N1 Neuraminidase Research Articles

Structure of an Influenza A virus N9 neuraminidase with a tetrabrachion-domain stalk.

The influenza neuraminidase (NA) is a homotetramer with head, stalk, transmembrane and cytoplasmic regions. The structure of the NA head with a stalk has never been determined. The NA head from an N9 subtype influenza A virus, A/tern/Australia/G70C/1975 (H1N9), was expressed with an artificial stalk derived from the tetrabrachion (TB) tetramerization domain from Staphylothermus marinus. The NA was successfully crystallized both with and without the TB stalk, and the structures were determined to 2.6 and 2.3 Å resolution, respectively. Comparisons of the two NAs with the native N9 NA structure from egg-grown virus showed that the artificial TB stalk maintained the native NA head structure, supporting previous biological observations.

Comprehensive profiling of translation initiation in influenza virus infected cells.

Translation can initiate at alternate, non-canonical start codons in response to stressful stimuli in mammalian cells. Recent studies suggest that viral infection and anti-viral responses alter sites of translation initiation, and in some cases, lead to production of novel immune epitopes. Here we systematically investigate the extent and impact of alternate translation initiation in cells infected with influenza virus. We perform evolutionary analyses that suggest selection against non-canonical initiation at CUG codons in influenza virus lineages that have adapted to mammalian hosts. We then use ribosome profiling with the initiation inhibitor lactimidomycin to experimentally delineate translation initiation sites in a human lung epithelial cell line infected with influenza virus. We identify several candidate sites of alternate initiation in influenza mRNAs, all of which occur at AUG codons that are downstream of canonical initiation codons. One of these candidate downstream start sites truncates 14 amino acids from the N-terminus of the N1 neuraminidase protein, resulting in loss of its cytoplasmic tail and a portion of the transmembrane domain. This truncated neuraminidase protein is expressed on the cell surface during influenza virus infection, is enzymatically active, and is conserved in most N1 viral lineages. We do not detect globally higher levels of alternate translation initiation on host transcripts upon influenza infection or during the anti-viral response, but the subset of host transcripts induced by the anti-viral response is enriched for alternate initiation sites. Together, our results systematically map the landscape of translation initiation during influenza virus infection, and shed light on the evolutionary forces shaping this landscape.

Influenza A Prevalence and Subtype Diversity in Migrating Teal Sampled Along the United States Gulf Coast.

Wild birds in the order Anseriformes are important reservoirs for influenza A viruses (IAVs); however, IAV prevalence and subtype diversity may vary by season, even at the same location. To better understand the ecology of IAV during waterfowl migration through the Gulf Coast of the United States (Louisiana and Texas), surveillance of blue-winged (Spatula discors) and American green-winged (Anas carolinensis) teal was conducted. The surveillance was done annually during the spring (live capture; 2012-17) and fall (hunter harvested; 2007-17) at times inferred to coincide with northward and southward movements, respectively, for these waterfowl species. During spring migration, 266 low pathogenicity (LP) IAV positive samples were recovered from 7547 paired cloacal-oropharyngeal (COP) samples (prevalence, 3.5%; annual range, 1.3%-8.4%). During fall migration, 650 LP IAV-positive samples were recovered from 9493 COP samples (prevalence, 6.8%; annual range, 0.4%-23.5%). Overall, 34 and 20 different IAV subtypes were recovered during fall and spring sampling, respectively. Consistent with previous results for fall migrating ducks, H3 and H4 hemagglutinin (HA) subtypes were most common; however, H4 subtype viruses predominated every year. This is in contrast to the predominance of LP H7 and H10 HA subtype viruses during spring. The N6 and N8 neuraminidase subtypes, which were usually associated with H4, were most common during fall; the N6 subtype was not recovered in the spring. These consistent seasonal trends in IAV subtype detection in teal are currently not understood and highlight the need for further research regarding potential drivers of spatiotemporal patterns of infection, such as population immunity.

