Background
The pandemic of 2009 highlighted the importance of global influenza viral surveillance for the detection of new virus variants and the need of antiviral medications to mitigate the public health impact of influenza. Oseltamivir is a drug commonly used for the prevention and treatment of influenza. During the 2009 influenza pandemic, oseltamivir was used worldwide and has been listed as a stockpiled drug in many countries in response to influenza pandemics [
1,
2].
After pandemic of 2009, influenza A (H1N1) pdm09 (abbreviated as H1N1pdm09 here after) viruses became one of the seasonal influenza viruses. Prior to 2013, less than 1 % of H1N1pdm09 viruses worldwide were oseltamivir-resistant and most came from patients who had received oseltamivir treatment before specimen collection [
3]. Most oseltamivir-resistant H1N1pdm09 viruses possessed histidine (H) to tyrosine (Y) change at amino acid position 275 of the NA genes [
4]. From November 2013 through February 2014, a cluster of H1N1pdm09 viruses with H275Y substitution were detected in Sapporo, Japan. No epidemiological link were identified among the patients except for one family infection, and almost all of the patients had no exposure to NAIs before specimen collection [
5], leading to concerns about local epidemics of oseltamivir-resistant viruses. In mainland China, the routine antiviral susceptibility surveillance to influenza virus with phenotypic method was established in 2010. During the influenza season from September 2013 through March 2014 in Mainland China, oseltamivir-resistant H1N1pdm09 viruses were much more frequently detected than ever since the appearance of the virus in 2009, and here we report the findings to provide data for global surveillance of antiviral-resistant influenza virus and guidance in the choice of antiviral drugs for clinical treatment.
Discussion
From September 2013 through March 2014, 24 H1N1pdm09 viruses with highly reduced sensitivity to oseltamivir were detected in mainland China. The proportion of resistant viruses was 2.14 % with highest proportion of 5.71 % in September, which is higher than the 1 % proportion worldwide in the previous year [
3], and similar to the proportion of 2 % for the circulating viruses tested during the 2013–2014 influenza season globally [
13]. Except for H275Y substitution in the NA protein, no other substitution previously reported to be related to reduced susceptibility to oseltamivir was detected in the 24 resistant viruses.
One limitation of the study is the lack of sequences obtained from corresponding clinical specimens, because only seasonal influenza virus isolates were submitted to Chinese National Influenza Center and clinical specimens were not available in the routine surveillance.
In terms of HA gene mutation, the H1N1pdm09 viruses with H275Y substitution isolated from mainland China were more similar to the clustered cases from the United States in 2013–2014. However, with regard to the NA gene phylogenetic tree, the H1N1pdm09 viruses isolated from mainland China were more similar to those from Japan, in 2013–2014. Thus, it is possible that the NA genes of the H1N1pdm09 viruses isolated from mainland China and Japan shared the same origin. The 24 viruses were isolated from 23 cases from 14 provinces or municipalities in mainland China with the first case detected in September 2013 in China. The first clustered case in Sapporo, Japan, was detected in November 2013. Therefore, it is plausible that this virus was introduced to Japan from China, and further mutation in the HA and internal genes of the Japanese oseltamivir resistant H1N1pdm09 viruses might enable this virus to have caused a limited human-to-human transmission in Sapporo, Japan and distinct from the sporadic H275Y mutant viruses in Japan during the same period [
5].
The seasonal influenza A (H1N1) viruses, which contain an H275Y substitution in the NA protein, were first detected in the Norway and subsequently elsewhere in Europe in the early 2008, and then spread globally within nine months, indicating that other amino acid substitutions may have compensate the destabilizing effect from H275Y substitutions, thus enabling the spread of viruses with the H275Y substitutions in the population [
9,
14]. After the H1N1pdm09 pandemic started, a widespread community cluster of an oseltamivir-resistant H1N1pdm09 virus occurred in 2011 in Newcastle, Australia [
8]. This resistant virus possessed the H275Y substitution and three additional substitutions, V241, N369K and N386S, in the NA protein. The V241I and N369K substitutions, were reported to confer robust viral fitness on the H275Y mutant virus [
15,
16]. The vast majority of recently circulating H1N1pdm09 viruses possessed two amino acid substitutions, V241I and N369K, in the NA protein [
3]. The N386S substitution, however, decreased the enzymatic activity and surface expression of NA in infected cell, suggesting a negative effect on virus fitness [
15]. Similarly, the 24 viruses from mainland China and the viruses from Sapporo, Japan possessed the V241I, N369K and N386K substitutions. However, most (>89 %) resistant-viruses from the United States during the same period did not have the N386K substitution [
7]. Before the 2013–2014 influenza season, H275Y mutant virus with V241I, N369K and N386K substitutions was not detected in Mainland China, or Japan [
17]. It was reported the N386K destabilizes the NA structure in the presence of the V241I and N369K substitutions, causing a negative effect on virus fitness [
5]. Therefore, the structure of the mutant NA molecule was less stable than that of the sensitive virus, presumably because of the N386K substitution. In addition to permissive NA mutations, other properties which might provide an advantage to oseltamivir-resistant viruses and facilitate their spread, should also be monitored.
Oseltamivir was widely used for influenza prevention and treatment in 2009, and has been listed as a stockpiled drug in response to influenza pandemics in many countries after the 2009 pandemic. Emergence of the clustered cases in Australia in 2011, in the United States and Japan in 2013–2014, as well as the rise in the proportion of oseltamivir-resistant H1N1pdm09 virus in Mainland China from September 2013 through March 2014, with most of the patients weren’t being treated with NAIs before sampling, has indicated that the human-to-human transmission capacity of the H1N1 pdm09 viruses with H275Y substitution is gradually increasing. Fortunately, in our study, the H1N1pdm09 viruses with H275Y substitution in NA protein were still sensitive to zanamivir. Therefore, in addition to strengthening the enhanced surveillance of antiviral susceptibility of influenza virus, we should pay attention to clinical drug choices, development of new drugs, and strive to improve capabilities for influenza prevention and control.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
WDY and SYL designed the research; WZ and XN received the viruses; TMJ, CHY, GJF and WHJ propagated in either MDCK cells or embryonated chicken eggs prior to testing; HWJ and LXY performed NI assay; LXY, CHY, ZX and LY performed sequencing; SYL contributed new reagents/analytic tools; HWJ analyzed data; HWJ wrote the manuscript; WDY and SYL revised the manuscript. All authors read and approved the final manuscript.