Mutant Hemagglutinin Research Articles

HA gene amino acid mutations contribute to antigenic variation and immune escape of H9N2 influenza virus

Based on differences in the amino acid sequence of the protein haemagglutinin (HA), the H9N2 avian influenza virus (H9N2 virus) has been clustered into multiple lineages, and its rapidly ongoing evolution increases the difficulties faced by prevention and control programs. The HA protein, a major antigenic protein, and the amino acid mutations that alter viral antigenicity in particular have always been of interest. Likewise, it has been well documented that some amino acid mutations in HA alter viral antigenicity in the H9N2 virus, but little has been reported regarding how these antibody escape mutations affect antigenic variation. In this study, we were able to identify 15 HA mutations that were potentially relevant to viral antigenic drift, and we also found that a key amino acid mutation, A180V, at position 180 in HA (the numbering for mature H9 HA), the only site of the receptor binding sites that is not conserved, was directly responsible for viral antigenic variation. Moreover, the recombinant virus with alanine to valine substitution at position 180 in HA in the SH/F/98 backbone (rF/HAA180V virus) showed poor cross-reactivity to immune sera from animals immunized with the SH/F/98 (F/98, A180), SD/SS/94 (A180), JS/Y618/12 (T180), and rF/HAA180V (V180) viruses by microneutralization (MN) assay. The A180V substitution in the parent virus caused a significant decrease in cross-MN titres by enhancing the receptor binding activity, but it did not physically prevent antibody (Ab) binding. The strong receptor binding avidity prevented viral release from cells. Moreover, the A180V substitution promoted H9N2 virus escape from an in vitro pAb-neutralizing reaction, which also slightly affected the cross-protection in vivo. Our results suggest that the A180V mutation with a strong receptor binding avidity contributed to the low reactors in MN/HI assays and slightly affected vaccine efficacy but was not directly responsible for immune escape, which suggested that the A180V mutation might play a key role in the process of the adaptive evolution of H9N2 virus.

Adaptation of the H7N2 Feline Influenza Virus to Human Respiratory Cell Culture.

During 2016–2017, the H7N2 feline influenza virus infected more than 500 cats in animal shelters in New York, USA. A veterinarian who had treated the cats became infected with this feline virus and showed mild respiratory symptoms. This suggests that the H7N2 feline influenza virus may evolve into a novel pandemic virus with a high pathogenicity and transmissibility as a result of mutations in humans. In this study, to gain insight into the molecular basis of the transmission of the feline virus to humans, we selected mutant viruses with enhanced growth in human respiratory A549 cells via successive passages of the virus and found almost all mutations to be in the envelope glycoproteins, such as hemagglutinin (HA) and neuraminidase (NA). The reverse genetics approach revealed that the HA mutations, HA1-H16Q, HA2-I47T, or HA2-Y119H, in the stalk region can lead to a high growth of mutant viruses in A549 cells, possibly by changing the pH threshold for membrane fusion. Furthermore, NA mutation, I28S/L, or three-amino-acid deletion in the transmembrane region can enhance viral growth in A549 cells, possibly by changing the HA–NA functional balance. These findings suggest that the H7N2 feline influenza virus has the potential to become a human pathogen by adapting to human respiratory cells, owing to the synergistic biological effect of the mutations in its envelope glycoproteins.

The primary structure of the cytoplasmic tail domain of influenza hemagglutinin regulates its co-clustering with phosphatidylinositol 4,5-bisphosphate

The influenza glycoprotein hemagglutinin (HA) is highly involved in the attachment, entry, and assembly stages of the viral life cycle. Previous work has observed co-localization and evidence for an interaction between HA and the host cell lipid phosphatidylinositol 4,5-bisphosphate (PIP2) (Curthoys et al. 2019 Biophysical J. 116:893), although the details of this interaction are not yet fully understood. While mutations in HA occur frequently, in general there are 3 cysteines and 1-2 basic residues in the cytoplasmic tail domain (CTD) which remain highly conserved.

Selection of an Optimal Recombinant Egyptian H9N2 Avian Influenza Vaccine Strain for Poultry with High Antigenicity and Safety.

