N1 Neuraminidase Research Articles

Study of Antibodies to Influenza Neuraminidase N2.

Humoral immunity to influenza neuraminidase (NA) was evaluated among different groups of people including patients with acute influenza infection and healthy people in different age groups using an enzyme linked lectin assay (ELLA). The amino acid composition of NA of seasonal influenza viruses A/Victoria/361/2011(H3N2) and A/Hong Kong/4801/2014(H3N2) differed by 2%, while cross-reacting neuraminidase-inhibiting (NI) antibodies to them in the same serum samples were detected in 10% of cases. Middle-aged patients born from 1977 to 2000 had a high level of hemagglutination-inhibiting (HI) antibodies to A/Hong Kong/4801/2014(H3N2), but almost no NI antibodies, which may indicate that in the case of a change in the hemagglutinin (HA) subtype, this age group will be susceptible to influenza A/H3N2 viruses. Therefore, it could mean there is a need for priority vaccination of this age group with a vaccine against the appropriate strain. It was shown that after intranasal administration of live influenza vaccine (LAIV) for the 2017–2018 season, serum antibody response was not lower compared to that during natural infection. In older people, antibodies to archival A/H2N2 viruses were detected more often than to modern A/H3N2. Since the conversion of antibodies to HA and NA often did not coincide, antibodies to NA can serve as an additional criterion for assessing the immunogenicity of influenza vaccines.

Characterization of a 2016-2017 Human Seasonal H3 Influenza A Virus Spillover Now Endemic to U.S. Swine.

ABSTRACTIn 2017, the Iowa State University Veterinary Diagnostic Laboratory detected a reverse-zoonotic transmission of a human seasonal H3 influenza A virus into swine (IAV-S) in Oklahoma. Pairwise comparison between the recently characterized human seasonal H3 IAV-S (H3.2010.2) hemagglutinin (HA) sequences detected in swine and the most similar 2016–2017 human seasonal H3 revealed 99.9% nucleotide identity. To elucidate the origin of H3.2010.2 IAV-S, 45 HA and 27 neuraminidase (NA) sequences from 2017 to 2020 as well as 11 whole-genome sequences (WGS) were genetically characterized. Time to most recent common human ancestor was estimated between August and September 2016. The N2 NA was of human origin in all but one strain from diagnostic submissions with NA sequences, and the internal gene segments from WGS consisted of matrix genes originating from the 2009 pandemic H1N1 and another 5 internal genes of triple reassortant swine origin (TTTTPT). Pigs experimentally infected with H3.2010.2 demonstrated efficient nasal shedding and replication in the lungs, mild pneumonia, and minimal microscopic lung lesions and transmitted the virus to indirect contact swine. Antigenically, H3.2010.2 viruses were closer to a human seasonal vaccine strain, A/Hong Kong/4801/2014, than to the H3.2010.1 human seasonal H3 viruses detected in swine in 2012. This was the second sustained transmission of a human seasonal IAV into swine from the 2010 decade after H3.2010.1. Monitoring the spillover and detection of novel IAV from humans to swine may help vaccine antigen selection and could impact pandemic preparedness.IMPORTANCE H3.2010.2 is a new phylogenetic clade of H3N2 circulating in swine that became established after the spillover of a human seasonal H3N2 from the 2016–2017 influenza season. The novel H3.2010.2 transmitted and adapted to the swine host and demonstrated reassortment with internal genes from strains endemic to pigs, but it maintained human-like HA and NA. It is genetically and antigenically distinct from the H3.2010.1 H3N2 introduced earlier in the 2010 decade. Human seasonal IAV spillovers into swine become established in the population through adaptation and sustained transmission and contribute to the genetic and antigenic diversity of IAV circulating in swine. Continued IAV surveillance is necessary to detect emergence of novel strains in swine and assist with vaccine antigen selection to improve the ability to prevent respiratory disease in swine as well as the risk of zoonotic transmission.

