Impact of Potential Permissive Neuraminidase Mutations on Viral Fitness of the H275Y Oseltamivir-Resistant Influenza A(H1N1)pdm09 VirusIn Vitro, in Mice and in Ferrets

Abstract

, and H275Y/V241I/N369K (a natural variant) NA mutants were generated by reverse genetics. Replication kinetics were performed by using ST6GalI-MDCK cells. Virulence was assessed in C57BL/6 mice, and contact transmission was evaluated in ferrets. The H275Y mutation significantly reduced viral titers during the first 12 to 36 h postinfection (p.i.) in vitro. Nevertheless, the WT and H275Y viruses induced comparable mortality rates, weight loss, and lung titers in mice. The T289M mutation eliminated the detrimental effect caused by the H275Y change in vitro while causing greater weight loss and mortality in mice, with significantly higher lung viral titers on days 3 and 6 p.i. than with the H275Y mutant. In index ferrets, the WT, H275Y, H275Y/T289M, and H275Y/V241I/N369K recombinants induced comparable fever, weight loss, and nasal wash viral titers. All tested viruses were transmitted at comparable rates in contact ferrets, with the H275Y/V241I/N369K recombinant demonstrating higher nasal wash viral titers than the H275Y mutant. Permissive mutations may enhance the fitness of A(H1N1)pdm09 H275Y viruses in vitro and in vivo. The emergence of such variants should be carefully monitored.

Characterization of a Large Cluster of Influenza A(H1N1)pdm09 Viruses Cross-Resistant to Oseltamivir and Peramivir during the 2013-2014 Influenza Season in Japan

“…However, the proportion of the early mutant A/Chiba/1017/2009 to that of wild-type virus decreased significantly, whereas the proportion of the mutant A/Sapporo/114/2013 from the cluster to that of wildtype virus was 84% at passage 1, decreasing to 69% and 39% at passages 2 and 3, respectively, indicating that the mutant A/Sapporo/114/2013 virus continued to replicate efficiently, competing with the wild-type virus at the initial cycle of infection. These results suggest that the Sapporo/Hokkaido cluster virus retained the ability to grow comparably to the wild-type virus and grew significantly better than did the early mutant virus, consistent with previous reports (13,14).…”

“…Previous reports suggested that the introduction of the V241I and N369K substitutions to the NA protein of the H275Y mutant A(H1N1)pdm09 virus resulted in greater viral fitness (13,14). Because all circulating viruses in Japan, including the cluster viruses, possessed the V241I and N369K substitutions (Table 1 and (Fig.…”

“…The H275Y substitution caused a detrimental effect on viral fitness by decreasing the stability of the NA protein (12). Three additional changes were thought to improve the stability of the mutant NA, thereby compensating for the negative effects of the H275Y substitution on the growth and transmissibility of the mutant virus (13,14). The Sapporo/Hokkaido cluster viruses also possessed three substitutions (Table 1 and Fig.…”

“…In 2011, a widespread cluster of the H275Y mutant A(H1N1)pdm09 virus occurred in Newcastle, Australia (12). The V241I and N369K substitutions in the NA of this mutant virus were reported to increase its replication and transmission fitness, contributing to its efficient transmission (13,14).…”

“…In 2011, a widespread cluster of the H275Y mutant A(H1N1)pdm09 virus occurred in Newcastle, Australia (12). The V241I and N369K substitutions in the NA of this mutant virus were reported to increase its replication and transmission fitness, contributing to its efficient transmission (13,14).Recently, we reported a community cluster of A(H1N1)pdm09 viruses exhibiting cross-resistance to oseltamivir and peramivir in Sapporo, the capital of Hokkaido, Japan (15). In November and December 2013, all six A(H1N1)pdm09 viruses isolated in Sapporo were resistant to oseltamivir and peramivir and possessed the H275Y substitution (15).…”