Computational assay of H7N9 influenza neuraminidase reveals R292K mutation reduces drug binding affinity.
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ABSTRACT:
SUBMITTER: Woods CJ
PROVIDER: S-EPMC3868970 | biostudies-literature | 2013
REPOSITORIES: biostudies-literature
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Project description:Neuraminidase inhibitors (NAIs) are the only available licensed therapeutics against human H7N9 influenza virus infections. The emergence of NAI-resistant variants of H7N9viruses with an NA R292K mutation poses a therapeutic challenge. A comprehensive understanding of the susceptibility of these viruses to clinically available NAIs, non-NAIs and their combinations is crucial for effective treatment. In this study, by using limited serial passage and plaque purification, an R292K variant of the Anhui1 lineage was isolated from a patient with clinical evidence of resistance to oseltamivir. In vitro and cell-based assays confirmed a high level of resistance conferred by the R292K mutation to oseltamivir carboxylate and a moderate level of resistance to zanamivir and peramivir. Non-NAI antivirals, such as T-705, ribavirin and NT-300, efficiently inhibited both the variant and the wild-type in cell-based assays. A combination of NAIs and non-NAIs did not exhibit a marked synergistic effect against the R292K variant. However, the combination of two non-NAIs (T-705 and ribavirin) exhibited significant synergism against the mutant virus. In experimentally infected mice, the variant showed delayed onset of symptoms, a reduced viral load and attenuated lethality compared with the wild-type. Our study suggested non-NAIs should be tested clinically for H7N9 patients with a sustained high viral load. Possible drug combination regimens, such as T-705 plus ribavirin, should be further tested in animal models. The pathogenicity and transmissibility of the R292K H7N9 variant should be further assessed with genetically well-characterized pairs of viruses and, most-desirably, with competitive fitness experiments.
| S-EPMC4274890 | biostudies-literature
Project description:In March 2013, a novel avian influenza A (H7N9) virus emerged in China. By March 2021, it had infected more than 1500 people, raising concerns regarding its epidemic potential. Similar to the highly pathogenic H5N1 virus, the H7N9 virus causes severe pneumonia and acute respiratory distress syndrome in most patients. Moreover, genetic analysis showed that this avian H7N9 virus carries human adaptation markers in the hemagglutinin and polymerase basic 2 (PB2) genes associated with cross-species transmissibility. Clinical studies showed that a single mutation, neuraminidase (NA) R292K (N2 numbering), induces resistance to peramivir in the highly pathogenic H7N9 influenza A viruses. Therefore, to evaluate the risk for human public health and understand the possible source of drug resistance, we assessed the impact of the NA-R292K mutation on avian H7N9 virus resistance towards peramivir using various molecular dynamics approaches. We observed that the single point mutation led to a distorted peramivir orientation in the enzyme active site which, in turn, perturbed the inhibitor's binding. The R292K mutation induced a decrease in the interaction among neighboring amino acid residues when compared to its wild-type counterpart, as shown by the high degree of fluctuations in the radius of gyration. MM/GBSA calculations revealed that the mutation caused a decrease in the drug binding affinity by 17.28 kcal/mol when compared to the that for the wild-type enzyme. The mutation caused a distortion of hydrogen bond-mediated interactions with peramivir and increased the accessibility of water molecules around the K292 mutated residue.
