A two-year survey of the oseltamivir-resistant influenza A(H1N1) virus in Yamagata, Japan and the clinical effectiveness of oseltamivir and zanamivir

Background Oseltamivir is the preferred antiviral drug for influenza, but oseltamivir-resistant A(H1N1) viruses have circulated worldwide since the 2007-2008 influenza season. We aimed to determine the rate of oseltamivir resistance among A(H1N1) isolates from Yamagata, Japan, to compare the virological characteristics between isolates from the 2007-2008 and 2008-2009 seasons, and to evaluate the clinical effectiveness of oseltamivir. Results Oseltamivir resistance, determined by detecting the H275Y mutation in the neuraminidase (NA) gene, was observed in 2.5% (2 of 79) and 100% (77 of 77) of isolates from the 2007-2008 and 2008-2009 seasons, respectively. Antigenic analysis suggested that antigenically different variants of A(H1N1) viruses circulated in the 2008-2009 season. Growth testing demonstrated that the ability of the 2008-2009 isolates to replicate in MDCK cells was similar to those of the oseltamivir-susceptible isolates from the 2007-2008 season. A phylogenetic analysis revealed that two oseltamivir-resistant viruses isolated in the 2007-2008 season were closely related to other oseltamivir-susceptible viruses in Yamagata but were different from oseltamivir-resistant viruses isolated in Europe and North America in the 2007-2008 season. The oseltamivir-resistant viruses isolated in Japan in the 2008-2009 season were phylogenetically similar to oseltamivir-resistant isolates from Europe and North America during the 2007-2008 season. Furthermore, the median duration of fever after the start of oseltamivir treatment was significantly longer in oseltamivir-resistant cases (2 days; range 1-6 days) than in oseltamivir-susceptible cases (1.5 days: range 1-2 days) (P = 0.0356). Conclusion Oseltamivir-resistant A(H1N1) isolates from Yamagata in the 2007-2008 season might have acquired resistance through the use of oseltamivir, and the 2008-2009 oseltamivir-resistant isolates might have been introduced into Japan and circulated throughout the country. Influenza surveillance to monitor oseltamivir-resistance would aid clinicians in determining an effective antiviral treatment strategy.


Background
During the 2007-2008 season, increased levels of resistance to oseltamivir among influenza A (H1N1) viruses were reported in Europe and North America [1][2][3][4][5][6], and oseltamivir-resistant viruses were also detected in the southern hemisphere [7,8]. The frequency of oseltamivir-resistance in A(H1N1) isolates was highest (67%) in Norway [9]. During the same season in Japan, it is estimated that up to 2.6% of all influenza A(H1N1) isolates were resistant to oseltamivir [10]. It was reported that some of the resistant viruses found in Japan during the 2007-2008 season were not phylogenetically related to those found in Europe and that these resistant isolates from Japan emerged independently in Japan [11,12]. Further, during the 2008-2009 season, the A(H1N1) virus was prominent in influenza outbreaks in Japan, and national surveillance showed that 99.6% of A(H1N1) isolates had the histidineto-tyrosine substitution at residue 275 (H275Y) of the neuraminidase (NA) gene; this mutation is associated with oseltamivir resistance [13]. Oseltamivir is widely used in clinical settings in Japan. Therefore, an increase in oseltamivir-resistant influenza viruses is an important problem that is likely to influence the treatment strategy for influenza virus infections.
The purposes of this study were to investigate the percentage of A(H1N1) isolates from Yamagata Prefecture during the 2007-2008 and 2008-2009 seasons that had the H275Y mutation in the NA gene and to compare the virological characteristics between the A(H1N1) viruses isolated in those seasons. Additionally, we evaluated the clinical effectiveness of oseltamivir and zanamivir against oseltamivir-resistant A(H1N1) virus infections.  (Table  2). However, the H275Y mutation had no impact on the susceptibility to zanamivir.

