Hemagglutinin Receptor Binding Site Research Articles

Computational screening of phytocompounds from C. amboinicus identifies potential inhibitors of influenza A (H3N2) virus by targeting hemagglutinin

The H3N2 subtype of the influenza A virus continues to be a notable public health issue due to its association with seasonal epidemics and severe human morbidity. The constrained effectiveness of current antiviral medications, combined with the inevitable emergence of drug-resistant variants, mandates the exploration of innovative therapeutic approaches. This study focuses on the identification of phytocompounds from Coleus amboinicus with the potential to target hemagglutinin, viral protein involved in viral entry by binding to sialyl glycoconjugates receptors on the surface of host cells. Molecular docking studies were carried out to assess the efficacy of C. amboinicus phytocompounds with hemagglutinin receptor-binding site. The study revealed that among the 84 signature phytocompounds, isosalvianolic acid and salvianolic acid C showed the highest docking scores and favourable intermolecular interactions. Pharmacokinetic analysis and Pan-assay interference compounds (PAINS) filtering confirmed that isosalvianolic acid meets the criteria outlined in Lipinski’s rule of five, exhibits favourable ADMET profiles and passes PAINS filters. Furthermore, the molecular dynamics simulations followed by radius of gyration (Rg), solvent accessible surface area (SASA), and MM-PBSA calculations for binding free energy, verified the stability of the docked complexes. Together, the study identifies isosalvianolic acid as a promising inhibitor of the H3N2 virus by binding to hemagglutinin, indicating its potential as a strategy for therapeutic intervention.

Genetic characteristics of influenza A and B viruses circulating in Russia in 2019-2023.

Relevance. Influenza viruses have a high potential for genetic change. These viruses are monitored annually around the world, including Russia, to determine the dominant genetic groups and select the strains to be included in influenza vaccines. Aims of the study include: analysis of the circulation of influenza viruses in Russia in 2019-2023, phylogenetic and molecular analysis of hemagglutinin (HA) sequences of influenza virus strains, detection of drug resistance mutations to neuraminidase (NA) inhibitors and M2-protein (M2) ion channel inhibitors. Materials and methods. Biological samples containing RNA of influenza viruses were studied: 417 A(H1N1)pdm09, 147 A(H3N2) and 167 B(Victoria). Sequencing of the PCR fragments was performed on the 3500xL Genetic Analyzer (Applied Biosytems). Data processing and analysis were carried out using DNASTAR, Nextclade, FluSurver and BioNumerics v.6.6 software. Results. Influenza A(H1N1)pdm09, A(H3N2), B(Victoria) viruses circulating in 2019-2023 were investigated. The highest variability of HA was observed in A(H3N2) viruses. 3% of A(H1N1)pdm09 viruses had the D222N mutation in the receptor-binding site of HA, which is associated with more severe disease. The oseltamivir and zanamivir resistance mutation H275Y in NA was detected in 2% of influenza A(H1N1)pdm09 viruses. No oseltamivir and zanamivir resistance mutations in NA were detected in all influenza A(H3N2) and B viruses tested. In all investigated influenza A(H1N1)pdm09 and A(H3N2) viruses the adamantane resistance mutation S31N in M2 was identified. Conclusions. The detection of amino acid substitutions in HA antigenic sites and resistance mutations in NA and M2 confirms the evolution of influenza viruses and the need for continuous genetic surveillance.

Natural variation in neuraminidase activity influences the evolutionary potential of the seasonal H1N1 lineage hemagglutinin.

The antigenic evolution of the influenza A virus hemagglutinin (HA) gene poses a major challenge for the development of vaccines capable of eliciting long-term protection. Prior efforts to understand the mechanisms that govern viral antigenic evolution mainly focus on HA in isolation, ignoring the fact that HA must act in concert with the viral neuraminidase (NA) during replication and spread. Numerous studies have demonstrated that the degree to which the receptor-binding avidity of HA and receptor-cleaving activity of NA are balanced with each other influences overall viral fitness. We recently showed that changes in NA activity can significantly alter the mutational fitness landscape of HA in the context of a lab-adapted virus strain. Here, we test whether natural variation in relative NA activity can influence the evolutionary potential of HA in the context of the seasonal H1N1 lineage (pdmH1N1) that has circulated in humans since the 2009 pandemic. We observed substantial variation in the relative activities of NA proteins encoded by a panel of H1N1 vaccine strains isolated between 2009 and 2019. We comprehensively assessed the effect of NA background on the HA mutational fitness landscape in the circulating pdmH1N1 lineage using deep mutational scanning and observed pronounced epistasis between NA and residues in or near the receptor-binding site of HA. To determine whether NA variation could influence the antigenic evolution of HA, we performed neutralizing antibody selection experiments using a panel of monoclonal antibodies targeting different HA epitopes. We found that the specific antibody escape profiles of HA were highly contingent upon NA background. Overall, our results indicate that natural variation in NA activity plays a significant role in governing the evolutionary potential of HA in the currently circulating pdmH1N1 lineage.