Broadened immunity against influenza by vaccination with computationally designed influenza virus N1 neuraminidase constructs

Split inactivated influenza vaccines remain one of the primary preventative strategies against severe influenza disease in the population. However, current vaccines are only effective against a limited number of matched strains. The need for broadly protective vaccines is acute due to the high mutational rate of influenza viruses and multiple strain variants in circulation at any one time. The neuraminidase (NA) glycoprotein expressed on the influenza virion surface has recently regained recognition as a valuable vaccine candidate. We sought to broaden the protection provided by NA within the N1 subtype by computationally engineering consensus NA sequences. Three NA antigens (NA5200, NA7900, NA9100) were designed based on sequence clusters encompassing three major groupings of NA sequence space; (i) H1N1 2009 pandemic and Swine H1N1, (ii) historical seasonal H1N1 and (iii) H1N1 viruses ranging from 1933 till current times. Recombinant NA proteins were produced as a vaccine and used in a mouse challenge model. The design of the protein dictated the protection provided against the challenge strains. NA5200 protected against H1N1 pdm09, a Swine isolate from 1998 and NIBRG-14 (H5N1). NA7900 protected against all seasonal H1N1 viruses tested, and NA9100 showed the broadest range of protection covering all N1 viruses tested. By passive transfer studies and serological assays, the protection provided by the cluster-based consensus (CBC) designs correlated to antibodies capable of mediating NA inhibition. Importantly, sera raised to the consensus NAs displayed a broader pattern of reactivity and protection than naturally occurring NAs, potentially supporting a predictive approach to antigen design.

Clinical management and viral genomic diversity analysis of a child's influenza A(H1N1)pdm09 infection in the context of a severe combined immunodeficiency

IntroductionA child with severe combined immunodeficiency (SCID) had an influenza A(H1N1)pdm09 infection with viral excretion longer than 6 months, during 2013–2014 influenza season, despite cord blood transplantation and antiviral treatments. MethodsConventional real-time RT-PCR methods were used to estimate viral load and to detect the presence of the common N1 neuraminidase (NA) H275Y substitution responsible for oseltamivir resistance. Next-generation sequencing (NGS) of influenza viruses was performed retrospectively to characterize viral quasispecies in specimens. ResultsThe patient was first treated with oral oseltamivir, leading to detection of low-levels of NA-H275Y substitution. Concomitant cord blood cell transplantation, intravenous administration of zanamivir and immunoglobulins led to an increase in white blood cells and influenza viral load decrease. A viral rebound occurred as soon as the antiviral treatment was discontinued. Eventually, influenza viral load was negated with immune reconstitution. NGS found influenza quasispecies harboring NA-E119A substitution (10.3%). Moreover, NGS showed that viral genomic diversity evolved under antiviral treatment and immune status. ConclusionsConventional virological techniques were sufficient for influenza infection follow-up but NGS performances allowed characterization of viral variants evolution in this specific case of prolonged influenza virus infection. New and efficient treatments against influenza in immunocompromised patients are needed.

Ancestral and Compensatory Mutations that Promote Antiviral Resistance in InfluenzaN1 Neuraminidase Revealed by a Phylonumerics Approach.

Implementation of a new phylonumerics approach to construct a mass tree representing over 6000 H1N1 human influenza strains has enabled ancestral and compensatory descendant mutations to be identified in N1 neuraminidase that promote antiviral resistance and restore viral fitness. Adjacent to the H275Y resistance mutation site, mutations S299A and S247N, respectively, lead the evolution of oseltamivir-resistant strains and restore viral fitness to those strains thereafter. Importantly the mass tree phylonumerics approach can identify such mutations globally, without any positional bias, so that functionally linked or compensatory mutations remote in the sequence or structure of the protein can be identified and interrogated. This is achieved using mass map datasets commonly employed for protein identification in proteomics applications, thus avoiding the need for either gene or protein sequences that are central to other phylogenetic methods.

