Oseltamivir-Resistant Influenza A Pandemic (H1N1) 2009 Virus, Hong Kong, China

Resistance to oseltamivir was observed in influenza A pandemic (H1N1) 2009 virus isolated from an untreated person in Hong Kong, China. Investigations showed a resistant virus with the neuraminidase (NA) 274Y genotype in quasi-species from a nasopharyngeal aspirate. Monitoring for the naturally occurring NA 274Y mutation in this virus is necessary.

Emergence of resistance to oseltamivir by seasonal infl uenza A virus (H1N1) was detected in Norway in 2007. This virus has evolved into the dominant infl uenza A virus (H1N1) in humans (6). This fi nding raises strong concerns that the 274Y resistant mutation in pandemic (H1N1) 2009 virus might circulate and become dominant. We report virologic investigation of the emergence of oseltamivir resistance in this virus in a patient from Hong Kong.

The Study
A 16-year-old previously healthy girl had a fever at the Hong Kong International Airport after her arrival from San Francisco, California, USA, on June 11, 2009. Physical examination showed a temperature of 38.3°C, a blood pressure of 117/66 mm Hg, a pulse rate of 94 beats/min, and an oxygen saturation of 99% at room air. Results of a complete blood count and liver and renal function tests were normal. She had a leukocyte count of 4.69 × 10 9 cells/L, an absolute neutrophil count of 2.36 × 10 9 cells/L, and a lymphocyte count of 1.74 × 10 9 cells/L. Findings on her chest radiograph were normal.
A nasopharyngeal aspirate (NPA) was positive for infl uenza A virus (H1N1) nucleoprotein by immunofl uorescence. NPA specimens on days 1 and 5 were positive for infl uenza A virus (H1N1) M gene and swine-specifi c specifi c H1 gene by reverse transcription-PCR (RT-PCR). Samples obtained on days 6-8 were negative. Serum and midstream urine specimens obtained on day 2 were negative for infl uenza A virus (H1N1) M gene by RT-PCR.
The patient refused antiviral therapy with oseltamivir because of fear of its potential side effects. She was then offered symptomatic treatment. Her clinical condition gradually improved and she was discharged on day 8 uneventfully.
Infl uenza A pandemic (H1N1) 2009 virus was cultured from NPA. Subsequent drug susceptibility testing showed that this isolate was resistant to oseltamivir (50% inhibitory concentration 197.5 nM), but susceptible to zanamivir, as determined by enzymatic assay (Table).
To confi rm whether the virus contained mutations associated with resistance to NA inhibitors, NA sequences from the day 1 NPA specimen and an MDCK cell isolate were examined. Viral RNA was extracted from NPA and MDCK cell supernatants by using reported procedures (7). RT-PCR was performed by using primers spanning position 274 of the NA gene (forward: 5′-ACACAAGAGTCTGAATGTGCATGT-3′; reverse: 5′-GTCTCCGAAAATCCCACTGCATAT-3′). Direct sequencing of PCR products was performed by using a Big-Dye Terminator v3.1 cycle sequencing reaction kit on an ABI PRISM 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA). Sequences indicated that the NA genes in the NPA and MDCK cell virus isolates contained an H→Y mutation at the NA 274 (H3 numbering, 275 in H1 numbering) residue (GenBank accession no. GQ351316). No other NA mutations known to be associated with oseltamivir resistance were observed. Further examination of sequences showed mixed populations (T/C) in the NA gene from the NPA specimen ( Figure, panel A).
Estimation of 274H and 274Y populations in the NPA specimen was performed by cloning and sequencing PCR products. The NPA specimen contained approximately equal proportions of 274Y and 274H (52.63% and 47.37%, respectively). Examination of sequences from the MDCK cell isolate showed predominantly the 274Y type, although a minor 274H peak was also observed (Figure, panel B). Cloning and sequencing of PCR products from the MDCK virus isolate showed that 97.92% of the NA genes were 274Y, which suggests that the 274Y population overtook the 274H population during MDCK cell culture.

Conclusions
Resistance to NA inhibitors among seasonal strains of human infl uenza viruses (A/H1N1, A/H3N2, and B) has been rare until recently. Development of resistance after oseltamivir treatment has occurred in 0.33%-5.5% of treated patients (8). Oseltamivir resistance associated with the NA 274Y genotype was also observed in human infections with avian infl uenza A virus (H5N1) (9,10). Low levels of 274Y quasi-species in avian infl uenza A viruses (H5N1) from avian hosts has been reported (11). Oseltamivir-resistant human infl uenza A viruses (H3N2 and H1N1) have been found to replicate less effi ciently than oseltamivir-susceptible strains in cell culture and animal models (12)(13)(14). However, the NA 274Y resistant mutant in highly pathogenic avian infl uenza A virus (H5N1) retained the high pathogenicity of wild-type virus in mammalian species (15).
In 2007, an NA H274Y oseltamivir-resistant variant of seasonal infl uenza A virus (H1N1) was detected in Norway (6). This virus has now become the dominant virus population globally, overtaking oseltamivir-susceptible infl uenza A virus (H1N1). The molecular basis for the 274Y variant in seasonal infl uenza A virus (H1N1) virus and the mechanism by which this resistant variant became the dominant population remain unknown.
Lack of general immunity to pandemic (H1N1) 2009 virus in the human population, combined with the inherent adamantane resistance of the virus, indicates that NA inhibitors constitute the primary treatment regimen for susceptible patient groups and those in whom severe diseases develop during the current pandemic. There is great concern that an oseltamivir-resistant variant of pandemic (H1N1) 2009 virus may emerge and circulate in a manner similar to oseltamivir-resistant seasonal infl uenza A virus (H1N1).
The patient in this study was not treated with oseltamivir. Therefore it is unlikely that the 274Y mutation was drug-induced. Detection of mixed populations of 274Y and 274H in the NPA specimen before antiviral treatment suggests that the mutation occurs naturally, either before or during infection. Although no experimental data exist that show the growth properties of this resistant variant, examination of the quasi-species population in the cell culture-