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. 2010 Nov;84(21):11219-26.
doi: 10.1128/JVI.01424-10. Epub 2010 Aug 25.

Oseltamivir-resistant variants of the 2009 pandemic H1N1 influenza A virus are not attenuated in the guinea pig and ferret transmission models

Christopher W Seibert  1 , Michael Kaminski, Jennifer Philipp, Dennis Rubbenstroth, Randy A Albrecht, Folker Schwalm, Silke Stertz, Rafael A Medina, Georg Kochs, Adolfo García-Sastre, Peter Staeheli, Peter Palese
Affiliations

Oseltamivir-resistant variants of the 2009 pandemic H1N1 influenza A virus are not attenuated in the guinea pig and ferret transmission models

Christopher W Seibert et al. J Virol. 2010 Nov.

Abstract

Oseltamivir is routinely used worldwide for the treatment of severe influenza A virus infection, and should drug-resistant pandemic 2009 H1N1 viruses become widespread, this potent defense strategy might fail. Oseltamivir-resistant variants of the pandemic 2009 H1N1 influenza A virus have been detected in a substantial number of patients, but to date, the mutant viruses have not moved into circulation in the general population. It is not known whether the resistance mutations in viral neuraminidase (NA) reduce viral fitness. We addressed this question by studying transmission of oseltamivir-resistant mutants derived from two different isolates of the pandemic H1N1 virus in both the guinea pig and ferret transmission models. In vitro, the virus readily acquired a single histidine-to-tyrosine mutation at position 275 (H275Y) in viral neuraminidase when serially passaged in cell culture with increasing concentrations of oseltamivir. This mutation conferred a high degree of resistance to oseltamivir but not zanamivir. Unexpectedly, in guinea pigs and ferrets, the fitness of viruses with the H275Y point mutation was not detectably impaired, and both wild-type and mutant viruses were transmitted equally well from animals that were initially inoculated with 1:1 virus mixtures to naïve contacts. In contrast, a reassortant virus containing an oseltamivir-resistant seasonal NA in the pandemic H1N1 background showed decreased transmission efficiency and fitness in the guinea pig model. Our data suggest that the currently circulating pandemic 2009 H1N1 virus has a high potential to acquire drug resistance without losing fitness.