Investigation on the Binding Properties of N1 Neuraminidase of H5N1 Influenza Virus in Complex with Fluorinated Sialic Acid Analog Compounds—a Study by Molecular Docking and Molecular Dynamics Simulations

Worldwide, only two types of antiviral inhibitors (M2 ion channel protein inhibitor and Neuraminidase inhibitors) are approved to treat the influenza viral infection. But the mutation of amino acid sequence in the viral membrane proteins creates the viral resistance to existing antiviral drugs or inhibitors. So the corresponding antiviral drugs have to be reformulated to match these antigenic variations. Fluorination on the carbon–based molecule significantly enriches its biological properties. Hence this study is motivated to design the fluorinated sialic acid (SIA) analog inhibitors for the neuraminidase of H5N1 influenza A virus by substituting fluorine atom at different hydroxyls (O2, O4, O7, O8, and O9) of sialic acid. 100 ns molecular dynamics simulations are carried out for each protein–ligand complex system. NAMD pair interaction energy and MM–PBSA binding free energy calculations predict two possible binding modes for N1–SIA_F2, N1–SIA_F4, and N1–SIA_F7 complexes and single binding mode for N1–SIA_F8 and N1–SIA_F9 complexes. RMSD, RMSF, and hydrogen bonding analyses are used to understand the conformational flexibility and structural stability of each complex system. It has been concluded that the fluorinated sialic acid drug candidates SIA_F2 and SIA_F7 have better inhibiting potency against the N1 neuraminidase of H5N1 influenza virus.

Spatial and temporal coevolution of N2 neuraminidase and H1 and H3 hemagglutinin genes of influenza A virus in US swine.

The neuraminidase (NA) and hemagglutinin (HA) are essential surface glycoproteins of influenza A virus (IAV). In this study, the evolution of subtype N2 NA paired with H1 and H3 subtype HA in swine was evaluated to understand if the genetic diversity of HA and NA were linked. Using time-scaled Bayesian phylodynamic analyses, the relationships of paired swine N2 with H1 or H3 from 2009 to 2018 were evaluated. These data demonstrated increased relative genetic diversity within the major N2 clades circulating in swine in the USA (N2.1998 between 2014 and 2017 and N2.2002 between 2010 and 2016). Preferential pairing was observed among specific NA and HA genetic clades. Gene reassortment between cocirculating influenza A strains resulted in novel pairings that persisted. The changes in genetic diversity in the NA gene were quantified using Bayesian phylodynamic analyses, and increases in diversity were observed subsequent to novel NA–HA reassortment events. The rate of evolution among NA–N2 clades and HA–H1 and HA–H3 clades were similar. Bayesian phylodynamic analyses demonstrated strong spatial patterns in N2 genetic diversity, but frequent interstate movement of rare N2 clades provided opportunity for reassortment and emergence of new N2–HA pairings. The frequent regional movement of pigs and their influenza viruses is an explanation for the documented patterns of reassortment and subsequent changes in gene diversity. The reassortment and evolution of NA and linked HA evolution may result in antigenic drift of both major surface glycoproteins, reducing vaccine efficacy, with subsequent impact on animal health.

Evolution and Antigenic Advancement of N2 Neuraminidase of Swine Influenza A Viruses Circulating in the United States following Two Separate Introductions from Human Seasonal Viruses.