| S-EPMC8912059 | biostudies-literature
Project description:BackgroundNeuraminidase (NA) inhibitors are the recommended antiviral medications for influenza treatment. However, their therapeutic efficacy can be compromised by NA changes that emerge naturally and/or following antiviral treatment. Knowledge of which molecular changes confer drug resistance of influenza A(H7N9) viruses (group 2NA) remains sparse.MethodsFourteen amino acid substitutions were introduced into the NA of A/Shanghai/2/2013(H7N9). Recombinant N9 (recN9) proteins were expressed in a baculovirus system in insect cells and tested using the Centers for Disease Control and Prevention standardized NA inhibition (NI) assay with oseltamivir, zanamivir, peramivir, and laninamivir. The wild-type N9 crystal structure was determined in complex with oseltamivir, zanamivir, or sialic acid, and structural analysis was performed.ResultsAll substitutions conferred either reduced or highly reduced inhibition by at least 1 NA inhibitor; half of them caused reduced inhibition or highly reduced inhibition by all NA inhibitors. R292K conferred the highest increase in oseltamivir half-maximal inhibitory concentration (IC50), and E119D conferred the highest zanamivir IC50. Unlike N2 (another group 2NA), H274Y conferred highly reduced inhibition by oseltamivir. Additionally, R152K, a naturally occurring variation at the NA catalytic residue of A(H7N9) viruses, conferred reduced inhibition by laninamivir.ConclusionsThe recNA method is a valuable tool for assessing the effect of NA changes on drug susceptibility of emerging influenza viruses.
| S-EPMC8580195 | biostudies-literature
Project description:Wild waterfowl is the natural reservoir of influenza A virus (IAV); hosted viruses are very variable and provide a source for genetic segments which can reassort with poultry or mammalian adapted IAVs to generate novel species crossing viruses. Additionally, wild waterfowl act as a reservoir for highly pathogenic IAVs. Exposure of wild birds to the antiviral drug oseltamivir may occur in the environment as its active metabolite can be released from sewage treatment plants to river water. Resistance to oseltamivir, or to other neuraminidase inhibitors (NAIs), in IAVs of wild waterfowl has not been extensively studied.In a previous in vivo Mallard experiment, an influenza A(H6N2) virus developed oseltamivir resistance by the R292K substitution in the neuraminidase (NA), when the birds were exposed to oseltamivir. In this study we tested if the resistance could be maintained in Mallards without drug exposure. Three variants of resistant H6N2/R292K virus were each propagated during 17 days in five successive pairs of naïve Mallards, while oseltamivir exposure was decreased and removed. Daily fecal samples were analyzed for viral presence, genotype and phenotype.Within three days without drug exposure no resistant viruses could be detected by NA sequencing, which was confirmed by functional NAI sensitivity testing. We conclude that this resistant N2 virus could not compete in fitness with wild type subpopulations without oseltamivir drug pressure, and thus has no potential to circulate among wild birds. The results of this study contrast to previous observations of drug induced resistance in an avian H1N1 virus, which was maintained also without drug exposure in Mallards. Experimental observations on persistence of NAI resistance in avian IAVs resemble NAI resistance seen in human IAVs, in which resistant N2 subtypes do not circulate, while N1 subtypes with permissive mutations can circulate without drug pressure. We speculate that the phylogenetic group N1 NAs may easier compensate for NAI resistance than group N2 NAs, though further studies are needed to confirm such conclusions.
| S-EPMC4589409 | biostudies-literature
Project description:Studies of pathogen-host specificity, virulence, and transmissibility are critical for basic research as well as for assessing the pandemic potential of emerging infectious diseases. This is especially true for viruses such as influenza, which continue to affect millions of people annually through both seasonal and occasional pandemic events. Although the influenza virus has been fairly well studied for decades, our understanding of host-cell binding and its relation to viral transmissibility and infection is still incomplete. Assessing the binding mechanisms of complex biological systems with atomic-scale detail is challenging given current experimental limitations. Much remains to be learned, for example, about how the terminal residue of influenza-binding host-cell receptors (sialic acid) interacts with the viral surface. Here, we present an integrative structural-modeling and physics-based computational assay that reveals the sialic acid association rate constants (k on) to three influenza sites: the hemagglutinin (HA), neuraminidase (NA) active, and NA secondary binding sites. We developed a series of highly detailed (atomic-resolution) structural models of fully intact influenza viral envelopes. Brownian dynamics simulations of these systems showed how structural properties, such as stalk height and secondary-site binding, affect sialic acid k on values. Comparing the k on values of the three sialic acid binding sites across different viral strains suggests a detailed model of encounter-complex formation and indicates that the secondary NA binding site may play a compensatory role in host-cell receptor binding. Our method elucidates the competition among these sites, all present on the same virion, and provides a new technology for directly studying the functional balance between HA and NA.