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Figures

FIG. 1.
FIG. 1.
Oseltamivir-resistant pandemic H1N1 mutants are selected in cell culture and carry the substitution H275Y. (A) Oseltamivir inhibited plaque formation of wild-type pandemic H1N1 virus (rHH-wt). Mutants selected by 10 serial passages on MDCK cells in the presence of increasing oseltamivir concentrations (HH-Os-P10) or generated by introduction of the H275Y substitution into NA (rHH-H275Y) were not affected by oseltamivir. Zanamivir inhibited plaque formation of all three viruses. (B) Passaging of pandemic A/HH/01/2009 on MDCK cells in the presence of escalating oseltamivir concentrations resulted in the occurrence of a H275Y mutation in the NA gene, starting at passage 8. This was paralleled by increased resistance to oseltamivir-mediated inhibition of NA activity. (C) Pandemic H1N1 with wild-type NA (rCal09-wt) showed significant reductions in plaque size, with increasing drug concentrations supplemented to the agar overlay. No significant differences in plaque size were observed for the rCal09-H275Y mutant and a rCal09(7:1)NY1326 virus at an oseltamivir concentration of 1.3 nM. No statistical difference in plaque sizes was observed between the rCal09-H275Y and the rCal09(7:1)NY1326 viruses at both the 1.3 nM (P value = 0.17) and 130 nM (P value = 0.34) drug concentrations. The mean plaque area is normalized to the untreated plaque area, and error bars denote the standard errors of the means (SEM). Statistical significance was determined using Student's t test. *, P < 0.05; ***, P < 0.001.
FIG. 2.
FIG. 2.
Oseltamivir-resistant pandemic H275Y mutants and a reassortant with a seasonal H1N1 virus are not attenuated in the MDCK cell culture. H275Y mutants of pandemic A/HH/01/2009 (HH-Os-P10 and rHH-H275Y) and A/California/04/2009 (rCal09-H275Y) showed multicycle growth kinetics comparable to those of the respective wild-type viruses (A/HH/01/2009, rHH-wt, and rCal09-wt). (A) Similar growth kinetics of the A/HH/01/2009 virus and H275Y mutant derivatives in MDCK cells infected at an MOI of 0.001. (B) Similar growth kinetics of the A/California/04/2009 virus and H275Y mutant derivatives in MDCK cells infected at an MOI of 0.004. Additionally, similar kinetics were also observed for a reassortant virus carrying NA derived from an oseltamivir-resistant seasonal H1N1 virus [rCal09(7:1)NY1326] in MDCK cells infected at an MOI of 0.004. Statistical significance was determined by using Student's t test. *, P < 0.05.
FIG. 3.
FIG. 3.
Wild-type and oseltamivir-resistant pandemic H1N1 viruses are transmitted efficiently by physical and aerosol contact in the guinea pig model. The pandemic H1N1 isolate (A/HH/01/2009) (A), its recombinant wild-type equivalent (rHH-wt) (B), and a H275Y NA-mutant (rHH-H275Y) (C) were transmitted from guinea pigs intranasally (i.n.) inoculated with 104 PFU to naïve guinea pigs exposed by physical contact, with transmission rates of 100%. Wild-type (rCal09-wt) (D) and H275Y mutant (rCal09-H275Y) (E) recombinants of a second pandemic H1N1 virus were transmitted with comparable efficacies from inoculated guinea pigs (103 PFU, i.n.) to aerosol-exposed animals. A reassortant virus carrying NA of an oseltamivir-resistant seasonal H1N1 strain [rCal09(7:1)NY1326] (F) was transmitted to only 50% of aerosol-exposed guinea pigs. Virus transmission was assessed by determining viral titers in nasal wash samples collected at the indicated times. Transmission rates for aerosol experiments were determined by two independent experiments performed at 20°C and 20% humidity in environmental chambers.
FIG. 4.
FIG. 4.
rCal09-H275Y virus is able to transmit as efficiently as rCal09-wt virus, whereas rCal09(7:1)NY1326 virus cannot transmit in competition experiments. Guinea pigs were intranasally inoculated with the indicated viruses mixed in a 1:1 ratio (103 PFU). (A, B) Nasal wash specimens collected from inoculated and aerosol-exposed animals were investigated for viral titers. (C, D) The presence or absence of each virus contained in the original inoculum was determined by restriction enzyme analysis. Digest assay-based banding patterns from the rCal09-wt versus rCal09-H275Y experiments illustrate that all of the inoculated guinea pigs and 3 of the 4 exposed guinea pigs were shedding a heterogeneous mixture of viruses. Restriction enzyme analysis of nasal wash samples obtained from exposed guinea pigs in the rCal09(7:1)NY1326 experiment revealed banding patterns corresponding to predominant transmission of the wild-type virus. GPs, guinea pigs.
FIG. 5.
FIG. 5.
Wild-type and oseltamivir-resistant pandemic H1N1 viruses are transmitted efficiently by physical and aerosol contact in the ferret transmission model. (A) Wild-type viruses (rHH01-wt and rCal09-wt) and H275Y mutants (rHH01-H275Y and rCal09-H275Y) reached comparable titers in nasal wash samples following intranasal inoculation with 104 PFU. They were readily transmitted to ferrets exposed by physical or aerosol contact. (B) Ferrets were intranasally inoculated with wild-type and H275Y mutant viruses mixed in a 1:1 ratio (104 PFU). (Top) Nasal wash samples collected from directly inoculated, contact-exposed, and aerosol-exposed animals were investigated for viral titers. (Bottom) Pyrosequencing-based single nucleotide polymorphism (SNP) analysis shows that wild-type and mutant viruses were transmitted by both routes.
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