Two separate introductions of human seasonal N2 neuraminidase genes were sustained in U.S. swine since 1998 (N2-98) and 2002 (N2-02). Herein, we characterized the antigenic evolution of the N2 of swine influenza A virus (IAV) across 2 decades following each introduction. The N2-98 and N2-02 expanded in genetic diversity, with two statistically supported monophyletic clades within each lineage. To assess antigenic drift in swine N2 following the human-to-swine spillover events, we generated a panel of swine N2 antisera against representative N2 and quantified the antigenic distance between wild-type viruses using enzyme-linked lectin assay and antigenic cartography. The antigenic distance between swine and human N2 was smallest between human N2 circulating at the time of each introduction and the archetypal swine N2. However, sustained circulation and evolution in swine of the two N2 lineages resulted in significant antigenic drift, and the N2-98 and N2-02 swine N2 lineages were antigenically distinct. Although intralineage antigenic diversity was observed, the magnitude of antigenic drift did not consistently correlate with the observed genetic differences. These data represent the first quantification of the antigenic diversity of neuraminidase of IAV in swine and demonstrated significant antigenic drift from contemporary human seasonal strains as well as antigenic variation among N2 detected in swine. These data suggest that antigenic mismatch may occur between circulating swine IAV and vaccine strains. Consequently, consideration of the diversity of N2 in swine IAV for vaccine selection may likely result in more effective control and aid public health initiatives for pandemic preparedness. IMPORTANCE Antibodies inhibiting the neuraminidase (NA) of IAV reduce clinical disease, virus shedding, and transmission, particularly in the absence of neutralizing immunity against hemagglutinin. To understand antibody recognition of the genetically diverse NA in U.S. swine IAV, we characterized the antigenic diversity of N2 from swine and humans. N2 detected in swine IAV were derived from two distinct human-to-swine spillovers that persisted, are antigenically distinct, and underwent antigenic drift. These findings highlight the need for continued surveillance and vaccine development in swine with increased focus on the NA. Additionally, human seasonal N2 isolated after 2005 were poorly inhibited by representative swine N2 antisera, suggesting a lack of cross-reactive NA antibody-mediated immunity between contemporary swine and human N2. Bidirectional transmission between humans and swine represents a One Health challenge, and determining the correlates of immunity to emerging IAV strains is critical to mitigating zoonotic and reverse-zoonotic transmission.

N1 neuraminidase of H5N1 avian influenza A virus complexed with sialic acid and zanamivir – A study by molecular docking and molecular dynamics simulation

Development of antiviral drugs is an urgent need to control and prevent the presently circulating H5N1 avian influenza virus which is affects the human respiratory tract. The complex crystal structure of N1–N–acetylneuranamic acid (sialic acid, SIA) is not available as complex and hence SIA and zanamivir (ZMR) are docked into the binding site of N1 neuraminidase. Based on the analysis, the initial complex structures have been simulated for 120 ns to get insight into the binding modes and interaction between protein–ligand complex systems. NAMD pair interaction energy and MM–PBSA binding free energy are calculated and show that there are two possible binding modes (BM1 and BM2) for N1–SIA and a single binding mode (BM1) for and N1–ZMR complex structures respectively. BM1 of N1–SIA is the most preferred binding mode. On contrary to the currently available drugs in which the chair conformation is distorted, in both the binding modes of N1–SIA, the binding pocket of N1 neuraminidase is able to accommodate SIA in 2C5 chair conformation which is the preferred conformation of SIA in solution state. In N1–ZMR complex, ZMR is bind in a distorted chair conformation. The neuraminidase binding pocket is also able to accommodate galactose of SIAα(2→3)GAL and SIAα(2→6)GAL. RMSD, RMSF and hydrogen bonding analyses have been carried out to identify the conformational flexibility and structural stability of each complex system. All the analyses show that SIA can be used as an inhibitor for N1 neuraminidase of H5N1 influenza viral infection. Communicated by Ramaswamy H. Sarma

Dynamic residue interaction network analysis of the oseltamivir binding site of N1 neuraminidase and its H274Y mutation site conferring drug resistance in influenza A virus.

BackgroundOseltamivir (OTV)-resistant influenza virus exhibits His-to-Tyr mutation at residue 274 (H274Y) in N1 neuraminidase (NA). However, the molecular mechanisms by which the H274Y mutation in NA reduces its binding affinity to OTV have not been fully elucidated.MethodsIn this study, we used dynamic residue interaction network (dRIN) analysis based on molecular dynamics simulation to investigate the correlation between the OTV binding site of NA and its H274Y mutation site.ResultsdRIN analysis revealed that the OTV binding site and H274Y mutation site of NA interact via the three interface residues connecting them. H274Y mutation significantly enhanced the interaction between residue 274 and the three interface residues in NA, thereby significantly decreasing the interaction between OTV and its surrounding loop 150 residues. Thus, we concluded that such changes in residue interactions could reduce the binding affinity of OTV to NA, resulting in drug resistant influenza viruses. Using dRIN analysis, we succeeded in understanding the characteristic changes in residue interactions due to H274Y mutation, which can elucidate the molecular mechanism of reduction in OTV binding affinity to influenza NA. Finally, the dRIN analysis used in this study can be widely applied to various systems such as individual proteins, protein-ligand complexes, and protein-protein complexes, to characterize the dynamic aspects of the interactions.

