Oseltamivir-Resistant Pandemic (H1N1) 2009 Virus, South Korea

To identify oseltamivir resistance, we analyzed neuraminidase H275Y mutations in samples from 10 patients infected with pandemic (H1N1) 2009 virus in South Korea who had influenza that was refractory to antiviral treatment with this drug. A neuraminidase I117M mutation that might influence oseltamivir susceptibility was detected in sequential specimens from 1 patient.

To identify oseltamivir resistance, we analyzed neuraminidase H275Y mutations in samples from 10 patients infected with pandemic (H1N1) 2009 virus in South Korea who had infl uenza that was refractory to antiviral treatment with this drug. A neuraminidase I117M mutation that might infl uence oseltamivir susceptibility was detected in sequential specimens from 1 patient.

S ince April 2009, pandemic (H1N1) 2009 has spread
worldwide and caused the fi rst infl uenza pandemic of the 21st century. Pandemic (H1N1) 2009 virus initially showed resistance to amantadine but susceptibility to oseltamivir (1). Thereafter, 285 cases of oseltamivir-resistant pandemic viral infection were reported worldwide on April 14, 2010 (2). However, information is limited about oseltamivir-resistant pandemic infl uenza in South Korea. Monitoring of community circulation of oseltamivir-resistant viruses has not yet detected any evidence of oseltamivir resistance in South Korea. To identify these viruses, we conducted specifi c surveillance of antiviral drug-resistant infection in patients whose illness did not resolve after antiviral treatment.

The Study
The study was reviewed and approved by ethics committees of relevant institutions and hospitals. After patients provided informed consent, we obtained >150 clinical specimens from patients in various hospitals in South Korea. Respiratory specimens (>60% nasopharyngeal swab specimens) were obtained during October 2009-January 2010 from patients whose illness had been clinically refractory to antiviral treatment since October 2009. Viral RNAs were isolated from specimens of 10 patients (Table 1) by using the QIAamp viral RNA Mini Kit (QIAGEN, Crawley, UK). PCR products of the neuramini-dase (NA) and matrix 2 (M2) genes were generated by reverse transcription-PCR with primers for NA (forward: 5′-AAATTAACGGGCAATTCCTCTCT-3′; reverse: 5′-CCGAAAATCCCACTGCATATGTAT-3′) and M2 (forward: 5′-CTAGCTCCAGTGCTGGTCTGA-3′; reverse: 5′-CTCAGGCACTCCTTCCGTAGA-3′). DNA sequences of NA and M2 reverse transcription-PCR products were analyzed by using the Big-Dye Terminator Sequencing Reaction Kit and an ABI 3700 DNA analyzer (Applied Biosystems, Foster City, CA, USA). A total of 58 NA sequences and 52 M2 sequences were obtained and analyzed by

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Learning Objectives
Upon completion of this activity, participants will be able to: Describe the pattern and characteristics using the sequence analysis tool in the Infl uenza Sequences and Epitopes Database for detecting oseltamivir-resistance mutations (3). Ten patients were detected who had oseltamivirresistant pandemic (H1N1) 2009 virus with the H275Y substitution in viral NA (Table 2). Oseltamivir resistance was associated with oseltamivir treatment on the basis of H275Y changes from the oseltamivir-sensitive genotypes to oseltamivir-resistant genotypes of viral NA in consecutive samples from the same patient. Furthermore, a novel NA (I117M) substitution that may be associated with oseltamivir resistance (4,5) was detected in specimens from 1 patient (patient G) who had myelodysplasia and received oseltamivir and peramivir (Tables 1, 2).
In addition, we cultured viral isolates from clinical specimens (patients A and C) and evaluated antiviral susceptibility by measuring the dose of oseltamivir and zanamivir required for 50% inhibition (IC 50 ) of NA activity. One isolate of pandemic (H1N1) 2009 virus with an oseltamivir-sensitive genotype (H275 in its NA) was susceptible to oseltamivir (IC 50 1.18 nmol/L) and zanamivir (IC 50 0.42 nmol/L). Viral isolates from patients A and C with an oseltamivir-resistant genotype (Y275 in NA) were resistant only to oseltamivir (IC 50 713.2 nmol/L and 359.4 nmol/L, respectively). Susceptibility to zanamivir was not altered whether NA contained Y275 or H275 (IC 50 0.13 nmol/L and 0.78 nmol/L, respectively).
Patients with oseltamivir-resistant pandemic (H1N1) 2009 were treated during hospitalization with oseltamivir alone or with a combination of other antiviral drugs ( Table  2). Active surveillance that evaluated spread of oseltamivirresistant viral infections among hospital staff, family members, and other patients who had contacted with or cared for the patients showed no evidence of virus transmission in the hospitals.