Epistasis mediates the evolution of the receptor binding mode in recent human H3N2 hemagglutinin

The receptor-binding site of influenza A virus hemagglutinin partially overlaps with major antigenic sites and constantly evolves. In this study, we observe that mutations G186D and D190N in the hemagglutinin receptor-binding site have coevolved in two recent human H3N2 clades. X-ray crystallography results show that these mutations coordinately drive the evolution of the hemagglutinin receptor binding mode. Epistasis between G186D and D190N is further demonstrated by glycan binding and thermostability analyses. Immunization and neutralization experiments using mouse and human samples indicate that the evolution of receptor binding mode is accompanied by a change in antigenicity. Besides, combinatorial mutagenesis reveals that G186D and D190N, along with other natural mutations in recent H3N2 strains, alter the compatibility with a common egg-adaptive mutation in seasonal influenza vaccines. Overall, our findings elucidate the role of epistasis in shaping the recent evolution of human H3N2 hemagglutinin and substantiate the high evolvability of its receptor-binding mode.

Molecular modeling and phylogenetic analyses highlight the role of amino acid 347 of the N1 subtype neuraminidase in influenza virus host range and interspecies adaptation.

The N1 neuraminidases (NAs) of avian and pandemic human influenza viruses contain tyrosine and asparagine, respectively, at position 347 on the rim of the catalytic site; the biological significance of this difference is not clear. Here, we used molecular dynamics simulation to model the effects of amino acid 347 on N1 NA interactions with sialyllacto-N-tetraoses 6'SLN-LC and 3'SLN-LC, which represent NA substrates in humans and birds, respectively. Our analysis predicted that Y347 plays an important role in the NA preference for the avian-type substrates. The Y347N substitution facilitates hydrolysis of human-type substrates by resolving steric conflicts of the Neu5Ac2-6Gal moiety with the bulky side chain of Y347, decreasing the free energy of substrate binding, and increasing the solvation of the Neu5Ac2-6Gal bond. Y347 was conserved in all N1 NA sequences of avian influenza viruses in the GISAID EpiFlu database with two exceptions. First, the Y347F substitution was present in the NA of a specific H6N1 poultry virus lineage and was associated with the substitutions G228S and/or E190V/L in the receptor-binding site (RBS) of the hemagglutinin (HA). Second, the highly pathogenic avian H5N1 viruses of the Gs/Gd lineage contained sporadic variants with the NA substitutions Y347H/D, which were frequently associated with substitutions in the HA RBS. The Y347N substitution occurred following the introductions of avian precursors into humans and pigs with N/D347 conserved during virus circulation in these hosts. Comparative evolutionary analysis of site 347 revealed episodic positive selection across the entire tree and negative selection within most host-specific groups of viruses, suggesting that substitutions at NA position 347 occurred during host switches and remained under pervasive purifying selection thereafter. Our results elucidate the role of amino acid 347 in NA recognition of sialoglycan substrates and emphasize the significance of substitutions at position 347 as a marker of host range and adaptive evolution of influenza viruses.

An epitope-enriched immunogen expands responses to a conserved viral site.

Pathogens evade host humoral responses by accumulating mutations in surface antigens. While variable, there are conserved regions that cannot mutate without compromising fitness. Antibodies targeting these conserved epitopes are often broadly protective but remain minor components of the repertoire. Rational immunogen design leverages a structural understanding of viral antigens to modulate humoral responses to favor these responses. Here, we report an epitope-enriched immunogen presenting a higher copy number of the influenza hemagglutinin (HA) receptor-binding site (RBS) epitope relative to other B cell epitopes. Immunization in a partially humanized murine model imprinted with an H1 influenza shows H1-specific serum and >99% H1-specific B cells being RBS-directed. Single B cell analyses show a genetically restricted response that structural analysis defines as RBS-directed antibodies engaging the RBS with germline-encoded contacts. These data show how epitope enrichment expands B cell responses toward conserved epitopes and advances immunogen design approaches for next-generation viral vaccines.