Substrate Binding by the Second Sialic Acid-Binding Site of Influenza A Virus N1 Neuraminidase Contributes to Enzymatic Activity.

The influenza A virus (IAV) neuraminidase (NA) protein plays an essential role in the release of virus particles from cells and decoy receptors. The NA enzymatic activity presumably needs to match the activity of the IAV hemagglutinin (HA) attachment protein and the host sialic acid (SIA) receptor repertoire. We analyzed the enzymatic activities of N1 NA proteins derived from avian (H5N1) and human (H1N1) IAVs and analyzed the role of the second SIA-binding site, located adjacent to the conserved catalytic site, therein. SIA contact residues in the second SIA-binding site of NA are highly conserved in avian, but not human, IAVs. All N1 proteins preferred cleaving α2,3- over α2,6-linked SIAs even when their corresponding HA proteins displayed a strict preference for α2,6-linked SIAs, indicating that the specificity of the NA protein does not need to fully match that of the corresponding HA protein. NA activity was affected by substitutions in the second SIA-binding site that are observed in avian and human IAVs, at least when multivalent rather than monovalent substrates were used. These mutations included both SIA contact residues and residues that do not directly interact with SIA in all three loops of the second SIA-binding site. Substrate binding via the second SIA-binding site enhanced the catalytic activity of N1. Mutation of the second SIA-binding site was also shown to affect virus replication in vitro Our results indicate an important role for the N1 second SIA-binding site in binding to and cleavage of multivalent substrates.IMPORTANCE Avian and human influenza A viruses (IAVs) preferentially bind α2,3- and α2,6-linked sialic acids (SIAs), respectively. A functional balance between the hemagglutinin (HA) attachment and neuraminidase (NA) proteins is thought to be important for host tropism. What this balance entails at the molecular level is, however, not well understood. We now show that N1 proteins of both avian and human viruses prefer cleaving avian- over human-type receptors although human viruses were relatively better in cleavage of the human-type receptors. In addition, we show that substitutions at different positions in the second SIA-binding site found in NA proteins of human IAVs have a profound effect on binding and cleavage of multivalent, but not monovalent, receptors and affect virus replication. Our results indicate that the HA-NA balance can be tuned via modification of substrate binding via this site and suggest an important role of the second SIA-binding site in host tropism.

Monoclonal antibody against N2 neuraminidase of cold adapted A/Leningrad/134/17/57 (H2N2) enables efficient generation of live attenuated influenza vaccines

Cold adapted influenza virus A/Leningrad/134/17/57 (H2N2) is a reliable master donor virus (Len/17-MDV) for preparing live attenuated influenza vaccines (LAIV). LAIVs are 6:2 reasortants that contain 6 segments of Len/17-MDV and the hemagglutinin (HA) and neuraminidase (NA) of contemporary circulating influenza A viruses. The problem with the classical reassortment procedure used to generate LAIVs is that there is limited selection pressure against NA of the Len/17-MDV resulting in 7:1 reassortants with desired HA only, which are not suitable LAIVs. The monoclonal antibodies (mAb) directed against the N2 of Len/17-MDV were generated. 10C4–8E7 mAb inhibits cell-to-cell spread of viruses containing the Len/17-MDV N2, but not viruses with the related N2 from contemporary H3N2 viruses. 10C4–8E7 antibody specifically inhibited the Len/17-MDV replication in vitro and in ovo but didn’t inhibit replication of H3N2 or H1N1pdm09 reassortants. Our data demonstrate that addition of 10C4–8E7 in the classical reassortment improves efficiency of LAIV production.