Introduction of Cysteines in the Stalk Domain of Recombinant Influenza Virus N1 Neuraminidase Enhances Protein Stability and Immunogenicity in Mice.

Influenza virus surface glycoproteins represent the main targets of the immune system during infection and vaccination. Current influenza virus vaccines rely mostly on the hemagglutinin, requiring a close match between the vaccine and circulating strains. Recently, the neuraminidase (NA) has become an attractive target; however low immunogenicity and stability in vaccine preparations remain an obstacles. Here, we took advantage of the hypervariable stalk domain of the NA to introduce cysteines at different positions and to produce more stable multimeric forms of the protein. We generated 11 N1 constructs and characterized the proteins by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis and by testing their enzymatic activity and representation of antigenic epitopes. Moreover, we evaluated their potential to induce a protective immune response in vivo and characterized the polyclonal antibody responses of immunized mice. We observed that the introduction of cysteines at certain positions led to the formation of stable N1 dimers, which are capable of inducing a strong antibody response characterized by neuraminidase inhibiting activity and protection of mice from high dose viral challenge. Overall, our results provide evidence for the feasibility of introducing stalk modifications to enhance the stability and immunogenicity of NA-based recombinant antigens.

Identification and Characterization of Novel Antibody Epitopes on the N2 Neuraminidase.

ABSTRACTThe influenza virus neuraminidase (NA) is becoming a focus for novel vaccine designs. However, the epitopes of human anti-NA antibodies have been poorly defined. Using a panel of 10 anti-N2 monoclonal antibodies (MAbs) that bind the H3N2 virus A/Switzerland/9715293/2013, we generated five escape mutant viruses. These viruses contained mutations K199E/T, E258K, A272D, and S331N. We found that mutations at K199 and E258 had the largest impact on MAb binding, NA inhibition and neutralization activity. In addition, a natural isolate from the 2017-2018 season was found to contain the E258K mutation and was resistant to numerous antibodies tested. The mutation S331N, was identified in virus passaged in the presence of antibody; however, it had little impact on MAb activity and greatly decreased viral fitness. This information aids in identifying novel human MAb epitopes on the N2 and helps with the detection of antigenically drifted NAs.IMPORTANCE The influenza virus neuraminidase is an emerging target for universal influenza virus vaccines. However, in contrast to influenza virus hemagglutinin, we know little about antibody epitopes and antigenic sites on the neuraminidase. Characterizing and defining these sites is aiding vaccine development and helping to understand antigenic drift of NA.

MD simulation of the interaction between sialoglycans and the second sialic acid binding site of influenza A virus N1 neuraminidase.

The neuraminidases (NAs) of avian influenza viruses (IAVs) contain a second sialic acid-binding site (2SBS), historically known as the hemadsorption site, which is separated from the sialyl-hydrolase catalytic site and serves to facilitate NA catalytic activity towards multivalent sialyl-capped glycoconjugates. Transmission and adaptation of avian IAVs to humans decreases hemadsorption and catalytic activities of the NA. Here, we report the molecular recognition features of the NA 2SBS of two pandemic H1N1 IAVs, A/Brevig Mission /1/1918 (BM18) and A/California/04/2009 (CA09), differing by their 2SBS activity. Using explicit solvent MD simulation, molecular mechanics, and glycosidic conformation analysis we initially analyzed the interactions of BM18 2SBS with two sialyllacto-N-tetraose pentasaccharides, 3'SLN-LC and 6'SLN-LC, which are models for the glycan receptors of IAVs in birds and humans, respectively. These studies characterize the binding specificity of BM18 2SBS towards human-type and avian-type receptors and identifies the key amino acids that affects binding. We next compared the interactions of the 2SBSs of BM18 and CA09 with 6'SLN-LC, revealing the critical effect of amino acid 372 on binding. Our results expand the current knowledge of the molecular features of NA 2SBSs and its alteration during the adaptation of avian IAVs to humans.