Substitutions near the HA receptor binding site explain the origin and major antigenic change of the B/Victoria and B/Yamagata lineages

Influenza B virus primarily infects humans, causing seasonal epidemics globally. Two antigenic variants-Victoria-like and Yamagata-like-were detected in the 1980s, of which the molecular basis of emergence is still incompletely understood. Here, the antigenic properties of a unique collection of historical virus isolates, sampled from 1962 to 2000 and passaged exclusively in mammalian cells to preserve antigenic properties, were determined with the hemagglutination inhibition assay and an antigenic map was built to quantify and visualize the divergence of the lineages. The antigenic map revealed only three distinct antigenic clusters-Early, Victoria, and Yamagata-with relatively little antigenic diversity in each cluster until 2000. Viruses with Victoria-like antigenic properties emerged around 1972 and diversified subsequently into two genetic lineages. Viruses with Yamagata-like antigenic properties evolved from one lineage and became clearly antigenically distinct from the Victoria-like viruses around 1988. Recombinant mutant viruses were tested to show that insertions and deletions (indels), as observed frequently in influenza B virus hemagglutinin, had little effect on antigenic properties. In contrast, amino-acid substitutions at positions 148, 149, 150, and 203, adjacent to the hemagglutinin receptor binding site, determined the main antigenic differences between the Early, Victoria-like, and Yamagata-like viruses. Surprisingly, substitutions at two of the four positions reverted in recent viruses of the Victoria lineage, resulting in antigenic properties similar to viruses circulating ∼50 y earlier. These data shed light on the antigenic diversification of influenza viruses and suggest there may be limits to the antigenic evolution of influenza B virus.

Haemagglutinin substitutions N125D, D127E, D222G and R223Q improve replicative fitness and vaccine effectiveness of an A/H1N1pdm09 live attenuated influenza vaccine virus by enhancing α-2,6 receptor binding.

During 2013–14 and 2015–16, A/H1N1pdm09 live attenuated influenza vaccine (LAIV) viruses replicated inefficiently in primary human nasal epithelial cells (hNEC). This led to reduced vaccine effectiveness (VE) in quadrivalent formulations, mediated by inter-strain competition. By mutating the haemagglutinin (HA) protein, we aimed to enhance hNEC replication of a novel A/H1N1pdm09 vaccine strain to overcome competition and improve VE. Combinations of N125D, D127E, D222G and R223Q substitutions were introduced to the HA protein of A/Slovenia/2903/2015 (A/SLOV15). A/SLOV15 S13, containing all four HA substitutions, produced approximately 1000-fold more virus than parental V1 during hNEC infection. Immunogenicity in ferrets was increased by approximately 10-fold, without compromising yield in eggs or antigenic match to wild-type (wt) reference strains. Despite S13 and V1 being antigenically similar, only S13 protected ferrets from wt virus shedding and fever post-challenge. Crucially, these data suggested that enhanced fitness allowed S13 to overcome inter-strain competition in quadrivalent LAIV (QLAIV). This improved efficacy was later validated by real-world VE data. S13 displayed increased binding avidity to a mammalian-like α-2,6 receptor analogue (6-SLN), relative to V1, while maintaining avian-like 3-SLN avidity. In silico modelling of the HA receptor binding site revealed additional interactions in the S13:6-SLN binding network and a mild increase in 6-SLN binding energy, indicating a possible mechanism for increased α-2,6 receptor-binding avidity. These data confirm that rational HA mutagenesis can be used to optimise hNEC replication and VE for A/H1N1pdm09 LAIV viruses.

H3N2 Influenza Viruses with 12- or 16-Amino Acid Deletions in the Receptor-Binding Region of Their Hemagglutinin Protein.

