Isolation Of
H5N1 Virus

From Patricia Doyle, PhD
ProMed Comment: Herewith is the text of the Brie Communications article published in Nature (ahead of print) that was not accessible at the time of posting of "Influenza viruses, drug resistance (04) 20051015.2999". - Mod.CP
Isolation Of Drug-Resistant H5N1 Virus
Nature Online - Brief Communications
The persistence of H5N1 avian influenza viruses in many Asian countries and their ability to cause fatal infections in humans have raised serious concerns about a global flu pandemic (1). Here we report the isolation of an H5N1 virus from a Vietnamese girl that is resistant to the drug oseltamivir (2), which is an inhibitor of the viral enzyme neuraminidase and is currently used for protection against and treatment of influenza. Further investigation is necessaryto determine the prevalence of oseltamivir-resistant H5N1 viruses among patients treated with this drug.
An H5N1 influenza virus, A/Hanoi/30408/ 2005, was isolated on 27 Feb 2005 from a 14 year old Vietnamese girl (patient 1) who had received a prophylactic dose (75mg once a day) of oseltamivir from 24 to 27 Feb 2005 and was given a therapeutic dose (75mg twice daily) for 7 days starting on 28 Feb. No virus was isolated from specimens after the administration of increased doses of oseltamivir. The patient recovered and was discharged from hospital on 14 Mar 2005.
Direct sequencing after amplification by polymerase chain reaction of the virus isolated from a specimen collected on 27 Feb 2005 indicated that some of the virus population had a histidine-to-tyrosine substitution at position 274 (represented as H274Y) in its neuraminidase protein, a mutation that confers resistance to oseltamivir (3,4,5). We therefore tested the sensitivity of the virus to oseltamivir carboxylate (6) -- the active form of the drug -- and found that the dose required for 50 per cent inhibition of neuraminidase activity (IC50) in the isolate was 90 nM, which exceeds the IC50 for oseltamivir-sensitive viruses (0.110 nM ) (7). We then plaque-purified the virus.
Ten viral clones, randomly picked from the resultant plaques, were classified into 3 groups according to their response to oseltamivir (see supplementary information): 6 were highly resistant to the drug (IC50 = 763 nM), 3 were slightly resistant (IC50 values between 7.1 and 12.5 nM) and one was highly sensitive (IIC50 = 0.6 nM). The highly resistant viruses had tyrosine at position 274 in their neuraminidase, whereas those showing only slight resistance had serine at position 294.
Patient 1 had not had any known direct contact with poultry, but had cared for her 21 year old brother (patient 2) while he had a documented H5N1 virus infection (for details of the disease course and treatment in these patients, see supplementary information). We found that the neuraminidase gene of the brother's virus was identical to clone 7 of the girl's virus (see supplementary information). Also, the haemagglutinin gene of the brother's virus was identical to clones 2 and 9 of the girl's virus, apart from a nucleotide change at position 271. The timing of infection in these 2 patients, together with the lack of known interaction of the girl with poultry, raises the possibility that the virus could have been transmitted from brother to sister.
We assessed the growth of a highly oseltamivir-resistant clone (H274Y,clone 9) and of an oseltamivir-sensitive clone (H274, clone 7) in ferrets (8). Viral titres were higher in animals infected with the oseltamivir-sensitive virus (See figure in original text). Oseltamivir treatment reduced viral titres in animals infected with the drug sensitive virus (See original text for figure), but not in animals infected with the resistant virus. However, all of the viral clones, including those highly resistant to oseltamivir, were sensitive to zanamivir (9, 10) (IC50 = 0.53.1 nM), another neuraminidase inhibitor. In ferrets, we found that zanamivir treatment reduced viral titres in animals infected with virus that was oseltamivir-sensitive or oseltamivir-resistant (see original figures).
We investigated how the viruses bound in vitro to different configurations of sialyl glycopolymers, similar to those on the host's cell-surface receptor (11). We compared binding by 2 of the viral clones (clones 7 and 9) with binding by an avian flu virus (A/duck/Mongolia/ 301/2001) and another human flu virus (A/Kawasaki/1/2001). We found that both H5N1 clones bound to -2,3-linked polymer and (less efficiently) to -2,6-linked polymer (seeoriginal text figure). The A/duck/Mongolia/301/2001 virus also bound -2,3-linked polymer but did not bind -2,6- linked polymer at all; the A/Kawasaki/1/2001 virus bound strongly to -2,6-linked polymer but only weakly to -2,3-linked polymer (results not shown). The broader binding properties of our H5N1 viral clones may reflect a degree of adaptation in human hosts.
Although our findings are based on a virus from only a single patient, they raise the possibility that it might be useful to stockpile zanamivir as well as oseltamivir in the event of an H5N1 influenza pandemic. They also highlight the importance of monitoring the emergence of drug resistance in H5N1 isolates from patients treated with neuraminidase inhibitors.
[Authored by Q. Mai Le and 15 others, at the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam; Division of Virology, Department of Microbiology and Immunology, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan; Daiichi Pharmaceutical, Tokyo 134-8630, Japan; National Institute for Clinical Research in Tropical Medicine, Hanoi, Vietnam; Department of Biochemistry, University of Shizuoka, School of Pharmaceutical Sciences and COE Program in the 21st Century, Yada, Shizuoka-shi 422-8526, Japan; and Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53706, USA.]
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(The Supplementary information referred to above accompanies this communication on Nature's website. - Mod.CP]
Patricia A. Doyle, PhD
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