For the development of an optimized Egyptian H9N2 vaccine candidate virus for poultry, various recombinant Egyptian H9N2 viruses generated by a PR8-based reverse genetics system were compared in terms of their productivity and biosafety since Egyptian H9N2 avian influenza viruses already possess mammalian pathogenicity-related mutations in the hemagglutinin (HA), neuraminidase (NA), and PB2 genes. The Egyptian HA and NA genes were more compatible with PR8 than with H9N2 AIV (01310) internal genes, and the 01310-derived recombinant H9N2 strains acquired the L226Q reverse mutation in HA after passages in eggs. Additionally, the introduction of a strong promoter at the 3′-ends of PB2 and PB1 genes induced an additional mutation of P221S. When recombinant Egyptian H9N2 viruses with intact or reverse mutated HA (L226Q and P221S) and NA (prototypic 2SBS) were compared, the virus with HA and NA mutations had high productivity in ECES but was lower in antigenicity when used as an inactivated vaccine due to its high binding affinity into non-specific inhibitors in eggs. Finally, we substituted the PB2 gene of PR8 with 01310 to remove the replication ability in mammalian hosts and successfully generated the best recombinant vaccine candidate in terms of immunogenicity, antigenicity, and biosafety.

Identification of Hemagglutinin Mutations Caused by Neuraminidase Antibody Pressure

ABSTRACTThe balance in the functions of hemagglutinin (HA) and neuraminidase (NA) plays an important role in influenza virus genesis. However, whether and how N2 neuraminidase-specific antibodies may affect the attributes of HA remains to be investigated. In this study, we examined the presence of amino acid mutations in the HA of mutants selected by incubation with N2-specific monoclonal antibodies (MAbs) and compared the HA properties to those of the wild-type (WT) A/Chicken/Jiangsu/XXM/1999 (XXM) H9N2 virus. The higher NA inhibition (NI) ability of N2-specific MAbs was found to result in greater proportions of mutations in the HA head. The HA mutations affected the thermal stability, switched the binding preferences from α2,6-linked sialic acid receptor to α2,3-linked sialic acid receptor, and promoted viral growth in mouse lungs. These mutations also caused significant HA antigenic drift as they decreased hemagglutination inhibition (HI) titers. The evolutionary analysis also proved that some HA mutations were highly correlated with NA antibody pressure. Our data demonstrate that HA mutations caused by NA-specific antibodies affect HA properties and may contribute to HA evolution.IMPORTANCE HA binds with the sialic acid receptor on the host cell and initiates the infection mode of influenza virus. NA cleaves the connection between receptor and HA of newborn virus at the end of viral production. The HA-NA functional balance is crucial for viral production and interspecies transmission. Here, we identified mutations in the HA head of H9N2 virus caused by NA antibody pressure. These HA mutations changed the thermal stability and switched the receptor-binding preference of the mutant virus. The HI results indicated that these mutations resulted in significant antigenic drift in mutant HA. The evolutionary analysis also shows that some mutations in HA of H9N2 virus may be caused by NA antibody pressure and may correlate with the increase in H9N2 infections in humans. Our results provide new evidence for HA-NA balance and an effect of NA antibody pressure on HA evolution.

Graphical Method to Determine Sequence Variation on NA Protein of H5N1 Virus using Discrete Wavelet Transform

Influenza A virus belongs to the Orthomyxoviridae family and its genome is divided in eight distinct linear segments of negative-sense single stranded ribonucleic acid (RNA). Of all the eight influenza protein sequences, mutations in hemagglutinin and neuraminidase proteins show significant variations in their sequences. The threat of Influenza pandemic is ever rising due to its constant antigenic drift. Thus there is a need to characterize the genomic information in these viruses and signal processing methods offer the advantage of faster analysis in comparison to conventional techniques. Genomic information is converted into digital form by representation of the nucleotide bases in the form of mathematical sequences. In this paper, sequence variations of H5N1 virus have been studied using wavelet transforms as a signal analysis technique. Nucleotide sequences of neuraminidase protein of influenza virus occurring in different regions, in different hosts and over different years have been downloaded from National Centre for Biotechnology Information (NCBI) database. Sequences are aligned and converted into mathematical sequences then transformed using wavelet transforms. Graphical representations of the transformed sequences have been used to localise the regions of mutations along the sequence length.

