprotein, with the neuraminidase (NA) glycoprotein being responsible for cleaving the receptor to allow virus release. Previous studies showed that the NA proteins of the N9 subtype can bind sialic acid via a separate binding site distinct from the sialidase active site. This study demonstrates for purified protein and virus that the NA of the zoonotic H7N9 viruses has a binding capacity via both the secondary binding site and unusual kinetic properties of the sialidase site which promote receptor binding via this site and which enhance binding to human-like receptors. This could have implications for understanding human-to-human transmission of these viruses.

Identification of Low- and High-Impact Hemagglutinin Amino Acid Substitutions That Drive Antigenic Drift of Influenza A(H1N1) Viruses.

Determining phenotype from genetic data is a fundamental challenge. Identification of emerging antigenic variants among circulating influenza viruses is critical to the vaccine virus selection process, with vaccine effectiveness maximized when constituents are antigenically similar to circulating viruses. Hemagglutination inhibition (HI) assay data are commonly used to assess influenza antigenicity. Here, sequence and 3-D structural information of hemagglutinin (HA) glycoproteins were analyzed together with corresponding HI assay data for former seasonal influenza A(H1N1) virus isolates (1997-2009) and reference viruses. The models developed identify and quantify the impact of eighteen amino acid substitutions on the antigenicity of HA, two of which were responsible for major transitions in antigenic phenotype. We used reverse genetics to demonstrate the causal effect on antigenicity for a subset of these substitutions. Information on the impact of substitutions allowed us to predict antigenic phenotypes of emerging viruses directly from HA gene sequence data and accuracy was doubled by including all substitutions causing antigenic changes over a model incorporating only the substitutions with the largest impact. The ability to quantify the phenotypic impact of specific amino acid substitutions should help refine emerging techniques that predict the evolution of virus populations from one year to the next, leading to stronger theoretical foundations for selection of candidate vaccine viruses. These techniques have great potential to be extended to other antigenically variable pathogens.