Competitive Fitness of Oseltamivir-Sensitive and -Resistant Highly Pathogenic H5N1 Influenza Viruses in a Ferret Model

Abstract

The fitness of oseltamivir-resistant highly pathogenic H5N1 influenza viruses has important clinical implications. We generated recombinant human A/Vietnam/1203/04 (VN; clade 1) and A/Turkey/15/06 (TK; clade 2.2) influenza viruses containing the H274Y neuraminidase (NA) mutation, which confers resistance to NA inhibitors, and compared the fitness levels of the wild-type (WT) and resistant virus pairs in ferrets. The VN-H274Y and VN-WT viruses replicated to similar titers in the upper respiratory tract (URT) and caused comparable disease signs, and none of the animals survived. On days 1 to 3 postinoculation, disease signs caused by oseltamivir-resistant TK-H274Y virus were milder than those caused by TK-WT virus, and all animals survived. We then studied fitness by using a novel approach. We coinoculated ferrets with different ratios of oseltamivir-resistant and -sensitive H5N1 viruses and measured the proportion of clones in day-6 nasal washes that contained the H274Y NA mutation. Although the proportion of VN-H274Y clones increased consistently, that of TK-H274Y virus decreased. Mutations within NA catalytic (R292K) and framework (E119A/K, I222L, H274L, and N294S) sites or near the NA enzyme active site (V116I, I117T/V, Q136H, K150N, and A250T) emerged spontaneously (without drug pressure) in both pairs of viruses. The NA substitutions I254V and E276A could exert a compensatory effect on the fitness of VN-H274Y and TK-H274Y viruses. NA enzymatic function was reduced in both drug-resistant H5N1 viruses. These results show that the H274Y NA mutation affects the fitness of two H5N1 influenza viruses differently. Our novel method of assessing viral fitness accounts for both virus-host interactions and virus-virus interactions within the host.

Zanamivir-resistant influenza viruses with Q136K or Q136R neuraminidase residue mutations can arise during MDCK cell culture creating challenges for antiviral susceptibility monitoring

“…Mutations at Q136 have also been detected in ferrets infected with influenza A(H5N1). The Q136L variant was detected in the nasal wash of a zanamivir treated ferret infected with an A(H5N1) virus [28], while a Q136H mutation was detected in an A(H5N1) virus from a ferret not being treated with an antiviral [29]. These reports demonstrate that viruses with these mutations have the potential to infect or replicate in vivo both in the presence or absence of zanamivir pressure.…”

Treatment Options for H5N1: Lessons Learned from the H1N1 Pandemic

“…Recent studies in ferrets with 2 different strains of H275Y oseltamivir-resistant influenza A (H5N1) virus showed that they had different competitive fitness when compared with the wild-type virus. 23 The effect of various NAI-resistant mutations on the pathogenicity of a strain of H5N1 was also studied in ferrets. 24 Two of the NAI-resistant mutants were associated with higher viral titers and more inflammation in the lungs of animals than the wild-type virus.…”

Fitness of neuraminidase inhibitor-resistant influenza A viruses

“…A clone of this mutant H5N1 virus with the N294S substitution also had lower levels of virus replication and pathogenicity in mice and ferrets and less susceptibility to oseltamivir than did the wild-type virus [59]. In contrast, neither the H274Y nor the N294S NA mutations compromised the lethality or pathogenicity of clade 1 A/Vietnam/1203/2004 (H5N1) virus in mice and ferrets [7, 60, 61], suggesting that oseltamivir-resistant variants of H5N1 subtype may possess an undiminished phenotype and exhibit high pathogenicity in mammalian species. Interestingly, oseltamivir resistance mutations substantially decrease the enzymatic activity of avian-like NA glycoprotein of A/Vietnam/1203/2004 (H5N1) virus but do not affect virus pathogenicity in mice and ferrets [7, 60].…”

“…In contrast, neither the H274Y nor the N294S NA mutations compromised the lethality or pathogenicity of clade 1 A/Vietnam/1203/2004 (H5N1) virus in mice and ferrets [7, 60, 61], suggesting that oseltamivir-resistant variants of H5N1 subtype may possess an undiminished phenotype and exhibit high pathogenicity in mammalian species. Interestingly, oseltamivir resistance mutations substantially decrease the enzymatic activity of avian-like NA glycoprotein of A/Vietnam/1203/2004 (H5N1) virus but do not affect virus pathogenicity in mice and ferrets [7, 60]. Thus, the extremely efficient replication complexes of highly pathogenic H5N1 influenza viruses can overcome deficiency in NA function, leading to fitness similar to that of wild-type viruses.…”

“…For example, the H274Y NA mutation does not affect the fitness of the A/Vietnam/1203/2004 (H5N1) virus [30, 60] but diminishes that of the less pathogenic A/Hanoi/30408/2005 (H5N1) virus [56]. Fitness of highly pathogenic H5N1 viruses depend on location of particular NA mutations: in the homogeneous background of clade 2.2 A/Turkey/15/2006 (H5N1)-like virus, the H274Y NA mutation did not affect virulence, and the N294S NA mutation caused significantly higher virus titers and inflammation in the lungs than the wild-type virus did [62].…”