Monitoring and Characterization of Oseltamivir-Resistant Pandemic (H1N1) 2009 Virus, Japan, 2009–2010

No evidence of sustained spread was found, but 2 incidents of human-to-human transmission were suspected.


I n March and early April of 2009, a new swine-origin
A/H1N1 infl uenza virus, now called pandemic (H1N1) 2009, emerged in Mexico and the United States and spread rapidly (1-3). On June 11, 2009, the World Health Organization (WHO) declared a phase-6 pandemic alert, indicating a global pandemic. The earliest virus isolates were sensitive to the neuraminidase inhibitors (NAIs) zanamivir and oseltamivir, but resistant to M2 inhibitors, such as amantadine and rimantadine (1, [3][4][5]. Thus, the NAIs have been used globally for treatment and prophylaxis of pandemic (H1N1) 2009 virus infl ection.
Oseltamivir-resistant (OR) pandemic (H1N1) 2009 was fi rst detected in Japan, Denmark, and Hong Kong during May-June 2009 and has since been sporadically identifi ed around the world (6)(7)(8). The OR pandemic (H1N1) 2009 viruses have a specifi c NA mutation, a histidine-to-tyrosine substitution at amino acid position 275 (N1 numbering, H275Y), that confers resistance to oseltamivir. In a report of 39 OR pandemic (H1N1) 2009 cases (as of October 22, 2009), 16 were associated with treatment, 13 were associated with postexposure prophylaxis, 3 were in NAIuntreated patients, and 7 were of unknown association (8). Preliminary global NAI surveillance showed 190 OR pandemic (H1N1) 2009 infections among >15,000 clinical specimens; thus, the global frequency of OR pandemic (H1N1) 2009 was <1.5% (as of January 8, 2010) (9). These reports indicated that human-to-human transmission of OR pandemic (H1N1) 2009 was limited but that oseltamivir treatment and prophylaxis could lead to emergence of OR pandemic (H1N1) 2009 virus.
A report for 1997-2007 showed that Japan accounted for ≈70% of the world's oseltamivir consumption (10). oseltamivir were supplied in Japan, ≈2.3× that of the 2008-09 seasons (data from Chugai Co. Ltd, Tokyo, Japan). Thus, Japan is a high-risk environment for the development of OR pandemic (H1N1) 2009 virus because of drug use pressure. The emergence of such resistance is alarming, because OR seasonal infl uenza A (H1N1) viruses can rapidly spread worldwide once they acquire the capacity for human-to-human transmission (11)(12)(13)(14)(15). Additionally, in the 2009-10 season in Japan, almost all cases of infl uenza were caused by pandemic (H1N1) 2009 viruses ( Figure 1). Thus, close surveillance must be maintained to detect pandemic (H1N1) 2009 and changes in its transmissibility and genetic and antigenic characteristics. We monitored and characterized 4,307 clinical specimens collected in Japan during May 2009-February 2010 from patients with OR pandemic (H1N1) 2009 by NA sequencing, NAI assay, or both. Of them, we found 61 OR pandemic (H1N1) 2009 viruses with the H275Y mutation.

Virus Testing
Infl uenza sentinel clinics and nonsentinel institutes send original samples to local public health laboratories for detection and virus isolation. In total, 4,307 clinical specimens, comprising both original samples (n = 440) and clinical isolates (n = 3,867), underwent either full or partial (nt 695-1110) NA sequencing to detect the H275Y mutation. Samples from 1,088 cases were collected before oseltamivir exposure, 516 were associated with oseltamivir use, 103 were associated with zanamivir use, and for 2,600, antivirual treatment status was unknown. We collected all OR pandemic (H1N1) 2009 isolates and randomly selected OS isolates (≈10%) from local public health laboratories. These representative OS and OR pandemic (H1N1) 2009 isolates underwent NA inhibition assay (421 OS and 61 OR viruses tested), full NA and hemagglutination (HA) sequencing (190 OS and 61 OR), internal gene (PB2/PB1/PA/NP/M/NS) sequencing (138 OS and 20 OR), and hemagglutination inhibition (HI) test (583 OS and 59 OR).