ABSTRACTHuman influenza viruses evade host immune responses by accumulating mutations around the receptor-binding region of the hemagglutinin (HA) protein, which is composed of three key elements, the 130-loop, the 190-helix, and the 220-loop. Here, we characterized two human H3N2 influenza viruses with 12- and 16-amino acid deletions around the HA receptor-binding site that were isolated after antigenic selection of mutated H3N2 viruses. Structural modeling suggested that the 12-amino acid deletion eliminated the 190-helix. The 16-amino acid deletion comprises two stretches of 11- and 5-amino acid deletions. As the result of a frameshift, “novel” amino acids (not found in wild-type HA at these positions) are encoded between the deleted regions. Interestingly, structural modeling predicted that the novel sequence forms a structure resembling the 190-helix. However, compared to wild-type HA, the 16-amino acid deletion mutant lacks two antiparallel beta-sheets that connect the 190-helix and the 220-loop in wild-type HA. Nonetheless, both HA deletion mutants replicated in mammalian cells, and the 16-amino acid deletion mutant (with a remodeled 190-helix) also replicated in Syrian hamsters, albeit at low titers. Wild-type virus bound preferentially to α2,6-linked sialic acids, whereas both mutants gained affinity for α2,3-linked sialic acids. Moreover, the 12- and 16-amino acid deletions may affect the antigenic properties of the viruses. Thus, viruses with sizeable deletions around the HA receptor-binding site are viable but may display altered sialic acid preferences, altered antigenic properties, and attenuated replicative ability in cultured cells and virulence in Syrian hamsters.

Altering the Immunogenicity of Hemagglutinin Immunogens by Hyperglycosylation and Disulfide Stabilization.

Influenza virus alters glycosylation patterns on its surface exposed glycoproteins to evade host adaptive immune responses. The viral hemagglutinin (HA), in particular the H3 subtype, has increased its overall surface glycosylation since its introduction in 1968. We previously showed that modulating predicted N-linked glycosylation sites on H3 A/Hong Kong/1/1968 HA identified a conserved epitope at the HA interface. This epitope is occluded on the native HA trimer but is likely exposed during HA “breathing” on the virion surface. Antibodies directed to this site are protective via an ADCC-mediated mechanism. This glycan engineering strategy made an otherwise subdominant epitope dominant in the murine model. Here, we asked whether cysteine stabilization of the hyperglycosylated HA trimer could reverse this immunodominance by preventing access to the interface epitope and focus responses to the HA receptor binding site (RBS). While analysis of serum responses from immunized mice did not show a redirection to the RBS, cysteine stabilization did result in an overall reduction in immunogenicity of the interface epitope. Thus, glycan engineering and cysteine stabilization are two strategies that can be used together to alter immunodominance patterns to HA. These results add to rational immunogen design approaches used to manipulate immune responses for the development of next-generation influenza vaccines.

Evaluation of Multivalent Sialylated Polyglycerols for Resistance Induction in and Broad Antiviral Activity against Influenza A Viruses.

The development of multivalent sialic acid-based inhibitors active against a variety of influenza A virus (IAV) strains has been hampered by high genetic and structural variability of the targeted viral hemagglutinin (HA). Here, we addressed this challenge by employing sialylated polyglycerols (PGs). Efficacy of prototypic PGs was restricted to a narrow spectrum of IAV strains. To understand this restriction, we selected IAV mutants resistant to a prototypic multivalent sialylated PG by serial passaging. Resistance mutations mapped to the receptor binding site of HA, which was accompanied by altered receptor binding profiles of mutant viruses as detected by glycan array analysis. Specifying the inhibitor functionalization to 2,6-α-sialyllactose (SL) and adjusting the linker yielded a rationally designed inhibitor covering an extended spectrum of inhibited IAV strains. These results highlight the importance of integrating virological data with chemical synthesis and structural data for the development of sialylated PGs toward broad anti-influenza compounds.

Sequence dynamics of three influenza A virus strains grown in different MDCK cell lines, including those expressing different sialic acid receptors.