Mutations of 127, 183 and 212 residues on the HA globular head affect the antigenicity, replication and pathogenicity of H9N2 avian influenza virus.

H9N2 avian influenza virus (AIV), one of the predominant subtypes devastating the poultry industry, has been circulating widely in the poultry population and causing huge economic losses. In this study, two H9N2 viruses with similar genetic backgrounds but different antigenicity were isolated from a poultry farm, namely A/chicken/Jiangsu/75/2018 (JS/75) and A/chicken/Jiangsu/76/2018 (JS/76). Sequence analysis revealed that their surface genes differed in three amino acid residues (127, 183 and 212) on the head of hemagglutinin (HA). To explore the differences between the two viruses in their biological features, six recombinant viruses, including the wild-type or mutant HA and NA of JS/75 and JS/76 were generated with A/Puerto Rico/8/1934 (PR8) backbone via reverse genetics. The chicken challenge study and HI assay data indicated that r-76/PR8 showed the most obvious antigen escape due to 127 and 183 amino acid substitutions in HA gene. Further studies verified that the 127N site was glycosylated in JS/76 and its mutants. Receptor-binding assays showed that all the recombination viruses were prone to bind the human-like receptors, except for the mutants which glycosylated 127N was deleted. Growth kinetics and mice challenge experiments indicated that 127N-glycosylated viruses showed less replication in A549 cells and lower pathogenicity in mice compared with wild-type viruses. Therefore, the glycosylation site and two amino acid alternations in the HA globular head were responsible for the differences in antigenicity and pathogenicity between the two H9N2 isolates. This study is significant in the research of the antigenic variation and vaccine updates for the H9N2 AIV. Also, highlighted the critical functions of glycosylation in the influenza virus on the pathogenicity against mammals.

Relationship between hemagglutinin stability and influenza virus persistence after exposure to low pH or supraphysiological heating.

The hemagglutinin (HA) surface glycoprotein is triggered by endosomal low pH to cause membrane fusion during influenza A virus (IAV) entry yet must remain sufficiently stable to avoid premature activation during virion transit between cells and hosts. HA activation pH and/or virion inactivation pH values less than pH 5.6 are thought to be required for IAV airborne transmissibility and human pandemic potential. To enable higher-throughput screening of emerging IAV strains for “humanized” stability, we developed a luciferase reporter assay that measures the threshold pH at which IAVs are inactivated. The reporter assay yielded results similar to TCID50 assay yet required one-fourth the time and one-tenth the virus. For four A/TN/09 (H1N1) HA mutants and 73 IAVs of varying subtype, virion inactivation pH was compared to HA activation pH and the rate of inactivation during 55°C heating. HA stability values correlated highly with virion acid and thermal stability values for isogenic viruses containing HA point mutations. HA stability also correlated with virion acid stability for human isolates but did not correlate with thermal stability at 55°C, raising doubt in the use of supraphysiological heating assays. Some animal isolates had virion inactivation pH values lower than HA activation pH, suggesting factors beyond HA stability can modulate virion stability. The coupling of HA activation pH and virion inactivation pH, and at a value below 5.6, was associated with human adaptation. This suggests that both virologic properties should be considered in risk assessment algorithms for pandemic potential.

Genetic Characterisation and Comparison of Mutations in Haemagglutinin & Neuraminidase Gene of Influenza A H1N1 pdm09 Between Two Outbreaks in South India

Genetic Characterisation and Comparison of Mutations in Haemagglutinin & Neuraminidase Gene of Influenza A H1N1 pdm09 Between Two Outbreaks in South India

Broadly Protective CD8+ T Cell Immunity to Highly Conserved Epitopes Elicited by Heat Shock Protein gp96-Adjuvanted Influenza Monovalent Split Vaccine.