Sequence Analysis
Phylogenetic trees of NA and HA genes were constructed by neighbor-joining method. A phylogenetic tree was constructed by using representative OR and OS pandemic (H1N1) 2009 isolates from several prefectures of Japan. Sequence information of pandemic (H1N1) 2009 from other countries was downloaded from the Global Initiative on Sharing Avian Infl uenza Data (GISAID) and GenBank. All amino acid positions in the phylogenetic tree were described by N1 numbering.

Statistical Analyses
Box-and-whisker plots were used to determine the cutoff value between NAI-resistant (outlier) and -sensitive viruses. The box contains 50% of the results, representing the middle 2 quartiles (25%-75%). The length of the box shows the interquartile range (IQR). The cutoff value was defi ned as the upper quartile + 3.0 × interquartile range from the 25th to 75th percentile. For statistical analyses, OR pandemic (H1N1) 2009 viruses with the H275Y mutation were excluded from the overall population.

HI Test
An HI test was performed to evaluate the reactivity of ferret antiserum against the 2009/10 vaccine strain A/ California/7/2009, as described in the WHO Manual (17). The effi cacy of ferret postinfection antiserum against egg-grown A/California/7/2009 was used as a reference. Antiserum was treated with receptor-destroying enzyme II (Denka Seiken, Tokyo, Japan) and adsorbed with turkey erythrocytes before testing, to prevent nonspecifi c reactions. A 0.5% suspension of turkey erythrocytes was used for the HI test.

Patient Treatment History and Epidemiologic Background
Of 4,307 case-patients, 516 had oseltamivir treatment, 103 had zanamivir treatment, 1,088 were NAI-untreated, and the treatment history of 2,600 was unknown. Of the 61 cases of OR pandemic (H1N1) 2009, 45 were associated with oseltamivir treatment, 10 with postexposure prophylaxis, and 6 occurred in NAI-untreated patients; thus, oseltamivir treatment and prophylaxis likely accelerated emergence of OR pandemic (H1N1) 2009. The relationship between time of sampling (days after oseltamivir treatment) and OR pandemic (H1N1) 2009 detection showed that OR pandemic (H1N1) 2009 was generally detected at least 4 days after oseltamivir treatment (Table 1).

Two Possible Cases of Human-to-Human Virus Transmission
Almost all OR pandemic (H1N1) 2009 cases emerged sporadically and were not epidemiologically linked. However, 2 cases of human-to-human transmission were suspected. One was observed in Niigata Prefecture where 2 children hospitalized in the same room were infected with OR pandemic (H1N1) 2009 virus within a few days. Symptoms developed fi rst in a 4-year-old girl on October 10, The other suspected instance of human-to-human transmission occurred in Tottori Prefecture. In a 9-yearold boy, symptoms developed on December 18, 2009, and OR pandemic (H1N1) 2009 virus was isolated from a sample collected on the same day, before oseltamivir use. However, the patient's 2 brothers were both infected with pandemic (H1N1) 2009 virus and had received oseltamivir since December 15. Although samples from these persons were not available, OR pandemic (H1N1) 2009 likely emerged in 1 patient and was transmitted to the other.

Case Unrelated to Oseltamivir Use
Detailed epidemiologic information was available for 2 of 6 persons with OR pandemic (H1N1) 2009 infections untreated by NAIs. Besides the case in Tottori Prefecture, another occurred in Oita Prefecture. The index patient had a mild cough beginning on July 12, and typical  *Of total 4,307 specimens tested, neuraminidase inhibitor treatment history was available for 1,707; of these specimens, 516 were from patients who had received oseltamivir treatment. †Parentheses indicate prophylactic use, e.g., 4 (1) = 1 of 4 total uses was for prophylaxis. ‡Day 0 represents the samples collected within 24 h after oseltamivir use.  Figure, www.cdc.gov/EID/content/17/3/470-appF.htm). Because calculating precise IC 50 values from a mixed population of NAI-resistant and -sensitive viruses is not possible (13,18), the 13 mixed isolates were excluded from the overall population for the purposes of the statistical analysis of OR.