Antigenic analysis
The antigenic analysis was performed by hemagglutination inhibition (HI) tests for reactivity with post-   Phylogenetic analysis of the HA and NA genes  (Table 4). We found no significant differences in the age, maximum temperature, or total febrile period of oseltamivir-treated children between cases of the oseltamivir-susceptible and oseltamivir-resistant influenza A(H1N1) virus infections. However, for the children treated with oseltamivir, the

Discussion
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    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] Figure  3) [12]. 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 [18]. 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 oseltamivirresistant 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.

Conclusions
Oseltamivir resistance was observed in 2.5% and 100% of A(H1N1) isolates from Yamagata from the 2007-2008 and 2008-2009 seasons, respectively. Oseltamivir-resistant isolates from Yamagata in the 2007-2008 season might have acquired resistance through the use of oseltamivir. In contrast, the 2008-2009 oseltamivir-resistant isolates from Yamagata were similar to viruses isolated in Europe and North America; therefore, they might have been introduced into Japan and circulated throughout the country. It is certain that influenza surveillance to monitor oseltamivir resistance will benefit clinicians in determining an effective antiviral treatment strategy. Timely monitoring and reporting of antiviral drug-resistance is important at the global, national, and the local community levels.

Specimen collection and virus isolation
Nasopharyngeal swab specimens from patients with acute respiratory infection were collected at pediatric clinics collaborating with the local health authorities in Yamagata Prefecture for the surveillance of viral diseases in Japan. Specimens were transported to the Yamagata Prefectural Institute of Public Health and were grown in a virus culture as previously described [19]. A total of 79 A(H1N1) viruses isolated from 690 specimens between December 2007 and March 2008 and a total of 77 A(H1N1) viruses isolated from 831 specimens between December 2008 and March 2009 were used in this study. Of the 156 isolates, 84 (53.8%) were isolated from children under 6 years of age, 55 (35.3%) were from children aged between 6 and 10 years, 15 (9.6%) were from children aged between 11 and 15 years, and 2 (1.3%) were from patients >15 years.

NA inhibition assay
A chemiluminescent NA inhibition assay was performed using a commercially available kit, NA-Star (Applied Biosystems, Foster City, CA), according to the manufacturer's protocol. The NA inhibitors (oseltamivir and zanamivir) susceptibility of influenza virus isolates was expressed as the concentration of NA inhibitor needed to inhibit the NA enzyme activity by 50% (IC 50 ). Oseltamivir carboxylate, the active form of the prodrug oseltamivir phosphate, was provided by F. Hoffmann-La Roche Ltd, Switzerland, and zanamivir was provided by GlaxoSmithKline Research and Development Ltd., United Kingdom.

Hemagglutination inhibition test
The hemagglutination inhibition (HI) test was performed in microtiter plates with 1% guinea pig erythro- vaccine strain] (Denka Seiken Co., Ltd., Tokyo, Japan), as described previously [20]. Sera were treated with receptor-destroying enzyme before use. For the vaccine antigen, purified HA protein (Denka Seiken Co., Ltd) was diluted in a hemagglutination titer of eight, while MDCK culture fluid was also diluted in hemagglutination titer of eight and used as the isolate antigen.
Growth of H1N1 isolates in culture MDCK cells were infected with an A (H1N1) isolate at an MOI of 0.001 and incubated for 72 hours at 37°C in the presence of 2 μg/ml trypsin. At 12 h, 24 h, 48 h, and 72 h post-infection, the supernatants were harvested and virus titers were determined by a plaque assay on MDCK cells. The growth test was performed in duplicate experiments, and the mean virus titer was calculated.

Clinical information
We used the clinical information of children who visited the Sanjoh Clinic and from whom A(H1N1) influenza virus was isolated. Data were obtained retrospectively from their medical records. To compare the data between oseltamivir-resistant and oseltamivir-susceptible A(H1N1) virus infections, we used the Mann-Whitney U test. A P value of < 0.05 was regarded as statistically significant.