Viruses are often cultured in cell lines for research and vaccine development, and those often differ from the natural hosts or tissues. Cell lines can also differ in the presence of virus receptors, such as the sialic acid (Sia) receptors used by influenza A viruses (IAV), which can vary in linkage (α2,3- or α2,6-linkage) and form (N-glycolylneuraminic acid [Neu5Gc] or N-acetylneuraminic acid [Neu5Ac]). The selective pressures resulting from passaging viruses in cell types with host-specific variations in viral receptors are still only partially understood. IAV are commonly cultured in MDCK cells which are both derived from canine kidney tubule epithelium and inherently heterogeneous. MDCK cells naturally present Neu5Ac and α2,3-linked Sia forms. Here, we examine natural MDCK variant lineages, as well as engineered variants that synthesize Neu5Gc and/or α2,6-linkages. We determined how viral genetic variation occurred within human H3N2, H1N1 pandemic and canine H3N2 IAV populations when serially passaged in MDCK cell lines that vary in cell type (MDCK-Type I or MDCK-Type II clones) and in Sia display. Deep sequencing of viral genomes showed small numbers of consensus-level mutations, mostly within the hemagglutinin (HA) gene. Both human IAV showed variants in the HA stem and the HA receptor-binding site of populations passaged in cells displaying Neu5Gc. Canine H3N2 showed variants near the receptor-binding site when passaged in cells displaying Neu5Gc or α2,6-linkages. Viruses replicated to low titres in MDCK-Type II cells, suggesting that not all cell types in heterogeneous MDCK cell populations are equally permissive to infection.

Sulfated glycans containing NeuAcα2-3Gal facilitate the propagation of human H1N1 influenza A viruses in eggs

When human influenza viruses are isolated and passaged in chicken embryos, variants with amino acid substitutions around the receptor binding site of hemagglutinin (HA) are selected; however, the mechanisms that underlie this phenomenon have yet to be elucidated. Here, we analyzed the receptor structures that contributed to propagation of egg-passaged human H1N1 viruses. The analysis included seasonal and 2009 pandemic strains, both of which have amino acid substitutions of HA found in strains isolated or passaged in eggs. These viruses exhibited high binding to sulfated glycans containing NeuAcα2-3Gal. In MDCK cells overexpressing the sulfotransferase that synthesize Galβ1-4(SO3--6)GlcNAc, production of human H1N1 viruses was increased up to 90-fold. Furthermore, these sulfated glycans were expressed on the allantoic and amniotic membranes of chicken embryos. These results suggest that 6-sulfo sialyl Lewis X and/or NeuAcα2-3Galβ1-4(SO3--6)GlcNAc are involved in efficient propagation of human H1N1 viruses in chicken embryos.

Virus Adaptation Following Experimental Infection of Chickens with a Domestic Duck Low Pathogenic Avian Influenza Isolate from the 2017 USA H7N9 Outbreak Identifies Polymorphic Mutations in Multiple Gene Segments.

In March 2017, highly pathogenic (HP) and low pathogenic (LP) avian influenza virus (AIV) subtype H7N9 were detected from poultry farms and backyard birds in several states in the southeast United States. Because interspecies transmission is a known mechanism for evolution of AIVs, we sought to characterize infection and transmission of a domestic duck-origin H7N9 LPAIV in chickens and genetically compare the viruses replicating in the chickens to the original H7N9 clinical field samples used as inoculum. The results of the experimental infection demonstrated virus replication and transmission in chickens, with overt clinical signs of disease and shedding through both oral and cloacal routes. Unexpectedly, higher levels of virus shedding were observed in some cloacal swabs. Next generation sequencing (NGS) analysis identified numerous non-synonymous mutations at the consensus level in the polymerase genes (i.e., PA, PB1, and PB2) and the hemagglutinin (HA) receptor binding site in viruses recovered from chickens, indicating possible virus adaptation in the new host. For comparison, NGS analysis of clinical samples obtained from duck specimen collected during the outbreak indicated three polymorphic sides in the M1 segment and a minor population of viruses carrying the D139N (21.4%) substitution in the NS1 segment. Interestingly, at consensus level, A/duck/Alabama (H7N9) had isoleucine at position 105 in NP protein, similar to HPAIV (H7N9) but not to LPAIV (H7N9) isolated from the same 2017 influenza outbreak in the US. Taken together, this work demonstrates that the H7N9 viruses could readily jump between avian species, which may have contributed to the evolution of the virus and its spread in the region.

Neutralizing antibody PR8-23 targets the footprint of the sialoglycan receptor binding site of H1N1 hemagglutinin.

