- 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
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- Nature Online - Brief Communications
10-14-5
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- 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.
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- 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.
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- 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.
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- 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.
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- 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.
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- 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.
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- 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.
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- [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.]
-
- References ----------
- 1. Enserink M. Science 2005; 309: 3701. 2. Treanor JJ,
et al. JAMA 2000; 283: 101624. 3. Gubareva LV, Kaiser L, Hayden FG. Lancet
2000; 355: 82735. 4. Gubareva LV, Kaiser L, Matrosovich MN, Soo-Hoo Y,
Hayden FG. J Infect Dis 2001; 183: 52331. 5. Zambon M, Hayden FG. Antiviral
Res 2000; 47: 117. 6. Potier M, Mameli L, Belisle M, Dallaire L, Melancon
SB. Anal Biochem 1979; 94: 28796. 7. Hurt AC, Barr IG, Gunter H, Hampson
AW. Antiviral Res 2004; 62: 3745. 8. Toms GLR, Bird RA, Kingsman SM, Sweet
C, Smith H. Br J Exp Pathol 1977; 57: 3748. 9. Varghese JN, Laver WG, Colman
PM. Nature 1983; 303: 3550. 10. Varghese JN, McKimm-Breschkin JL, Caldwell
JB, Kortt AA, Colman PM. Proteins 1992; 11: 4956. 11. Shinya K, et al.
J Virol 2005; 79: 992632.
-
- (The Supplementary information referred to above accompanies
this communication on Nature's website. - Mod.CP]
-
- http://www.nature.com/nature/journal/vaop/ncurrent/pdf/4371108a.pdf
-
-
- Patricia A. Doyle, PhD
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