| S-EPMC6276040 | biostudies-literature
Project description:The QFlu prototype bioluminescence-based neuraminidase (NA) inhibition (NI) assay kit was designed to detect NA inhibitor (NAI)-resistant influenza viruses at point of care. Here, we evaluated its suitability for drug susceptibility assessment at a surveillance laboratory. A comprehensive panel of reference viruses (n = 14) and a set of 90 seasonal influenza virus A and B isolates were included for testing with oseltamivir and/or zanamivir in the QFlu assay using the manufacturer-recommended protocol and a modified version attuned to surveillance requirements. The 50% inhibitory concentrations (IC50s) generated were compared with those of NI assays currently used for monitoring influenza drug susceptibility, the fluorescent (FL) and chemiluminescent (CL) assays. To provide proof of principle, clinical specimens (n = 235) confirmed by real-time reverse transcription (RT)-PCR to contain influenza virus A(H1N1)pdm09 and prescreened for the oseltamivir resistance marker H275Y using pyrosequencing were subsequently tested in the QFlu assay. All three NI assays were able to discriminate the reference NA variants and their matching wild-type viruses based on the difference in their IC50s. Unless the antigenic types were first identified, certain NA variants (e.g., H3N2 with E119V) could be detected among seasonal viruses using the FL assays only. Notably, the QFlu assay identified oseltamivir-resistant A(H1N1)pdm09 viruses carrying the H275Y marker directly in clinical specimens, which is not feasible with the other two phenotypic assays, which required prior virus culturing in cells. Furthermore, The QFlu assay allows detection of the influenza virus A and B isolates carrying established and potential NA inhibitor resistance markers and may become a useful tool for monitoring drug resistance in clinical specimens.
| S-EPMC3811246 | biostudies-literature
Project description:The H7N9 influenza virus has been circulating in China for more than six years. The neuraminidase (NA) has gained great concern for the development of antiviral drugs, therapeutic antibodies, and new vaccines. In this study, we screened seven mouse monoclonal antibodies (mAbs) and compared their protective effects against H7N9 influenza virus. The epitope mapping from escape mutants showed that all the seven mAbs could bind to the head region of the N9 NA close to the enzyme activity sites, and four key sites of N9 NA were reported for the first time. The mAbs D3 and 7H2 could simultaneously inhibit the cleavage of the sialic acid of fetuin protein with large molecular weight and NA-XTD with small molecule weight in the NA inhibition experiment, prevent the formation of virus plaque at a low concentration, and effectively protect the mice from the challenge of the lethal dose of H7N9 virus.
| S-EPMC6968527 | biostudies-literature
Project description:BACKGROUND: Since late March 2013, there has been another global health concern with a sudden wave of flu infections by a novel strain of avian influenza A (H7N9) virus in China. To-date, there have been more than 100 infections with 23 deaths. It is more worrying as this viral strain has never been detected in humans and only been found to be of low-pathogenicity. Currently, there are 3 effective neuraminidase inhibitors for this H7N9 virus strain, i.e. oseltamivir, zanamivir, and peramivir. These drugs have been used for treatment of the H7N9 influenza in China. However, how these inhibitors work and affect the binding cavity of the novel H7N9 neuraminidase in the presence of potential mutations has not been disclosed. In our study, we investigate steric effects and subsequently show the conformational restraints of the inhibitor-binding site of the non-mutated and mutated H7N9 neuraminidase structures to different drug compounds. RESULTS: Combination of molecular docking and Molecular Dynamics simulation reveal that zanamivir forms more favorable and stable complex than oseltamivir and peramivir when binding to the active site of the H7N9 neuraminidase. And it is likely that the novel influenza A (H7N9) virus adopts a higher probability to acquire resistance to peramivir than the other two inhibitors. Conformational changes induced by the mutation R289K causes loss of number of hydrogen bonds between the inhibitors and the H7N9 viral neuraminidase in 2 out of 3 complexes. In addition, our results of binding-affinity relationships of the 3 inhibitors with the viral neuraminidase proteins of previous pandemics (H1N1, H5N1) and the current novel H7N9 reflected the extent of binding effectiveness of the 3 inhibitors to the novel H7N9 neuraminidase. CONCLUSIONS: The results are novel and specific for the A/Hangzhou/1/2013(H7N9) influenza strain. Furthermore, the protocol could be useful for further drug-binding analysis and prediction of future viral mutations to which the virus evolves through adaptation and acquires resistance to the current available drugs.