Molecular epidemiology and glycomics of swine influenza viruses circulating in commercial swine farms in the southeastern and midwest United States

Tracking the genetic diversity and spread of swine influenza viruses (SIVs) in commercial swine farms is central for control and to reduce the potential emergence of SIV reassortants. We analyzed the diversity of SIVs in nasal washes or oral fluids from commercial swine farms in North Carolina using influenza M qRT-PCR and hemagglutinin (HA) and neuraminidase (NA) subtyping. We found a predominance of H1 HAs and N2 NAs in the samples examined. The majority of the H1 HAs could be further classified into gamma and delta subclusters. We also identified HAs of the H1 alpha cluster, and those of human novel pandemic origin. Glycan binding profiles from a representative subset of these viruses revealed broad α2,6 sialylated glycan recognition, though some strains exhibited the ability to bind to α2,3 sialic acid. These data show that SIV surveillance can aid our understanding of viral transmission dynamics and help uncover the diversity at the human-swine interface.

Development of real-time RT-PCR for N2 subtype avian influenza RNA-virus detection

Currently, N2 subtype avian influenza (AI) virus actively circulates in domestic and wild bird populations and is regularly detected in China, other Asian countries and Russia, particularly in combination with H9 hemagglutinin. Therefore, a method for rapid detection of the said infectious agent is urgently required. Data on oligonucleotide primer selection and reverse transcription real-time polymerase chain reaction condition optimization for N2 AI virus detection are presented in the paper. Modified primers and probe proposed by B. Hoffmann in 2006 as well as original primers and probes with the viruses available in the Laboratory working collection and selected during testing were assessed for N2 neuraminidase gene fragment amplification. Optimal concentrations of real-time RT-PCR master mix components and temperature-time mode were determined. Various combinations of primers were tested against ten N2 avian influenza virus isolates that genetically differed from each other in N gene. Nine viruses were isolated from birds in the Russian Federation regions and classified to different genetic groups. The real-time RT-PCR assay was tested for its specificity using AI virus isolates of different neuraminidase subtypes (H5N8, H3N6, H4N6, H5N1, H10N7) as well as samples containing other RNA-viruses: Newcastle disease virus, infectious bronchitis virus and infectious bursal disease virus. As a result of the testing, real-time RT-PCR conditions providing high sensitivity and specificity of the assay were selected and optimized.

Correctly folded - but not necessarily functional - influenza virus neuraminidase is required to induce protective antibody responses in mice

The influenza virus neuraminidase (NA) plays an integral role in the influenza virus life cycle through the release of virions from infected cells. NA-specific antibodies can impede virus replication by binding to the NA and blocking its enzymatic activity, providing significant protection from influenza-associated morbidity and mortality. NA included in current seasonal influenza virus vaccines exhibits low immunogenicity, potentially caused by compromised antigenic integrity during vaccine production. To determine how certain types of “stress” could influence the antigenicity of NA we performed a series of in vitro experiments where we treated NA with formalin, EDTA or heat and measured the impact of these treatments on NA enzymatic activity and structural integrity. We found that increasing concentrations of formalin or EDTA and increasing temperature abolished the enzymatic activity of both H1N1, H3N2, and influenza B purified viruses and recombinant NA proteins. However, formalin and EDTA treatment did not drastically affect conformational epitopes found on the NA, whereas heat treatment abolished conformational epitopes. We next performed a vaccination experiment, where mice were vaccinated with recombinant N2 NA treated with 0.3% formalin or 0.125 M EDTA (which both inactivated NA activity) were protected from virus challenge while animals vaccinated with heat treated NA were not. We next tested the protective effect of monomeric (no enzymatic activity) versus tetrameric (highly active) N1 NA. Again, only the tetrameric form protected mice from challenge while the monomeric form did not. Together, our data demonstrate that enzymatically active NA is not required to induce protective antibody responses as a vaccine, however a correctly folded NA is essential.