Influenza virus cause seasonal influenza epidemic and seriously sporadic influenza pandemic outbreaks. Hemagglutinin (HA) is an important target in the therapeutic treatment and diagnostic detection of the influenza virus. Variation in the sialic acid receptor binding site leads to strain-specific binding and results in different binding modes to the host receptors. Here, we evaluated the neutralizing activity and hemagglutination inhibition activity of a prepared murine anti-H1N1 monoclonal antibody PR8-23. Then we identified the epitope peptide of antibody PR8-23 by phage display technique from phage display peptide libraries. The identified epitope, 63-IAPLQLGKCNIA-74, containing two α-helix and two β-fold located at the footprint of the sialoglycan receptor on the RBS in the globular head domain of HA. It broads the growing arsenal of motifs for the amino acids on the globular head domain of HA in sialic acid receptor binding site and neutralizing antibody production.

Genetic determinants of receptor-binding preference and zoonotic potential of H9N2 avian influenza viruses.

Receptor recognition and binding is the first step of viral infection and a key determinant of host specificity. The inability of avian influenza viruses to effectively bind human-like sialylated receptors is a major impediment to their efficient transmission in humans and pandemic capacity. Influenza H9N2 viruses are endemic in poultry across Asia and parts of Africa where they occasionally infect humans and are therefore considered viruses with zoonotic potential. We previously described H9N2 viruses, including several isolated from human zoonotic cases, showing a preference for human-like receptors. Here we take a mutagenesis approach, making viruses with single or multiple substitutions in H9 haemagglutinin and test binding to avian and human receptor analogues using biolayer interferometry. We determine the genetic basis of preferences for alternative avian receptors and for human-like receptors, describing amino acid motifs at positions 190, 226 and 227 that play a major role in determining receptor specificity, and several other residues such as 159, 188, 193, 196, 198 and 225 that play a smaller role. Furthermore, we show changes at residues 135, 137, 147, 157, 158, 184, 188, and 192 can also modulate virus receptor avidity and that substitutions that increased or decreased the net positive charge around the haemagglutinin receptor-binding site show increases and decreases in avidity, respectively. The motifs we identify as increasing preference for the human-receptor will help guide future H9N2 surveillance efforts and facilitate our understanding of the emergence of influenza viruses with increased zoonotic potential.IMPORTANCE As of 2020, over 60 infections of humans by H9N2 influenza viruses have been recorded in countries where the virus is endemic. Avian-like cellular receptors are the primary target for these viruses. However, given that human infections have been detected on an almost monthly basis since 2015, there may be a capacity for H9N2 viruses to evolve and gain the ability to target human-like cellular receptors. Here we identify molecular signatures that can cause viruses to bind human-like receptors, and we identify the molecular basis for the distinctive preference for sulphated receptors displayed by the majority of recent H9N2 viruses. This work will help guide future surveillance by providing markers that signify the emergence of viruses with enhanced zoonotic potential as well as improving understanding of the basis of influenza virus receptor-binding.

Phylogenetic Analysis of Hemagglutinin Gene and Evaluation of the Viral Shedding of H9N2 Avian Influenza Viruses Using Real-time RT-PCR in SPF Chickens.

In recent years, the H9N2 influenzavirus has been circulating widely in poultry farms causing extensive damage. The hemagglutinin (HA) genes of the two virus isolates of H9N2 subtype in specific pathogen-free chickens were studied to determine the shedding rate in the host’s oropharyngeal and cloacal routes and their genetic relationship. The sequence analysis and phylogenetic study of the samples were performed by comparing each isolate with other H9N2 isolates in the gene bank. In the present study, the chickens were inoculated with low pathogenic avian influenza virus (LPAIV) (A/Chicken/Iran/ZMT-101/1998 [H9N2]) through the intranasal route. Oropharyngeal and cloacal swabs were collected from the chickens within 1-10 days after inoculation. The rate of viral shedding was measured within the previous 10 days by the real-time reverse transcriptase polymerase chain reaction molecular technique. No clinical symptoms were observed during the experiment in the chickens. The results obtained from this technique showed that the main route of shedding for LPAIV was oropharyngeal areas (p <0.05). Both isolates had a similar proteolytic R-S-S-R sequence at the cleavage site of the HA gene and contained glutamine (Q) amino acid at position 226 of the HA receptor-binding site, indicating that these isolates were nonpathogenic. Phylogenetic analysis demonstrated that both isolates belonged to the Eurasian clade. The comparison of these isolates with other isolates in the gene bank showed that they had the greatest similarity with the isolates in clade 1 and the least homology with the isolates in clade 4.

High-Speed AFM Reveals Molecular Dynamics of Human Influenza A Hemagglutinin and Its Interaction with Exosomes.