Conclusions
During pandemic (H1N1) 2009, oseltamivir-resistant viral infections were found mainly in immunocompromised patients who were treated with antiviral drugs and chemoprophylaxis (6). In our study, genetic characteristics of oseltamivir resistance in virus isolates from all 10 patients showed the H275Y substitution in NA, resulting in phenotypic resistance to oseltamivir but not to zanamivir. In addition, a novel NA mutation (I117M) was detected in virus from patient G (Tables 1, 2). The NA 117 residue is near R118 and may have an effect on this residue, 1 of 3 arginine residues that bind the carboxylate region of the sialic acid substrate (5,7). This fi nding implies that changes at NA residue 117 may infl uence oseltamivir susceptibility of the virus. For example, the NA I117V mutation in avian infl uenza virus (H5N1) was reported to increase resistance to oseltamivir (5,6). The 117 residue is also highly conserved in all N1 typed viruses (5). These results indicate that the I117M substitution in NA of pandemic (H1N1) 2009 virus may infl uence oseltamivir resistance of this virus.
Multiple specimens were available from patients D, E, and G for analyzing antiviral resistance mutations in NA. Analysis of viruses from these patients showed that the H275Y mutation emerged in the later stages of viral infection, during oseltamivir treatment; infective viruses initially had an oseltamivir-sensitive genotype (H275) ( Table 2; Figure). A mixture of oseltamivir-sensitive (H275 in NA) and oseltamivir-resistant (Y275 in NA) viruses was observed in the second specimen from patient E and the third specimen from patient G (Figure). These specimens showed double signals (nucleotides T and C) for TAC and CAC encoding tyrosine (Y) and histidine (H), respectively. There are 2 possible explanations for mixtures of oseltamivir-resistant and oseltamivir-sensitive viral genotypes. The fi rst explanation is reinfection with Y275 virus after a previous infection with H275 virus. The second explanation is an H275Y mutation caused by selective pressure from oseltamivir treatment. Our molecular epidemiologic data (Table 2) support the second explanation that selective pressure from oseltamivir treatment caused the H275Y substitution in NA because sequential viruses from the same patient had identical HA sequences in the absence of additional infection (8).
Multiple specimens from the same patient showed identical hemagglutinin sequences despite amino acid changes (H to Y) at position 275 of NA (hemagglutinin sequences of patients D, E, and G) ( Table 2). This fi nding indicates that temporally sequential viruses from a patient were likely generated from the same viral progenitor without further viral infection. Conversely, our fi ndings also support absence of epidemiologic links among current cases of oseltamivir resistance, given that viruses from 9 patients in this study had their own signature hemagglutinin amino acid sequences (Table 2).
Spreading and clustering of oseltamivir-resistant strains in the general population has not been observed in South Korea. However, in the present study, a comparatively high rate of emergence (19%) of oseltamivir-resistant viral infections was observed in unhealthy patients treated with antiviral drugs. We also detected a novel substitution (I117M) in NA, which might infl uence susceptibility to NA inhibitors. Our fi ndings demonstrate that oseltamivir-resistant quasispecies of pandemic (H1N1) 2009 viruses can be generated. This suggestion indicates that continuous surveillance is required to evaluate emergence and circulation of drug-resistant pandemic (H1N1) 2009 viruses and possible reassortment with other viruses that have oseltamivir resistance.