Currently, immunization with inactivated influenza virus vaccines is the most prevalent method to prevent infections. However, licensed influenza vaccines provide only strain-specific protection and need to be updated and administered yearly; thus, new vaccines that provide broad protection against multiple influenza virus subtypes are required. In this study, we demonstrated that intradermal immunization with gp96-adjuvanted seasonal influenza monovalent H1N1 split vaccine could induce cross-protection against both group 1 and group 2 influenza A viruses in BALB/c mouse models. Vaccination in the presence of gp96 induced an apparently stronger antigen-specific T cell response than split vaccine alone. Immunization with the gp96-adjuvanted vaccine also elicited an apparent cross-reactive CD8+ T cell response that targeted the conserved epitopes across different influenza virus strains. These cross-reactive CD8+ T cells might be recalled from a pool of memory cells established after vaccination and recruited from extrapulmonary sites to facilitate viral clearance. Of note, six highly conserved CD8+ T epitopes from the viral structural proteins hemagglutinin (HA), M1, nucleoprotein (NP), and PB1 were identified to play a synergistic role in gp96-mediated cross-protection. Comparative analysis showed that most of conservative epitope-specific cytotoxic T lymphocytes (CTLs) apparently induced by heterologous virus infection were also activated by gp96-adjuvanted vaccine, thus resulting in broader protective CD8+ T cell responses. Our results demonstrated the advantage of adding gp96 to an existing seasonal influenza vaccine to improve its ability to provide better cross-protection.IMPORTANCE Owing to continuous mutations in hemagglutinin (HA) or neuraminidase (NA) or recombination of the gene segments between different strains, influenza viruses can escape the immune responses developed by vaccination. Thus, new strategies aimed to efficiently activate immune response that targets to conserved regions among different influenza viruses are urgently needed in designing broad-spectrum influenza vaccine. Heat shock protein gp96 is currently the only natural T cell adjuvant with special ability to cross-present coupled antigen to major histocompatibility complex class I (MHC-I) molecule and activate the downstream antigen-specific CTL response. In this study, we demonstrated the advantages of adding gp96 to monovalent split influenza virus vaccine to improve its ability to provide cross-protection in the BALB/c mouse model and proved that a gp96-activated cross-reactive CTL response is indispensable in our vaccine strategy. Due to its unique adjuvant properties, gp96 might be a promising adjuvant for designing new broad-spectrum influenza vaccines.

Can molecular dynamics explain decreased pathogenicity in mutant camphecene-resistant influenza virus?

ABSTARCT The development of new anti-influenza drugs remains an active area, and efforts in this direction will likely continue far into the future. In this paper, we present the results of a theoretical study explaining the mechanisms behind the antiviral activity of camphor derivatives. These include camphecene and a number of its analogues. The compounds tested can inhibit hemagglutinin (HA) by binding to it at two possible sites. Moreover, the binding site located at the site of proteolysis is the most important. Serial passaging of influenza in the presence of camphecene leads to the formation of mutation-associated resistance. Specifically, camphecene causes a significant mutation in HA (V615L). This substitution likely reduces the affinity of the compound for the binding site due to steric restriction of the positioning of camphecene in the binding cavity. Molecular dynamics (MD) simulation results show that the mutant HA is a more stable structure in terms of thermodynamics. In other words, launching conformational rearrangements preceding the transition from pre- to post-fusion requires more energy than in wild type HA. This may well explain the lower virulence seen with the camphecene-resistant strain.

Characterization of mutations of Haemagglutinin (HA) and Neuraminidase (NA) genes of Influenza A H1N1pdm09 isolated during the 2018 outbreak in India

Background: The influenza virus is a segmented RNA virus, has higher propensity to undergo mutations, which carry major implications, both clinically and epidemiologically. Mutations in HA and NA are of particular importance. Changes in the HA region are associated with increased virulence, antigenicity and host specificity as it is involved in receptor binding and membrane fusion. Hence is a major target for vaccine development. The NA gene is involved in virus release as well as drug susceptibility. These changes can trigger the emergence of new strains, which could lead to epidemics and pandemics. Therefore, it is important to monitor and characterize the mutations closely. Thus, this study was undertaken to understand the genetic variation in Influenza H1N1 pdm09 for the outbreak in 2018. Methods and materials: This study conducted in Regional Influenza Laboratory, Puducherry on the isolates positive for Influenza A pdm09 by real time RT PCR. Conventional PCR was done using the WHO primers. The PCR positive products were then subjected to Sanger sequencing in ABI3500. The sequences were then analysed on the MEGA X software and Flusurver software for detection of the mutations in HA and NA. Results: Phylogenetic analysis was performed for both HA and NA gene along with the clade-specific reference strains. All our isolates clustered along with the clade 6B.1. In HA region, of the 28 mutations documented, the most common mutation was R240Q (100%) followed by S181T, S200P and S91R. The mutation of N173K was detected for the 1st time globally in the antigenic site. Of the 12 mutations in the HA stem region, 4 new mutations documented – K71Q, V466L, R467T and Q529R. The most common mutations in NA observed were G77R, I188T, S200G/V, F74S/L and V81A. We documented 4 new mutations that could be associated with drug resistance – W179G, R225K, V267L and R293K. Conclusion: Continuous surveillance of Influenza virus is important to monitor the changes in the HA genes for providing recommendations for vaccine strain. Further, as the treatment options for influenza is limited, it makes it all the more important to have continuous surveillance to track the antiviral resistance.

