PT  - JOURNAL ARTICLE
AU  - Tamura, Daisuke
AU  - Nguyen, Ha T.
AU  - Sleeman, Katrina
AU  - Levine, Marnie
AU  - Mishin, Vasiliy P.
AU  - Yang, Hua
AU  - Guo, Zhu
AU  - Okomo-Adhiambo, Margaret
AU  - Xu, Xiyan
AU  - Stevens, James
AU  - Gubareva, Larisa V.
TI  - Cell culture-selected substitutions in influenza A(H3N2) neuraminidase affect drug susceptibility assessment
AID  - 10.1128/AAC.01364-13
DP  - 2013 Sep 30
TA  - Antimicrobial Agents and Chemotherapy
PG  - AAC.01364-13
4099  - http://aac.asm.org/content/early/2013/09/24/AAC.01364-13.short
4100  - http://aac.asm.org/content/early/2013/09/24/AAC.01364-13.full
AB  - 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-grown isolate of this virus exhibited highly reduced inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir (8005-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 zanamivir-IC50 and had no effect on inhibition by others. The T148I substitution reduced NA activity by 50%, most likely by affecting positioning of the 150-loop at the NA catalytic site. Using the pyrosequencing, changes at T148 were detected in 35 (23%) of 150 MDCK-grown A(H3N2) viruses tested, which was lower than the frequency of changes at D151 (85%), a 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 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.