Cell Culture-Selected Substitutions in Influenza A(H3N2) Neuraminidase Affect Drug Susceptibility Assessment

Abstract

Assessment of drug susceptibility has become an integral part of influenza virus surveillance. In this study, we describe the drug resistance profile of influenza A(H3N2) virus, A/Mississippi/05/2011, collected from a patient treated with oseltamivir and detected via surveillance. An MDCK cell-grown isolate of this virus exhibited highly reduced inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir (8,005-fold), zanamivir (813-fold), peramivir (116-fold), and laninamivir (257-fold) in the NA inhibition assay. Sequence analysis of its NA gene revealed a known oseltamivir-resistance marker, the glutamic acid-to-valine substitution at position 119 (E119V), and an additional change, threonine to isoleucine at position 148 (T148I). Unlike E119V, T148I was not detected in the clinical sample but acquired during viral propagation in MDCK cells. Using recombinant proteins, T148I by itself was shown to cause only a 6-fold increase in the zanamivir 50% inhibitory concentration (IC 50 ) and had no effect on inhibition by other drugs. The T148I substitution reduced NA activity by 50%, most likely by affecting the positioning of the 150 loop at the NA catalytic site. Using pyrosequencing, changes at T148 were detected in 35 (23%) of 150 MDCK cell-grown A(H3N2) viruses tested, which was lower than the frequency of changes at D151 (85%), an NA residue previously implicated in cell selection. We demonstrate that culturing of the A(H3N2) viruses (n ‫؍‬ 11) at a low multiplicity of infection delayed the emergence of the NA variants with changes at position 148 and/or 151, especially when conducted in MDCK-SIAT1 cells. Our findings highlight the current challenges in monitoring susceptibility of influenza A(H3N2) viruses to the NAI class of antiviral drugs.

Antiviral Susceptibility of Variant Influenza A(H3N2)v Viruses Isolated in the United States from 2011 to 2013

“…The effect of S247P on the enzymatic activity on the recNA was next investigated as previously described (32). When expressed either alone or together with S245N, S247P reduced by 60% the recNA's ability to cleave a small synthetic substrate MUNANA, compared to the wild-type recNA (data not shown).…”

Standardizing the influenza neuraminidase inhibition assay among United States public health laboratories conducting virological surveillance

“…The present study also showed no significant variations in IC 50 s arising from virus propagation. Nevertheless, participating laboratories were advised by the CDC to limit the number of virus passages since it could lead to emergence of NA changes resulting in elevated IC 50 s (Tamura et al, 2013;Mishin et al, 2014).…”

“…The assay was performed using the NA-Fluor™ Influenza Neuraminidase Assay Kit (Applied Biosystems/Life Technologies, Carlsbad, CA) in 96-well opaque black flat-bottom microplates (Corning Inc., Corning, NY), according to the CDC protocol, which can be requested by email (fluantiviral@cdc.gov). The CDC fluorescent NI assay protocol is optimized to the needs of virological surveillance (OkomoAdhiambo et al, 2013) and somewhat differs from the manufacturer's instructions in the NA-Fluor™ kit manual. Briefly, viruses were diluted at concentrations corresponding to the target fluorescence signal generated by 1000 pmol/well of 4-methylumbelliferone (4-MU) standard.…”

“…Later, monitoring NAIsusceptibility became an integral part of virological surveillance within the WHO Global Influenza Surveillance and Response System (WHO-GISRS), where both functional (NA inhibition) and sequence-based (pyrosequencing, real time RT-PCR, Sanger) assays have been utilized to conduct drug susceptibility monitoring worldwide (Monto et al, 2006;Meijer et al, 2014). Following the FDA's approval of zanamivir and oseltamivir, CDC implemented the NI assay, first using a chemiluminescence-based (Mungall et al, 2004), then a fluorescence-based methodology (OkomoAdhiambo et al, 2013). The NI assay testing is done for the purpose of monitoring changes in the baseline NAI susceptibility of circulating influenza viruses.…”

Detection of a Transient R292K Mutation in Influenza A/H3N2 Viruses Shed for Several Weeks by an Immunocompromised Patient

“…Culturing A/H3N2 influenza viruses has been shown to alter the influenza antiviral drug susceptibility profile as assessed in NA inhibition assays. When the NA of cultured virus is subsequently sequenced, reduced or additional variant populations may be observed (25,26). This highlights one of the difficulties in assessing the influenza antiviral susceptibility profiles of patients undergoing antiviral treatment and emphasizes the importance of the direct analysis of specimens with multiple testing methods (27).…”