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

Abstract

Between September 2013 and July 2014, 2,482 influenza 2009 pandemic A(H1N1) [A(H1N1)pdm09] viruses were screened in Japan for the H275Y substitution in their neuraminidase (NA) protein, which confers cross-resistance to oseltamivir and peramivir. We found that a large cluster of the H275Y mutant virus was present prior to the main influenza season in Sapporo /: Hokkaido, with the detection rate for this mutant virus reaching 29% in this area. Phylogenetic analysis suggested the clonal expansion of a single mutant virus in Sapporo /: Hokkaido. To understand the reason for this large cluster, we examined the in vitro and in vivo properties of the mutant virus. We found that it grew well in cell culture, with growth comparable to that of the wild-type virus. The cluster virus also replicated well in the upper respiratory tract of ferrets and was transmitted efficiently between ferrets by way of respiratory droplets. Almost all recently circulating A(H1N1)pdm09 viruses, including the cluster virus, possessed two substitutions in NA, V241I and N369K, which are known to increase replication and transmission fitness. A structural analysis of NA predicted that a third substitution (N386K) in the NA of the cluster virus destabilized the mutant NA structure in the presence of the V241I and N369K substitutions. Our results suggest that the cluster virus retained viral fitness to spread among humans and, accordingly, caused the large cluster in Sapporo/Hokkaido. However, the mutant NA structure was less stable than that of the wild-type virus. Therefore, once the wild-type virus began to circulate in the community, the mutant virus could not compete and faded out.

FIG 1

Detection of influenza viruses between September 2013 and May 2014 in Japan. (A and B) Weekly reports of influenza virus isolation/detection from local public health institutes as part of the National Epidemiological Surveillance of Infectious Diseases. (C and D) Number of A(H1N1)pdm09 viruses with the H275Y substitution. The viruses in Hokkaido were reported by the Sapporo City Institute of Public Health and the Hokkaido Institute of Public Health.

FIG 2

Distribution of the H275Y mutant influenza A(H1N1)pdm09 viruses in Hokkaido. The cities and towns where the mutant viruses were detected are shown in black.

FIG 3

Phylogenetic analysis of the neuraminidase (A) and hemagglutinin (B) genes of the H275Y mutant influenza A(H1N1)pdm09 viruses detected in Japan, China, and the United States in 2013/2014 and in the Australian cluster in 2011. The gene sequences were downloaded from the GISAID EpiFlu database (www.gisaid.org). The phylogenetic trees were constructed using the MEGA 6 software with the neighbor-joining method. The mutant viruses isolated in Japan, China, the United States, and Australia are shown in red, purple, green, and blue, respectively. Wild-type viruses are shown in black. The amino acid substitutions relative to the A/California/07/2009 virus are shown to the left of the nodes. The scale bar indicates the nucleotide substitutions per site. *, Sapporo/Hokkaido cluster viruses detected outside Hokkaido.

FIG 3

Phylogenetic analysis of the neuraminidase (A) and hemagglutinin (B) genes of the H275Y mutant influenza A(H1N1)pdm09 viruses detected in Japan, China, and the United States in 2013/2014 and in the Australian cluster in 2011. The gene sequences were downloaded from the GISAID EpiFlu database (www.gisaid.org). The phylogenetic trees were constructed using the MEGA 6 software with the neighbor-joining method. The mutant viruses isolated in Japan, China, the United States, and Australia are shown in red, purple, green, and blue, respectively. Wild-type viruses are shown in black. The amino acid substitutions relative to the A/California/07/2009 virus are shown to the left of the nodes. The scale bar indicates the nucleotide substitutions per site. *, Sapporo/Hokkaido cluster viruses detected outside Hokkaido.

FIG 4

Susceptibilities of the H275Y mutant influenza A(H1N1)pdm09 viruses to neuraminidase inhibitors. The IC₅₀s of the mutant viruses from the Sapporo/Hokkaido cluster and other sporadic cases to oseltamivir, peramivir, zanamivir, and laninamivir were determined by the use of a fluorescent neuraminidase inhibition assay. Box-and-whisker plots of the IC₅₀s (medians and interquartile ranges) are shown. The numbers at the bottom of each box-and-whisker plot indicate the fold change in IC₅₀ compared with the median IC₅₀ of wild-type viruses isolated in Japan during the same period.

FIG 5

Competitive growth capabilities of the H275Y mutant influenza A(H1N1)pdm09 virus and the wild-type virus. MDCK-AX4 cells overexpressing the β-galactoside α2,6-sialyltransferase I gene were coinfected in triplicate with mutant A/Sapporo/114/2013 or A/Chiba/1017/2009, and with wild-type A/Shizuoka-c/99/2013 at a multiplicity of infection of 0.01 PFU/cell for each virus. At 2 days postinfection, the culture supernatant was harvested and serially passaged three times at a multiplicity of infection of 0.01 PFU/cell. Each culture supernatant was subjected to the allelic discrimination assay to determine the relative proportion of each genotype. The error bars indicate the standard deviations.

FIG 6

Three-dimensional structure of mutant A/Sapporo/144/2013 NA. A molecular model of the NA protein of the mutant virus was constructed by means of homology modeling and refined by using MOE. Single-point substitutions were generated on the NA model.