Antiviral Drug Susceptibility
NAI data are summarized in Table 3

Antigenic Characterization
The HI test was performed to estimate the reactivity of OS (n = 583) and OR pandemic (H1N1)

Discussion
The data presented here provide no evidence of sustained spread of OR pandemic (H1N1) 2009 in Japan.  ‡IC 50 values of 13 mixed samples with H275 and Y275 were excluded from overall population in statistical analysis of OR isolates. §Mean SD IC 50 values of control seasonal influenza A (H1N1) viruses were determined from 10 independent experiments for oseltamivir and 2 for zanamivir.
In this study, clinical specimens were collected from both NAI-untreated and NAI-treated patients, so later samples were collected after the exertion of selective pressure by drug treatment. However, frequency of detection of OR pandemic (H1N1) 2009 was low (1.4%). Because OR and OS pandemic (H1N1) 2009 isolates were genetically and antigenically indistinguishable, the current 2009-10 vaccine would be expected to be effective against recent OR pandemic (H1N1) 2009. No evidence of reassortment with seasonal infl uenza A (H1N1) virus was detected. Immunocompetent patients infected with OR pandemic (H1N1) 2009 showed typical uncomplicated infl uenza symptoms, similar to those caused by OS pandemic (H1N1) 2009 (19).
Early reports suggested that ≈70% of the worldwide consumption of oseltamivir occurs in Japan (10), but long-term NAI surveillance in Japan from 1996 to 2007 (10) and previous NAI surveillance (16,20,21) showed a low frequency of resistant viruses, suggesting that the transmissibility of OR viruses selected by drug pressure was remarkably reduced. However, beginning in November 2007, an unexpectedly high frequency of OR seasonal infl uenza A (H1N1) viruses with the H275Y mutation was detected in Europe (11)(12)(13)(14)(15). Most were isolated from NAIuntreated patients and were more transmissible than OS infl uenza A (H1N1), resulting in rapid global dissemination (15). In contrast, even in the 2007-08 season, OR infl uenza A (H1N1) was detected only rarely (1.5%-2.6%) in Japan, despite the high level of oseltamivir use (13,22). However, OR infl uenza A (H1N1) virus was detected at a far higher frequency (≈100%) the next year (13). Thus, the pattern of oseltamivir use did not correspond to the emergence and widespread distribution of OR infl uenza A (H1N1) viruses.
In contrast, this study and a recent report (8) found that OR pandemic (H1N1) 2009 has been detected predominantly in isolates from oseltamivir recipients. Unlike recent OR infl uenza A (H1N1) viruses, such OR pandemic (H1N1) 2009 viruses seemed to have restricted transmissibility among humans. These fi ndings indicated that oseltamivir use was responsible for the emergence of OR pandemic (H1N1) 2009 viruses, but perhaps not for the widespread distribution of OR pandemic (H1N1) 2009.
Although the reason why recent OR seasonal infl uenza A (H1N1) isolates did not lose fi tness remains unclear, a functional defect in NA proteins caused by H275Y may be counteracted by permissive secondary mutations. Two such mutations, R222Q and V234M, have been identifi ed in seasonal infl uenza (H1N1) (23). Although whether the amino acids of the corresponding positions of pandemic (H1N1) 2009 play a similar role is unknown, the NA protein of this virus does have A and V residues at positions 222 and 234, respectively; a V at position 234 was identical to that in a nonpermissive amino acid sequence. Other sporadic and some shared amino acid change(s) were observed in the NA protein of OR pandemic (H1N1) 2009 viruses, but these changes apparently did not restore viral fi tness, because no effi ciently transmissible OR pandemic (H1N1) virus was found. These observations suggest that the NA proteins of recent OR pandemic (H1N1) 2009 isolates likely did not possess such permissive secondary mutation(s) (online Appendix Table).
However, all recent animal studies of OR pandemic (H1N1) 2009 virus have shown that viral fi tness and transmissibility did not differ from those of OS pandemic (H1N1) 2009 virus (24)(25)(26) and had a potential to supersede OS pandemic (H1N1) 2009 virus. Nevertheless, OR pandemic (H1N1) 2009 did not supersede OS pandemic (H1N1) 2009 in humans. This inconsistency may be explained by differences in infectious dose used in the animal models.
