Characterization of neuraminidase inhibitor-resistant influenza A(H1N1)pdm09 viruses isolated in four seasons during pandemic and post-pandemic periods in Japan

Abstract

Background/objectives: Japan has the highest frequency of neuraminidase (NA) inhibitor use against influenza in the world. Therefore, Japan could be at high risk of the emergence and spread of NA inhibitor-resistant viruses. The aim of this study was to monitor the emergence of NA inhibitor-resistant viruses and the possibility of human-to-human transmission during four influenza seasons in Japan.

Methods: To monitor antiviral-resistant A(H1N1)pdm09 viruses, we examined viruses isolated in four seasons from the 2008-2009 season through the 2011-2012 season in Japan by allelic discrimination, NA gene sequencing, and NA inhibitor susceptibility.

Results: We found that 157 (1·3%) of 12 026 A(H1N1)pdm09 isolates possessed an H275Y substitution in the NA protein that confers about 400- and 140-fold decreased susceptibility to oseltamivir and peramivir, respectively, compared with 275H wild-type viruses. The detection rate of resistant viruses increased from 1·0% during the pandemic period to 2·0% during the post-pandemic period. The highest detection rate of the resistant viruses was found in patients who were 0-9 years old. Furthermore, among the cases with resistant viruses, the percentage of no known exposure to antiviral drugs increased from 16% during the pandemic period to 44% during the post-pandemic period, implying that suspected human-to-human transmission of the resistant viruses gradually increased in the post-pandemic period.

Conclusions: A(H1N1)pdm09 viruses resistant to oseltamivir and peramivir were sporadically detected in Japan, but they did not spread throughout the community. No viruses resistant to zanamivir and laninamivir were detected.

Keywords: Influenza A(H1N1)pdm09 virus; neuraminidase inhibitor resistant; neuraminidase inhibitor susceptibility.

Figure 1

Detection of A(H1N1)pdm09 viruses with the H275Y substitution during the 2008–2012 influenza seasons in Japan. (A) Monthly reports of A(H1N1)pdm09 virus isolation/detection by local public health institutes under the National Epidemiological Surveillance of Infectious Diseases. (B) Number of A(H1N1)pdm09 viruses tested in this study. (C) Number of A(H1N1)pdm09 viruses with the H275Y substitution. Closed bars relate to the left y‐axis and open bars to the right y‐axis.

Figure 2

Susceptibility of A(H1N1)pdm09 viruses with the H275Y substitution to NA inhibitors. IC ₅₀ values of A(H1N1)pdm09 viruses to oseltamivir, zanamivir, peramivir, and laninamivir were determined by NA inhibition assay. Viruses were from the 2008–2009 (A), the 2009–2010 (B), and the 2010–2011 (C) influenza seasons. Box‐and‐whisker plots of IC ₅₀ values are shown. Numbers on top of each box‐and‐whisker plot indicate the mean IC ₅₀ value ± 1 standard deviation.

Figure 3

Age distribution of patients with oseltamivir‐ and peramivir‐resistant A(H1N1)pdm09 viruses. Age distribution of patients during the 2008–2009, the 2009–2010, and the 2010–2011 influenza seasons. (A) The proportion of patients infected with A(H1N1)pdm09 viruses. (B) The proportion of patients with resistant viruses.

Figure 4

Phylogenetic trees of the A(H1N1)pdm09 virus NA and HA1 genes. Phylogenetic trees of NA (A) and HA1 (B) genes were constructed by the neighbor‐joining method with bootstrap values of 1000 replicates. Amino acid substitutions relative to the A/California/07/2009 virus are shown on the left side of the node. Bootstrap values are indicated only when greater than 50%. Viruses isolated in the 2008–2009, the 2009–2010, the 2010‐2011, and the 2011–2012 influenza seasons are colored in green, yellow, orange, and blue, respectively. The blue circles indicate the H275Y mutant viruses in the cases with no known exposure to antiviral drug. The H275Y mutant viruses in the cases associated with antiviral drug use are represented by red circles. The scale bar shows the nucleotide substitutions per site.