| S-EPMC3853198 | biostudies-literature
Project description:A novel reassortant avian influenza A virus (H7N9) emerged in humans in Eastern China in late February 2013. All virus strains were resistant to adamantanes (amantadine and rimantadine), but susceptible to neuraminidase inhibitors (NAIs) (oseltamivir and zanamivir). One strain (A/shanghai/1/2013) contained the R294K substitution in the neuraminidase (NA) gene, indicating resistance to oseltamivir. Pyrosequencing has proven to be a useful tool in the surveillance of drug resistance in influenza A viruses. Here, we describe a reverse transcription (RT)-PCR assay coupled with pyrosequencing to identify the NA residues E120, H276, and R294 (N9 numbering) of H7N9 viruses. A total of 43 specimens (26 clinical samples and 17 isolates) were tested. Only one isolate containing the E120V heterogenic mutation was detected by pyrosequencing and confirmed by Sanger sequencing. However, this mutation was not detected in the original clinical specimen. Since virus isolation might lead to the selection of variants that might not fully represent the virus population in the clinical specimens, we suggest that using pyrosequencing to detect NAI resistance in H7N9 viruses directly from clinical specimens rather than from cultured isolates. No cross-reactions with other types of influenza virus and respiratory tract viruses were found, and this assay has a sensitivity of 100 copies of synthetic RNA for all three codons. The high sensitivity and specificity of the assay should be sufficient for the detection of positive clinical specimens. In this study, we provide a rapid and reliable method for the characterization of NAI resistance in H7N9 viruses.
| S-EPMC7126809 | biostudies-literature
Project description:Neuraminidase (NA) immunity leads to decreased viral shedding and reduced severity of influenza disease; however, NA content in influenza vaccines is currently not regulated, resulting in inconsistent quality and quantity of NA that can vary from manufacturer to manufacturer, from year to year, and from lot to lot. To address this problem, we have developed an assay for NA quantification that could be used by the industry to move toward developing influenza vaccines that induce a predictable immune response to NA. The VaxArray Influenza Seasonal NA Potency Assay (VXI-sNA) is a multiplexed sandwich immunoassay that relies on six subtype-specific monoclonal antibodies printed in microarray format and a suite of fluor-conjugated "label" antibodies. The performance of the assay as applied to a wide range of influenza vaccines is described herein. The assay demonstrated high NA subtype specificity and high sensitivity, with quantification limits ranging from 1 to 60 ng/mL and linear dynamic ranges of 24-500-fold. When compared to an enzymatic activity assay for samples exposed to thermal degradation conditions, the assay was able to track changes in protein stability over time and exhibited good correlation with enzyme activity. The assay also demonstrated excellent analytical precision with relative error ranging from 6 to 12% over day-to-day, user-to-user, and lot-to-lot variation. The high sensitivity and reproducibility of the assay enabled robust detection and quantification of NA in crude in-process samples and low-dose, adjuvanted vaccines with an accuracy of 100 ± 10%.
| S-EPMC6342948 | biostudies-literature