In an NAI assay of zanamivir susceptibility, statistical analysis identifi ed 3 outliers. One OS pandemic (H1N1) 2009 possessed the D151D/N mutation, which has been reported to affect susceptibility to zanamivir in seasonal viruses (20,21). Two OR pandemic (H1N1) 2009 isolates had the substitutions I34V and I195V in NA; however, whether these affect zanamivir susceptibility is unclear. We also assayed peramivir susceptibility in representative OR and OS pandemic (H1N1) 2009 isolates. Data suggested that OR pandemic (H1N1) 2009 virus, which contained the H275Y substitution, possessed cross-resistance to peramivir, as reported by another group (27).
Both case reports and preliminary NAI surveillance (8,19,(28)(29)(30)(31)(32) have indicated 2 groups are at high risk for the generation of resistant viruses. The fi rst is patients with severely compromised or suppressed immune systems, who shed virus for prolonged periods and thus have an increased chance of developing resistant virus (33,34). WHO reported that 25% of 285 resistant cases (as of April 17, 2010) occurred in immunocompromised patients (35). The second group is persons who are receiving postexposure prophylaxis, who take a subtherapeutic dose of 75 mg 1×/day (treatment dosage is 75 mg 2×/day). This regimen may only partially inhibit viral replication, thus facilitating the emergence of OR pandemic (H1N1) 2009. WHO recommends chemoprophylaxis only for persons who have a higher risk for severe or complicated illness (19). These groups were observed in our study.
We found that most OR pandemic (H1N1) 2009 virus was detected in samples collected at least 4 days after oseltamivir treatment or prophylaxis (Table 1). However, the frequency of OR pandemic (H1N1) 2009 in each day of treatment with oseltamivir could not be calculated because of a lack of treatment history data. This timing is consistent with that for OR seasonal infl uenza A (H1N1, H3N2) and pandemic (H1N1) 2009 viruses, which typically emerge 3-6 days after oseltamivir treatment (36)(37)(38). Additionally, we also observed the rapid emergence of OR pandemic (H1N1) 2009 virus within 48 hours of oseltamivir exposure (39). Nontheless, 6 cases occurred in untreated patients. Indeed, the OR pandemic (H1N1) 2009 in the Oita case may likely be a natural occurrence. We are aware of only 2 other reports, one from Vietnam and one from Hong Kong, of naturally occurring OR pandemic (H1N1) 2009 in untreated patients (7,40).
The greatest concern regarding OR pandemic (H1N1) 2009 is that drug-resistant viruses will acquire the ability to be transmitted effi ciently among humans as has recent OR seasonal infl uenza A (H1N1). Two hospital outbreaks in the United Kingdom and the United States have been reported (31,32). In both, the immune systems of all patients were severely compromised or suppressed, indicating that these patients had an increased risk for not only the emergence of OR pandemic (H1N1) 2009 virus, but also OR seasonal infl uenza (H1N1) virus (31,32). In contrast, particular attention should be paid to the Vietnamese case because a naturally occurring OR pandemic (H1N1) 2009 virus caused a cluster of 7 cases in immunocompetent patients with no history of oseltamivir use (40).
Despite the high level of oseltamivir use in Japan, prevalence of OR pandemic (H1N1) 2009 remains low (1.4%). Thus, oseltamivir remains the fi rst option for treating pandemic (H1N1) 2009, but zanamivir should be considered for immunocompromised patients. Additionally, as fi rst priority for prophylaxis of both OR and OS pandemic (H1N1) 2009 infection should be vaccination, but not antiviral agents. Conversely, a preclinical animal model showed that OR pandemic (H1N1) 2009 had high potential to acquire transmissibility without losing viral fi tness (24)(25)(26). Whether and how OR pandemic (H1N1) 2009 may acquire effi cient transmissibility among humans are not known. Thus, vigilant monitoring of OR pandemic (H1N1) 2009 infection and alterations in its transmissibility and antigenic and genetic characteristics is essential.