Unique Determinants of Neuraminidase Inhibitor Resistance among N3, N7, and N9 Avian Influenza Viruses

Abstract

Human 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 fluorescencebased 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. IMPORTANCE The 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.

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

“…As a continuation of our previous molecular-based resistance screening, which identified unique NA substitutions that confer reduced NAI susceptibility in AIVs of the N3, N7, and N9 subtypes (15), we report here the identification of 16 novel or previously reported NAI resistance-associated substitutions in AIVs of the N4, N5, N6, and N8 NA subtypes, with biosafety protocols conducted in the previous study maintained (15). These subtypes were already known to have resulted in human infections or represent a potential infection risk to humans.…”

“…The G¡N change at residue 147, conferring NAI resistance, was not reported previously in clinical samples after NAI treatment (28,29). Although the G147R substitution found in the N9 subtype conferred NAI susceptibility similar to that of the parental virus (15), the novel G147V in virus of the N5 subtype and N147I in virus of the N8 subtype have reduced inhibition by ZA (Tables 2 and 4). However, the essential role of G/N147 in the formation of the 150 cavity could potentially lead to genetic instability of the change and the possibility of return to the intrinsic amino acid (Tables 2 and 4).…”

“…N7, and N9 NA subtypes (15), we screened for molecular markers that confer NAI resistance in the N4, N5, N6, and N8 AIV subtypes. To study the N4 AIV subtype, random mutations were introduced into the catalytic domain of the NA gene of A/EM/Korea/ W140/2006 (H10N4) virus, followed by generation of a random mutant virus library in the background of PR8 virus (7ϩ1; NA gene is from avian H10N4 virus and the remaining 7 genes are from PR8 virus) by reverse genetics (RG).…”

Two Outbreak Sources of Influenza A (H7N9) Viruses Have Been Established in China

“…A total of 5.4% (19/ 352) of the H7N9 viruses possessed substitutions associated with drug resistance in the NA protein, indicating the sensitivity of neuraminidase inhibitors. More specifically, a total of 4, 1, and 14 human-origin H7N9 viruses possessed either E119V, A246T, or R292K substitutions in NA protein, respectively (21). None of the mutations occurred in avian-origin H7N9 viruses.…”

Drug Repurposing Identifies Inhibitors of Oseltamivir‐Resistant Influenza Viruses

“…This proof of principle study demonstrates that drug resistance caused by as pecific influenza N1 NA mutation (H275Y in this case) can be circumvented by using drug repositioning and structure-based computational methods to identify alternative agents.A lthough it is difficult to predict the site(s) of future NA mutations that may cause oseltamivir resistance, [16] the conformational changes induced by these mutations can be predicted after the NA is sequenced. [5b] Therefore,o ur procedure can be used to circumvent future NA mutations that cause oseltamivir resistance,a nd may be useful in rapidly controlling the spread of oseltamivir-resistant pandemic influenza viruses.…”