Characterization of Novel Cross-Reactive Influenza B Virus Hemagglutinin Head Specific Antibodies That Lack Hemagglutination Inhibition Activity.

Humoral immune responses to influenza virus vaccines in elderly individuals are poorly adapted toward new antigenically drifted influenza virus strains. Instead, older individuals respond in an original antigenic sin fashion and produce much more cross-reactive but less potent antibodies. Here, we investigated four influenza B virus hemagglutinin (HA) head specific, hemagglutination inhibition-inactive monoclonal antibodies (MAbs) from elderly individuals. We found that they were broadly reactive within the B/Victoria/2/1987-like lineage, and two were highly cross-reactive with B/Yamagata/16/1988-like lineage viruses. The MAbs were found to be neutralizing, to utilize Fc effector functions, and to be protective against lethal viral challenge in a mouse model. In order to identify residues on the influenza B virus hemagglutinin interacting with the MAbs, we generated escape mutant viruses. Interestingly, escape from these MAbs led to numerous HA mutations within the head domain, including in the defined antigenic sites. We observed that each individual escape mutant virus was able to avoid neutralization by its respective MAb along with other MAbs in the panel, although in many cases binding activity was maintained. Point mutant viruses indicated that K90 is critical for the neutralization of two MAbs, while escape from the other two MAbs required a combination of mutations in the hemagglutinin. Three of four escape mutant viruses had increased lethality in the DBA2/J mouse model. Our work indicates that these cross-reactive antibodies have the potential to cause antigenic drift in the viral population by driving mutations that increase virus fitness. However, binding activity and cross-neutralization were maintained by a majority of antibodies in the panel, suggesting that this drift may not lead to escape from antibody-mediated protection.IMPORTANCE Understanding the immune response that older individuals mount to influenza virus vaccination and infection is critical in order to design better vaccines for this age group. Here, we show that older individuals make broadly neutralizing antibodies that have no hemagglutination-inhibiting activity and are less potent than strain-specific antibodies. These antibodies could drive viral escape from neutralization but did not result in escape from binding. Given their different mechanisms of action, they might retain protective activity even against escape variants.

Molecular evolution and characterization of hemagglutinin and neuraminidase of influenza A(H1N1)pdm09 viruses isolated in Beijing, China, during the 2017-2018 and 2018-2019 influenza seasons.

We investigated and analysed the molecular evolution of hemagglutinin (HA) and neuraminidase (NA) of influenza A(H1N1)pdm09 virus during the 2017-2018 and 2018-2019 influenza seasons in Beijing, China. We collected and extracted RNA from influenza A(H1N1)pdm09 strains from Peking University People's Hospital and analyzed their HA and NA genes by RT-PCR and sequencing. Phylogenetic analysis of HA and NA sequences was used to compare the amino acid sequences of 51 strains with those of reference strains. All strains belonged to subclade 6B.1, with S162N and I216T substitutions (H1 numbering). Our strains differed from strain A/Michigan/45/2015, with the substitutions S91R, S181T and I312V in the HA antigenic epitope. An E189G mutation was detected in the 190 helix of the receptor binding region of HA. A new potential glycosylation site, 179 (NQT), which was not detected before the 2015 influenza season, was identified. Two strains were mutated at I223, the NA inhibitor resistance site. During 2012-2019, amino acids of HA and NA mutated over time. Co-occurrence mutations N146D, S200P, S202I and A273T in HA appeared along with Q51K, F74S and D416N in NA in six strains during two influenza seasons. Our work reveals the molecular changes and phylogenetic characteristics of influenza A(H1N1)pdm09 virus and suggests that a vaccine probably provides suboptimal protection. The biological characteristics of the new glycosylation and drug-resistance sites detected in this work need to be studied further. The co-occurrence of mutations in HA and NA might affect the characteristics of the virus and need to be given more attention.

