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

Abstract

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. 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.

Genetic Characterization of a Novel North American-Origin Avian Influenza A (H6N5) Virus Isolated from Bean Goose of South Korea in 2018

“…Additionally, the NAs of the H6N5 isolates had no amino acid deletion in the stalk region [ 26 ]. However, the E119V mutation in NA was detected in all H6N5 isolates, suggesting their susceptibility to neuraminidase inhibitors, such as Oseltamivir, Peramivir, and Zanamivir [ 27 ]. Mutations in internal genes, also presented in Table 2 , might imply an increase in viral replication efficiency, along with virulence, in mammals.…”

Zoonotic Potential of Influenza A Viruses: A Comprehensive Overview

“…The catalytic site cleaves the α2-3-SA and α2-6-SA on the host cell membrane to release progeny virions from host cell. Therefore, inhibition of NA activity was thought to be an ideal strategy for antiviral drug development [ 288 , 290 , 291 ]. Neuraminidase inhibitors (NAIs), such as (1) orally administered oseltamivir (Tamiflu); (2) inhaled zanamivir (Relenza); and (3) intravenously applied peramivir (Rapivab), are FDA-approved anti-influenza drugs that act by inhibiting the sialidase activity of the IAV/IBV NA, thereby inhibiting the release of progeny virions from infected host cells [ 292 ].…”

Five Novel Non-Sialic Acid-Like Scaffolds Inhibit In Vitro H1N1 and H5N2 Neuraminidase Activity of Influenza a Virus

“…As an asset for ORP counts the observation that its receptor affinity was not affected during docking simulations with mutated neuraminidases with respect to wild type NA. The simulated viral mutant types were studied because they cause known drug resistance to oseltamivir and zanamivir [ 55 , 56 , 57 ].…”