Development and evaluation of a TaqMan MGB RT-PCR assay for detection of H5 and N8 subtype influenza virus

BackgroundHighly pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. Thus, a specific, rapid and sensitive method for detection is urgently needed.MethodsIn the present study, TaqMan minor groove binder probes and multiplex real-time RT-PCR primers were designed to target the H5 hemagglutinin and N8 neuraminidase genes. A total of 38 strains of avian influenza viruses and other viruses were selected to test the performance of the assay.ResultsThe results showed that only H5 and N8 avian influenza viruses yielded a positive signal, while all other subtypes avian influenza viruses and other viruses were negative. High specificity, repeatability, and sensitivity were achieved, with a detection limit of 10 copies per reaction.ConclusionsThe developed assay could be a powerful tool for rapid detection of H5N8 influenza viruses in the future.

Negligible effect of chicken cytokine IL-12 integration into recombinant fowlpox viruses expressing avian influenza virus neuraminidase N1 on host cellular immune responses.

In comparison to the extensive characterization of haemagglutinin antibodies of avian influenza virus (AIV), the role of neuraminidase (NA) as an immunogen is less well understood. This study describes the construction and cellular responses of recombinant fowlpox viruses (rFWPV) strain FP9, co-expressing NA N1 gene of AIV A/Chicken/Malaysia/5858/2004, and chicken IL-12 gene. Our data shows that the N1 and IL-12 proteins were successfully expressed from the recombinants with 48 kD and 70 kD molecular weights, respectively. Upon inoculation into specific-pathogen-free (SPF) chickens at 105 p.f.u. ml−1, levels of CD3+/CD4+ and CD3+/CD8+ populations were higher in the wild-type fowlpox virus FP9 strain, compared to those of rFWPV-N1 and rFWPV-N1-IL-12 at weeks 2 and 5 time points. Furthermore, rFWPV-N1-IL-12 showed a suppressive effect on chicken body weight within 4 weeks after inoculation. We suggest that co-expression of N1 with or without IL-12 offers undesirable quality as a potential AIV vaccine candidate.

Use of traditional serological methods and oral fluids to assess immunogenicity in children aged 2–16 years after successive annual vaccinations with LAIV

BackgroundThe UK introduced quadrivalent live attenuated influenza vaccine (qLAIV) for children in 2013/2014. The impact of annual vaccination on effectiveness and immunogenicity is being assessed. MethodA phase III/IV open-label study of the immunogenicity of annual vaccination with qLAIV (Fluenz™) was conducted over three consecutive years (2014/15–2016/17) in 254, 249 and 162 children respectively. Serum responses to vaccine components were measured by Haemagglutination Inhibition (HAI) and anti-A(H1N1)pdm09 Neuraminidase (NAI) assays, stratified according to previous receipt of AS03B-adjuvanted A(H1N1)pdm09 pandemic vaccine in 2009/10. Antibody levels to the A(H1N1)pdm09 and H3N2 vaccine components in oral fluids (OF) were explored using an ELISA. FindingsMore paired pre- and post-vaccination oral fluids (96%) than paired sera (87%) were obtained. Geometric mean titre rises using HAI assays were limited, with maximum rises seen in year one for both influenza B strains when 39% and 43% of subjects seroconverted (95% confidence interval 33–46% and 36–50%, respectively) and year two for influenza H3N2, when 40% (33–46%) individuals seroconverted. Prior pandemic vaccine receipt resulted in higher pre- and post-vaccination A(H1N1)pdm09 HAI titres and lower pre-and post-vaccination NAI (N1 neuraminidase) titres in all three years. OF results were congruent with HAI results; assay specificity compared to HAI was 88.1 and 71.6 percent, and sensitivity was 86.4 and 74.8 percent respectively for A(H1N1)pdm09 and H3N2. ConclusionIn all three study years, vaccination with qLAIV resulted in poor antibody responses. However, OFs are an alternative specimen type that allows self sampling, can easily be obtained from children, and their analysis leads to similar conclusions as classic serology by HAI. Their suitability for seroprevalence studies should be investigated. We demonstrated a sustained effect from prior receipt of the AS03B-adjuvanted A(H1N1)pdm09 vaccine, even after repeat vaccination with qLAIV indicating that early exposure to influenza antigens has a significant long lasting effect.