Multi-clade H5N1 virus-like particles: Immunogenicity and protection against H5N1 virus and effects of beta-propiolactone

During the past decade, H5N1 highly pathogenic avian influenza (HPAI) viruses have diversified genetically and antigenically, suggesting the need for multiple H5N1 vaccines. However, preparation of multiple vaccines from live H5N1 HPAI viruses is difficult and economically not feasible representing a challenge for pandemic preparedness. Here we evaluated a novel multi-clade recombinant H5N1 virus-like particle (VLP) design, in which H5 hemagglutinins (HA) and N1 neuraminidase (NA) derived from four distinct clades of H5N1 virus were co-localized within the VLP structure. The multi-clade H5N1 VLPs were prepared by using a recombinant baculovirus expression system and evaluated for functional hemagglutination and neuraminidase enzyme activities, particle size and morphology, as well as for the presence of baculovirus in the purified VLP preparations. To remove residual baculovirus, VLP preparations were treated with beta-propiolactone (BPL). Immunogenicity and efficacy of multi-clade H5N1 VLPs were determined in an experimental ferret H5N1 HPAI challenge model, to ascertain the effect of BPL on immunogenicity and protective efficacy against lethal challenge. Although treatment with BPL reduced immunogenicity of VLPs, all vaccinated ferrets were protected from lethal challenge with influenza A/VietNam/1203/2004 (H5N1) HPAI virus, indicating that multi-clade VLP preparations treated with BPL represent a potential approach for pandemic preparedness vaccines.

Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6, and N8 Neuraminidase Subtypes.

Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs.IMPORTANCE The frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIVs of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated, and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes; some are novel and subtype specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza virus and help lead to the development of drugs that bind to alternative interaction motifs.

Antibodies Directed toward Neuraminidase N1 Control Disease in a Mouse Model of Influenza.

There is increasing evidence to suggest that antibodies directed toward influenza A virus (IAV) neuraminidase (NA) are an important correlate of protection against influenza in humans. Moreover, the potential of NA-specific antibodies to provide broader protection than conventional hemagglutinin (HA) antibodies has been recognized. Here, we describe the isolation of two monoclonal antibodies, N1-7D3 and N1-C4, directed toward the N1 NA. N1-7D3 binds to a conserved linear epitope in the membrane-distal, carboxy-terminal part of the NA and reacted with the NA of seasonal H1N1 isolates ranging from 1977 to 2007 and the 2009 H1N1pdm virus, as well as A/Vietnam/1194/04 (H5N1). However, N1-7D3 lacked NA inhibition (NI) activity and the ability to protect BALB/c mice against a lethal challenge with a range of H1N1 viruses. Conversely, N1-C4 bound to a conformational epitope that is conserved between two influenza virus subtypes, 2009 H1N1pdm and H5N1 IAV, and displayed potent in vitro antiviral activity mediating both NI and plaque size reduction. Moreover, N1-C4 could provide heterosubtypic protection in BALB/c mice against a lethal challenge with 2009 H1N1pdm or H5N1 virus. Glutamic acid residue 311 in the NA was found to be critical for the NA binding and antiviral activity of monoclonal antibody N1-C4. Our data provide further evidence for cross-protective epitopes within the N1 subtype and highlight the potential of NA as an important target for vaccine and therapeutic approaches.IMPORTANCE Influenza remains a worldwide burden on public health. As such, the development of novel vaccines and therapeutics against influenza virus is crucial. Human challenge studies have recently highlighted the importance of antibodies directed toward the viral neuraminidase (NA) as an important correlate of reduced influenza-associated disease severity. Furthermore, there is evidence that anti-NA antibodies can provide broader protection than antibodies toward the viral hemagglutinin. Here, we describe the isolation and detailed characterization of two N1 NA-specific monoclonal antibodies. One of these monoclonal antibodies broadly binds N1-type NAs, and the second displays NA inhibition and in vitro and in vivo antiviral activity against 2009 H1N1pdm and H5N1 influenza viruses. These two new anti-NA antibodies contribute to our understanding of the antigenic properties and protective potential of the influenza virus NA antigen.

