Oseltamivir is widely used for patients with influenza A and B infections in clinical settings in Japan. Therefore, oseltamivir-resistant viruses induced by oseltamivir treatment emerge in Japan more readily than in other countries where oseltamivir is not so widely used. It has been documented that the infectivity and replicative ability of neuraminidase inhibitor-resistant viruses are compromised [14, 15]; therefore, until recently it was thought that oseltamivir-resistant influenza A(H1N1) viruses were unlikely to circulate among humans.
Antigenic and phylogenetic analyses in this study revealed that two oseltamivir-resistant influenza A(H1N1) virus strains isolated in the 2007-2008 season (A/Yamagata/66/2008 and A/Yamagata/68/2008) were closely related to other oseltamivir-susceptible A(H1N1) viruses isolated in Yamagata but were different from the resistant viruses found in Europe. It appears that oseltamivir-resistant viruses found during the 2007-2008 season in Japan emerged independently among persons treated with oseltamivir and were isolated in various communities, as previously described [11, 12]. Two oseltamivir-resistant viruses in the present study have HA and NA genes sequences that are 100% identical between the two isolates; these viruses were isolated on February 29 and March 3 from two different children from the same elementary school who had not been treated with oseltamivir. In this school, an outbreak of influenza occurred on February 29, 2008, and 28 (22%) of 129 students showed influenza-like symptoms between February 29 and March 17, 2008. Although only two samples were collected from which oseltamivir-resistant viruses were isolated, there is a possibility that the transmission of an oseltamivir-resistant virus had occurred in this school. It was also reported that an outbreak of an oseltamivir-resistant A(H1N1) virus occurred in an elementary school in Yokohama City in Japan during the same season (A/Yokohama/22/2008 and A/Yokohama/35/2008 in the NA gene tree of Figure 3) . Thus, it is likely that oseltamivir-resistant viruses posses the ability to be transmitted among humans. However, because the level of oseltamivir resistance remained at 2.5% in Yamagata (2.6% in Japan), it is apparent that the oseltamivir-resistant viruses did not spread among humans as easily as the oseltamivir-susceptible viruses.
The oseltamivir-resistant viruses isolated during the 2008-2009 season in Yamagata were antigenically and phylogenetically different from those isolated during the 2007-2008 season in Yamagata but were phylogenetically similar to viruses isolated in Europe and North America during the 2007-2008 season. Thus, it seems that oseltamivir-resistant viruses were imported into Japan, where they spread across the country during the 2008-2009 season. Some reports have suggested that the emergence of oseltamivir-resistant viruses in the 2007-2008 season in Europe was not related to the use of oseltamivir [16, 17], and that natural genetic variations may have resulted in the change in sensitivity to oseltamivir . It is likely that influenza A(H1N1) viruses having an epidemiological advantage over previous viruses emerged with natural resistance to oseltamivir and spread throughout the world. Two representative viruses isolated during the 2008-2009 season in Yamagata (A/Yamagata/126/2008 and A/Yamagata/128/2008) showed the same level of growth in MDCK cells as the oseltamivir-susceptible 2007-2008 isolates. The maintenance of replicative ability and the acquirement of antigenic differences are vital for viruses to continue transmission among humans. Hereafter, A(H1N1) viruses might continue to circulate with the H275Y mutation in the NA gene preserved. The pandemic H1N1 viruses emerged in 2009 were oseltamivir-susceptible without H275Y mutation in the NA gene. Cocirculation of oseltamivir-resistant seasonal A(H1N1) viruses and the novel H1N1 pandemic viruses may give rise to the potential risk of genetic reassortment acquiring the H275Y resistance mutation in the NA gene.
There have been few reports concerning the clinical effectiveness of oseltamivir against oseltamivir-resistant influenza viruses. We found that the duration of fever after the start of oseltamivir treatment was significantly longer in children with oseltamivir-resistant influenza A(H1N1) virus infections than in children with oseltamivir-susceptible A(H1N1) virus infections. This result suggests that oseltamivir-resistant A(H1N1) viruses circulating during the 2008-2009 season were resistant to oseltamivir not only in vitro but also in vivo. Zanamivir, however, reduced the duration of fever in oseltamivir-resistant cases. In Japan, zanamivir is used as well as oseltamivir, especially for the treatment of patients older than five years. Thus, in cases of oseltamivir-resistant influenza virus infection, zanamivir would be a more appropriate treatment than oseltamivir.