Fate of Neuraminidases of Influenza A Viruses

The current COVID-19 pandemic creates the biggest health and economic challenges to the world. However, not much knowledge is available about this coronavirus, SARS-CoV-2, because of its novelty. Indeed, it necessarily knows the fate of proteins generated by SARS-CoV-2. Anyway, before a large-scale study on proteins from SARS-CoV-2, it would be better to conduct a small-scale study on a well-known protein from influenza A viruses, because both are positive-sense RNA viruses. Thus, we applied a simple method of amino-acid pair probability to analyze 94 neuraminidases of influenza A viruses for better understanding of their fate. The results demonstrate three features of these neuraminidases: (i) the N1 neuraminidases are more susceptible to mutations, which is the current state of the neuraminidases; (ii) the N1 neuraminidases have undergone more mutations in the past, which is the history of the neuraminidases; and (iii) the N1 neuraminidases have a larger potential towards future mutations, which is the future of the neuraminidases. Moreover, our study reveals two clues on the mutation tendency, i.e. the mutations represent a degeneration process, and chickens, ducks and geese are rendered more susceptive to mutation. We hope to apply this approach to study the proteins from SARS-CoV-2 in near future.

Emerging from the Shadow of Hemagglutinin: Neuraminidase Is an Important Target for Influenza Vaccination

Gilchuk etal. (2019) and Zhu etal. (2019) provide detailed structural information about epitopes on influenza virus N9 neuraminidase and characterize breadth, potency, mechanism of action, and invivo protective effects of human antibodies targeting these epitopes. The findings highlight the importance of neuraminidase as a target for improved influenza virus vaccines.

Influenza H7N9 Virus Neuraminidase-Specific Human Monoclonal Antibodies Inhibit Viral Egress and Protect from Lethal Influenza Infection in Mice

H7N9 avian influenza virus causes severe infections and might have the potential to trigger a major pandemic. Molecular determinants of human humoral immune response to N9 neuraminidase (NA) proteins, which exhibit unusual features compared with seasonal influenza virus NA proteins, are ill-defined. We isolated 35 human monoclonal antibodies (mAbs) from two H7N9 survivors and two vaccinees. These mAbs react to NA in a subtype-specific manner and recognize diverse antigenic sites on the surface of N9 NA, including epitopes overlapping with, or distinct from, the enzyme active site. Despite recognizing multiple antigenic sites, the mAbs use a common mechanism of action by blocking egress of nascent virions from infected cells, thereby providing an antiviral prophylactic and therapeutic protection invivo in mice. Studies of breadth, potency, and diversity of antigenic recognition from four subjects suggest that vaccination with inactivated adjuvanted vaccine induce NA-reactive responses comparable to that of H7N9 natural infection.

Mechanisms of antiviral resistance in influenza neuraminidase revealed by a mass spectrometry based phylonumerics approach

A mass based phylonumerics approach is shown to be able to investigate the origins of the emergence of antiviral resistance mutations in influenza neuraminidase through a global view of mutational trends. Frequent ancestral and descendant mutations that precede and follow the manifestation of antiviral resistance mutations are identified in N2 neuraminidase. The majority occur in the head region around the active site and drive hydrophilicity changes, primarily through the incorporation or loss of hydroxyl groups. These increase or reduce the accessibility of the site to the bulk solvent. The most frequent ancestral mutations that occur on at least two occasions are I/L307M, G/A414S/T, I312T, I/L307S, P386S and S367N; the latter introducing a glycosylation site. The most frequent descendant mutation, after incorporation of an antiviral resistance mutation, is D/E401G/A. Together with others observed, this restore the protein’s hydrophobicity about the active site region that limits entry of a sialic acid or inhibitor molecule and reduces viral fitness. The results of this global in silico phylonumerics study demonstrate that evolutionary mechanisms and functionally linked or compensatory mutations, remote in the sequence or structure, can be identified and interrogated at a molecular level.