Insights into genetic diversity and biological propensities of potentially zoonotic avian influenza H9N2 viruses circulating in Egypt

Low pathogenic avian influenza (LPAI) H9N2 viruses have established endemic status in Egyptian poultry populations since 2012. Recently, four cases of human H9N2 virus infections in Egypt demonstrated the zoonotic potential of these viruses. Egyptian H9N2 viruses obtained from 2011 to 2014 phylogenetically grouped into three clusters (1−3) within subclade B of the G1 lineage. Antigenically, a close clustering of the Egyptian H9N2 viruses with other recent G1-B like H9N2 strains and a significant antigenic distance from viruses outside the G1-B lineage was evident. Recent Egyptian LPAIV H9N2 showed a tendency to increased binding with erythrocytes expressing α 2,6-linked sialic acid which correlated with the Q226L amino acid substitution at the receptor binding unit of the hemagglutinin (Q234L, H9 numbering). Sequence analyses of the N2 neuraminidase (NA) revealed substitutions in the NA hemadsorption site similar to the N2 of prepandemic H3N2/1968, but no distinct antigenic or functional characteristics of the H9N2 NA associated with increased zoonotic potential could be identified.

Screening Viral Neuraminidase Inhibitors Using a Glycosylated N1 Structure Model Derived From Molecular Dynamics Simulations

We present a novel drug screening approach for discovering neuraminidase inhibitors, in which viral neuraminidase is a well‐characterized target to develop anti‐influenza pharmaceuticals. In this study, a series of molecular dynamics‐driven structures for a glycosylated form of the neuraminidase N1 was introduced during an in silico screening process. The efficiency of this approach was confirmed by activity‐based in vitro screening for inhibitory chemicals of the neuraminidase from H1N1 (A/California/04/2009). This is the first report on a virtual screening method for neuraminidase using a computational glycosylated structure model for N1.

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

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

Analysis of Anti-Influenza Virus Neuraminidase Antibodies in Children, Adults, and the Elderly by ELISA and Enzyme Inhibition: Evidence for Original Antigenic Sin.

ABSTRACTAntibody responses to influenza virus hemagglutinin provide protection against infection and are well studied. Less is known about the human antibody responses to the second surface glycoprotein, neuraminidase. Here, we assessed human antibody reactivity to a panel of N1, N2, and influenza B virus neuraminidases in different age groups, including children, adults, and the elderly. Using enzyme-linked immunosorbent assays (ELISA), we determined the breadth, magnitude, and isotype distribution of neuraminidase antibody responses to historic, current, and avian strains, as well as to recent isolates to which these individuals have not been exposed. It appears that antibody levels against N1 neuraminidases were lower than those against N2 or B neuraminidases. The anti-neuraminidase antibody levels increased with age and were, in general, highest against strains that circulated during the childhood of the tested individuals, providing evidence for “original antigenic sin.” Titers measured by ELISA correlated well with titers measured by the neuraminidase inhibition assays. However, in the case of the 2009 pandemic H1N1 virus, we found evidence of interference from antibodies binding to the conserved stalk domain of the hemagglutinin. In conclusion, we found that antibodies against the neuraminidase differ in magnitude and breadth between subtypes and age groups in the human population. (This study has been registered at ClinicalTrials.gov under registration no. NCT00336453, NCT00539981, and NCT00395174.)

Exploring docking methods for virtual screening: application to the identification of neuraminidase and Ftsz potential inhibitors

Virtual screenings based on molecular docking play a major role in medicinal chemistry for the identification of new bioactive molecules. For this purpose, several docking methods can be used. Here, using Arguslab as software and a Gold Platinum subset library of commercially available compounds from Asinex, two docking methods associated to the scoring function Ascore were employed to investigate virtual screenings. One method is based on a genetic algorithm and the other based on a shape-based method. As case studies, both docking techniques were explored by targeting the PC190723 binding site of FtsZ protein from Staphylococcus aureus and the active site of N8 neuraminidase from Influenza virus. Following four docking sequences for each docking engine, the genetic algorithm led to multiple docking results, whereas the shape-based method gave reproducible results. The present study shows that the stochastic nature of the genetic algorithm will require the biological evaluation of more compounds than the shape-based method. This study showed that both methods are complementary and also led to the identification of neuraminidase and FtsZ potential inhibitors.