The Effect of I155T, K156Q, K156E and N186K Mutations in Hemagglutinin on the Virulence and Reproduction of Influenza A/H5N1 Viruses.

—The continued circulation of influenza A virus subtype H5 may cause the emergence of new potential pandemic virus variants, which can be transmitted from person to person. The occurrence of such variants is mainly related to mutations in hemagglutinin (HA). Previously we discovered mutations in H5N1 influenza virus hemagglutinin, which contributes to virus immune evasion. The purpose of this work was to study the role of these mutations in changing other, non-antigenic properties of the virus and the possibility of their maintenance in the viral population. Mutations were introduced into the HA gene of a recombinant H5N1 influenza A virus (VNH5N1-PR8/CDC-RG) using site-specific mutagenesis. The “variant” viruses were investigated and compared with respect to replication kinetics in chicken embryos, thermostability, reproductive activity at different temperatures (33, 37 and 40°C), and virulence for mice. Amino acid substitutions I155T, K156Q, K156E+V138A, N186K led to a decrease in thermal stability, replication activity of the mutant viruses in chicken embryos, and virulence for mice, although these effects differed between the variants. The K156Q and N186K mutations reduced viral reproduction at elevated temperature (40°C). The analysis of the frequency of these mutations in natural isolates of H5N1 influenza viruses indicated that the K156E/Q and N186K mutations have little chance to gain a foothold during evolution, in contrast to the I155T mutation, which is the most responsible for antigenic drift. The A138V and N186K mutations seem to be adaptive in mammalian viruses.

The Effects of Genetic Variation on H7N9 Avian Influenza Virus Pathogenicity.

Since the H7N9 avian influenza virus emerged in China in 2013, there have been five seasonal waves which have shown human infections and caused high fatality rates in infected patients. A multibasic amino acid insertion seen in the HA of current H7N9 viruses occurred through natural evolution and reassortment, and created a high pathogenicity avian influenza (HPAI) virus from the low pathogenicity avian influenza (LPAI) in 2017, and significantly increased pathogenicity in poultry, resulting in widespread HPAI H7N9 in poultry, which along with LPAI H7N9, contributed to the severe fifth seasonal wave in China. H7N9 is a novel reassorted virus from three different subtypes of influenza A viruses (IAVs) which displays a great potential threat to public health and the poultry industry. To date, no sustained human-to-human transmission has been recorded by the WHO. However, the high ability of evolutionary adaptation of H7N9 and lack of pre-existing immunity in humans heightens the pandemic potential. Changes in IAVs proteins can affect the viral transmissibility, receptor binding specificity, pathogenicity, and virulence. The multibasic amino acid insertion, mutations in hemagglutinin, deletion and mutations in neuraminidase, and mutations in PB2 contribute to different virological characteristics. This review summarized the latest research evidence to describe the impacts of viral protein changes in viral adaptation and pathogenicity of H7N9, aiming to provide better insights for developing and enhancing early warning or intervention strategies with the goal of preventing highly pathogenic IAVs circulation in live poultry, and transmission to humans.

Measuring Site-specific Glycosylation Similarity between Influenza a Virus Variants with Statistical Certainty

Influenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information may likely improve the ability to design vaccines with broader effectiveness against evolving strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between WT IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.

Mutation of the second sialic acid-binding site of influenza A virus neuraminidase drives compensatory mutations in hemagglutinin.

Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics. Most pandemics occurred upon adaptation of avian IAVs to humans. This adaptation includes a hallmark receptor-binding specificity switch of hemagglutinin (HA) from avian-type α2,3- to human-type α2,6-linked sialic acids. Complementary changes of the receptor-destroying neuraminidase (NA) are considered to restore the precarious, but poorly described, HA-NA-receptor balance required for virus fitness. In comparison to the detailed functional description of adaptive mutations in HA, little is known about the functional consequences of mutations in NA in relation to their effect on the HA-NA balance and host tropism. An understudied feature of NA is the presence of a second sialic acid-binding site (2SBS) in avian IAVs and absence of a 2SBS in human IAVs, which affects NA catalytic activity. Here we demonstrate that mutation of the 2SBS of avian IAV H5N1 disturbs the HA-NA balance. Passaging of a 2SBS-negative H5N1 virus on MDCK cells selected for progeny with a restored HA-NA balance. These viruses obtained mutations in NA that restored a functional 2SBS and/or in HA that reduced binding of avian-type receptors. Importantly, a particular HA mutation also resulted in increased binding of human-type receptors. Phylogenetic analyses of avian IAVs show that also in the field, mutations in the 2SBS precede mutations in HA that reduce binding of avian-type receptors and increase binding of human-type receptors. Thus, 2SBS mutations in NA can drive acquisition of mutations in HA that not only restore the HA-NA balance, but may also confer increased zoonotic potential.

Retrospective Assessment of the Antigenic Similarity of Egg-Propagated and Cell Culture-Propagated Reference Influenza Viruses as Compared with Circulating Viruses across Influenza Seasons 2002-2003 to 2017-2018.

Suboptimal vaccine effectiveness against seasonal influenza is a significant public health concern, partly explained by antigenic differences between vaccine viruses and viruses circulating in the environment. Haemagglutinin mutations within vaccine viruses acquired during serial passage in eggs have been identified as a source of antigenic variation between vaccine and circulating viruses. This study retrospectively compared the antigenic similarity of circulating influenza isolates with egg- and cell-propagated reference viruses to assess any observable trends over a 16-year period. Using annual and interim reports published by the Worldwide Influenza Centre, London, for the 2002–2003 to 2017–2018 influenza seasons, we assessed the proportions of circulating viruses which showed antigenic similarity to reference viruses by season. Egg-propagated reference viruses were well matched against circulating viruses for A/H1N1 and B/Yamagata. However, A/H3N2 and B/Victoria cell-propagated reference viruses appeared to be more antigenically similar to circulating A/H3N2 and B/Victoria viruses than egg-propagated reference viruses. These data support the possibility that A/H3N2 and B/Victoria viruses are relatively more prone to egg-adaptive mutation. Cell-propagated A/H3N2 and B/Victoria reference viruses were more antigenically similar to circulating A/H3N2 and B/Victoria viruses over a 16-year period than were egg-propagated reference viruses.

Genetic Sequencing of Influenza A (H1N1) pdm09 Isolates from South India, Collected between 2011 and 2015 to Detect Mutations Affecting Virulence and Resistance to Oseltamivir

AbstractBackground: Influenza A viruses evolve continuously and the two surface antigens, hemagglutinin (HA) and neuraminidase (NA) have been the target proteins for research as they are vital components in determining the virulence, immune effectiveness, pathogenicity, transmission and resistance. Methods: Both HA and NA (partial genes) of 45 pandemic influenza A(H1N1)pdm09 isolates were sequenced. Phylogenetic analysis was performed with reference to representative global isolates retrieved from Influenza Virus Resource (IVR), GISAID EpiFluTM and GenBank and evolutionary analyses. Nucleotide and amino acid sequences were aligned using ClustalW/Clustal Omega/MEGA version 6 with reference to vaccine strain (A/California/07/2009). Results: All the isolates clustered along with the clade 7 virus, irrespective of the year of isolation. The study isolates exhibited 98.5% and 98.8% nucleotide homology to the reference strain A/California/07/2009(H1N1) for HA and NA, respectively. Overall, there was limited genetic diversity observed over a period of 3 years (2012-2015). Two samples collected from expired patients had D239N (D222G or D225G) mutation in HA. This mutation which is associated with dual-binding specificity of the virus has been well-correlated with severe disease outcomes. All the study isolates possessed H274 residue and 7 strains had N295S, the next most common mutation found in oseltamivir-resistant variants. Conclusion: In this study, although H274Y mutation associated with oseltamivir resistance has not been noted, significant mutations have been noted in both HA and NA genes including D239N, N295S, V106I, Q136K, N248D, V267A. In both HA and NA gene analysis, multiple mutations were found more in 2015 strains when compared to 2012 strains. Hence such accumulation of mutations has to be monitored continuously to determine the efficacy of annual flu vaccines and anti-influenza drugs.