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Functional neuraminidase inhibitor resistance motifs in avian influenza A(H5Nx) viruses.

ABSTRACT: Neuraminidase inhibitors (NAIs) are antiviral agents recommended worldwide to treat or prevent influenza virus infections in humans. Past influenza virus pandemics seeded by zoonotic infection by avian influenza viruses (AIV) as well as the increasing number of human infections with AIV have shown the importance of having information about resistance to NAIs by avian NAs that could cross the species barrier. In this study we introduced four NAI resistance-associated mutations (N2 numbering) previously found in human infections into the NA of three current AIV subtypes of the H5Nx genotype that threaten the poultry industry and human health: highly pathogenic H5N8, H5N6 and H5N2. Using the established MUNANA assay we showed that a R292K substitution in H5N6 and H5N2 viruses significantly reduced susceptibility to three licenced NAIs: oseltamivir, zanamivir and peramivir. In contrast the mutations E119V, H274Y and N294S had more variable effects with NAI susceptibility being drug- and strain-specific. We measured the replicative fitness of NAI resistant H5N6 viruses and found that they replicated to comparable or significantly higher titres in primary chicken cells and in embryonated hens' eggs as compared to wild type - despite the NA activity of the viral neuraminidase proteins being reduced. The R292K and N294S drug resistant H5N6 viruses had single amino acid substitutions in their haemagglutinin (HA): Y98F and A189T, respectively (H3 numbering) which reduced receptor binding properties possibly balancing the reduced NA activity seen. Our results demonstrate that the H5Nx viruses can support drug resistance mutations that confer reduced susceptibility to licenced NAIs and that these H5N6 viruses did not show diminished replicative fitness in avian cell cultures. Our results support the requirement for on-going surveillance of these strains in bird populations to include motifs associated with human drug resistance.

SUBMITTER: Bialy D

PROVIDER: S-EPMC7534037 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Functional neuraminidase inhibitor resistance motifs in avian influenza A(H5Nx) viruses.

Bialy Dagmara D Shelton Holly H

Antiviral research 20200801

Neuraminidase inhibitors (NAIs) are antiviral agents recommended worldwide to treat or prevent influenza virus infections in humans. Past influenza virus pandemics seeded by zoonotic infection by avian influenza viruses (AIV) as well as the increasing number of human infections with AIV have shown the importance of having information about resistance to NAIs by avian NAs that could cross the species barrier. In this study we introduced four NAI resistance-associated mutations (N2 numbering) prev ...[more]

PMID: 32750468

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Project description:UnlabelledHuman infections with avian influenza viruses are a serious public health concern. The neuraminidase (NA) inhibitors (NAIs) are the frontline anti-influenza drugs and are the major option for treatment of newly emerging influenza. Therefore, it is essential to identify the molecular markers of NAI resistance among specific NA subtypes of avian influenza viruses to help guide clinical management. NAI-resistant substitutions in NA subtypes other than N1 and N2 have been poorly studied. Here, we identified NA amino acid substitutions associated with NAI resistance among influenza viruses of N3, N7, and N9 subtypes which have been associated with zoonotic transmission. We applied random mutagenesis and generated recombinant influenza viruses carrying single or double NA substitution(s) with seven internal genes from A/Puerto Rico/8/1934 (H1N1) virus. In a fluorescence-based NA inhibition assay, we identified three categories of NA substitutions associated with reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir): (i) novel subtype-specific substitutions in or near the enzyme catalytic site (R152W, A246T, and D293N, N2 numbering), (ii) subtype-independent substitutions (E119G/V and/or D and R292K), and (iii) substitutions previously reported in other subtypes (Q136K, I222M, and E276D). Our data show that although some markers of resistance are present across NA subtypes, other subtype-specific markers can only be determined empirically.ImportanceThe number of humans infected with avian influenza viruses is increasing, raising concerns of the emergence of avian influenza viruses resistant to neuraminidase (NA) inhibitors (NAIs). Since most studies have focused on NAI-resistance in human influenza viruses, we investigated the molecular changes in NA that could confer NAI resistance in avian viruses grown in immortalized monolayer cells, especially those of the N3, N7, and N9 subtypes, which have caused human infections. We identified not only numerous NAI-resistant substitutions previously reported in other NA subtypes but also several novel changes conferring reduced susceptibility to NAIs, which are subtype specific. The findings indicate that some resistance markers are common across NA subtypes, but other markers need to be determined empirically for each subtype. The study also implies that antiviral surveillance monitoring could play a critical role in the clinical management of influenza virus infection and an essential component of pandemic preparedness.

Project description: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.

Dataset Information

Functional neuraminidase inhibitor resistance motifs in avian influenza A(H5Nx) viruses.

Publications

Functional neuraminidase inhibitor resistance motifs in avian influenza A(H5Nx) viruses.

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