Influenza A hemagglutinin (HA) is one of the crucial virulence factors that mediate host tropism and viral infectivity. Presently, the mechanism of the fusogenic transition of HA remains elusive. Here, we used high-speed atomic force microscopy (HS-AFM) to decipher the molecular dynamics of HA and its interaction with exosomes. Our data reveal that the native conformation of HA in the neutral buffer is ellipsoidal, and HA undergoes a conformational change in an acidic buffer. Real-time visualization of the fusogenic transition by HS-AFM suggests that the mechanism is possibly fit to the "uncaging" model, and HA intermediate appears as Y-shaped. A firm interaction between the HA and exosome in an acidic buffer indicates the insertion of a fusion peptide into the exosomal layer and subsequently destabilizes the layer, resulting in the deformation or rupture of exosomes, releasing exosomal contents. In contrast, the HA-exosome interaction is weak in a neutral buffer because the interaction is mediated by weak bonds between the HA receptor-binding site and receptors on the exosome.

Multi-task learning sparse group lasso: a method for quantifying antigenicity of influenza A(H1N1) virus using mutations and variations in glycosylation of Hemagglutinin

BackgroundIn addition to causing the pandemic influenza outbreaks of 1918 and 2009, subtype H1N1 influenza A viruses (IAVs) have caused seasonal epidemics since 1977. Antigenic property of influenza viruses are determined by both protein sequence and N-linked glycosylation of influenza glycoproteins, especially hemagglutinin (HA). The currently available computational methods are only considered features in protein sequence but not N-linked glycosylation.ResultsA multi-task learning sparse group least absolute shrinkage and selection operator (LASSO) (MTL-SGL) regression method was developed and applied to derive two types of predominant features including protein sequence and N-linked glycosylation in hemagglutinin (HA) affecting variations in serologic data for human and swine H1N1 IAVs. Results suggested that mutations and changes in N-linked glycosylation sites are associated with the rise of antigenic variants of H1N1 IAVs. Furthermore, the implicated mutations are predominantly located at five reported antibody-binding sites, and within or close to the HA receptor binding site. All of the three N-linked glycosylation sites (i.e. sequons NCSV at HA 54, NHTV at HA 125, and NLSK at HA 160) identified by MTL-SGL to determine antigenic changes were experimentally validated in the H1N1 antigenic variants using mass spectrometry analyses. Compared with conventional sparse learning methods, MTL-SGL achieved a lower prediction error and higher accuracy, indicating that grouped features and MTL in the MTL-SGL method are not only able to handle serologic data generated from multiple reagents, supplies, and protocols, but also perform better in genetic sequence-based antigenic quantification.ConclusionsIn summary, the results of this study suggest that mutations and variations in N-glycosylation in HA caused antigenic variations in H1N1 IAVs and that the sequence-based antigenicity predictive model will be useful in understanding antigenic evolution of IAVs.

Structural insights into the potential changes in receptor binding site found in the 1998–2018 influenza B/Yamagata hemagglutinin: A putative correlation between receptor binding site structural variability and seasonal infection

Influenza B virus has two distinct lineages (Victoria and Yamagata) and are associated with seasonal influenza epidemics that cause respiratory illness. Influenza B hemagglutinin (HA) is a major surface glycoprotein with the receptor-binding site (RBS) primarily involved in viral pathogenesis. Generally, influenza B exclusively infects the human population which would insinuate that the structural variability of the influenza B HA RBS rarely changes. However, to our knowledge, the potential impact of variations in the influenza B HA RBS structural variability was not fully elucidated. Throughout this study, we generated models from the transitioning (evolving viral lineage) 1998–2018 influenza B/Yamagata HA, verified the quality of each HA model, performed HA RBS structural variability measurements, superimposed varying HA models for comparison, and designed a phylogenetic tree network for further analyses. We found that measurements of the transitioning HA RBS structural variability were generally maintained and, similarly, measurements of the altered (years that differed from the evolving viral lineage, specifically 2003, 2007, 2017) HA RBS structural variability differed from the transitioning HA RBS. Moreover, we observed that the altered HA RBS structural variability favored the formation of a putative Y202–H191 hydrogen bond which we postulate may increase structural stability, thereby, allowing for a winter infection of the virus. Furthermore, we established that changes in HA RBS structural variability does not influence viral evolution, but putatively seasonal infection.