An Amino Acid in the Stalk Domain of N1 Neuraminidase Is Critical for Enzymatic Activity.

Neuraminidase (NA) is a sialidase that is one of the major surface glycoproteins of influenza A viruses and the target for the influenza drugs oseltamivir and zanamivir. NA is important as it releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. Mutations in the globular head domain that decrease enzymatic activity but confer resistance to NA inhibitors have been characterized; however, the importance of specific mutations in the stalk domain is unknown. We identified 66Y (N1 numbering), a highly conserved amino acid that was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.

The molecular mechanism of the ligand exchange reaction of an antibody against a glutathione-coated gold cluster

The labeling of proteins with heavy atom clusters is of paramount importance in biomedical research, but its detailed molecular mechanism remains unknown. Here we uncover it for the particular case of the anti-influenza N9 neuraminidase NC10 antibody against a glutathione-coated gold cluster by means of ab initio QM/MM calculations. We show that the labeling reaction follows an associative double SN2-like reaction mechanism, involving a proton transfer, with low activation barriers only if one of the two distinct peptide/peptidic ligands (the one that occupies the side position) is substituted. Positively charged residues in the vicinity of the incoming thiol result in strong interactions between the antibody and the AuMPC, favoring the ligand exchange reaction for suitable protein mutants. These results pave the way for future investigations aimed at engineering biomolecules to increase their reactivity towards a desired gold atom cluster.

In-silico Virtual Screening and ADMET Study to Find Novel Neuraminidase N1 Inhibitors Extended to the 150-Cavity

Neuraminidase (NA) is the major surface protein of the influenza virus. It extracellularly acts by cleaving the terminal neuraminic acid from cellular receptors recognized by the Hemagglutinin. Then, it facilitates the release of newly formed visions from the host cell surface to the neighboring cells, thereby facilitating the spread of the virus. A 150-cavity adjacent to the active conservative site is possessed by the group-1 neuraminidase, thereby rendering conformational change from open to close form when the ligand binds to the enzyme. In the present study, we reported an in silico virtual screening and docking analysis for potential neuraminidase inhibitors of various ligands obtained from the ZINC database using Autodock Vina against the 3TI6 protein. Analysis of 850 screened ligands reveal that five compounds with free binding energies of -11.2, -10.9, -10.4, -10.4, and -10.1 kcal/mol (ZINC03260201, ZINC09153352, ZINC09460395, ZINC13128611, and ZINC20605436, respectively) showed interaction with the protein at the known active site, as well as with the 150-cavity creating a stronger interaction between the ligand and the protein. Furthermore, lower binding energy is exhibited compared with the co-crystallized drug oseltamivir. In silico absorption, distribution, metabolism, and excretion (ADMET) prediction revealed that best compounds show comparative results with oseltamivir. Novel compounds interacting with the 150-cavity were successfully identified using this approach; such compounds could serve as a potential lead compound for developing a new anti-neuraminidase drug.

Virus-like particles displaying H5, H7, H9 hemagglutinins and N1 neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens

Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained influenza N1 neuraminidase and retroviral gag protein. The H5/H7/H9/N1/gag VLPs were prepared using baculovirus expression. Biochemical, functional and antigenic characteristics were determined including hemagglutination and neuraminidase enzyme activities. VLPs were further evaluated in a chicken AI challenge model for safety, immunogenicity and protective efficacy against heterologous AI viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenges with H5N2 and H7N3 highly pathogenic AI (HPAI) viruses, while all controls died. Immune response was also detectable after challenge with low pathogenicity AI (LPAI) H9N2 virus suggesting that H5/H7/H9/N1/gag VLPs represent a promising approach for the development of broadly protective AI vaccine.