Oseltamivir-Resistant Pandemic A/H1N1 Virus Is as Virulent as Its Wild-Type Counterpart in Mice and Ferrets

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Abstract
The neuraminidase inhibitor oseltamivir is currently used for treatment of patients infected with the pandemic A/H1N1 (pH1N1) influenza virus, although drug-resistant mutants can emerge rapidly and possibly be transmitted. We describe the characteristics of a pair of oseltamivir-resistant and oseltamivir-susceptible pH1N1 clinical isolates that differed by a single change (H274Y) in the neuraminidase protein. Viral fitness of pH1N1 isolates was assessed in vitro by determining replication kinetics in MDCK α2,6 cells and in vivo by performing experimental infections of BALB/c mice and ferrets. Despite slightly reduced propagation of the mutant isolate in vitro during the first 24 h, the wild-type (WT) and mutant resistant viruses induced similar maximum weight loss in mice and ferrets with an identical pyrexic response in ferrets (AUC of 233.9 and 233.2, P = 0.5156). Similarly, comparable titers were obtained for the WT and the mutant strains on days 1, 3, 6 and 9 post-infection in mouse lungs and on days 1–7 in ferret nasal washes. A more important perivascular (day 6) and pleural (days 6 and 12) inflammation was noted in the lungs of mice infected with the H274Y mutant, which correlated with increased pulmonary levels of IL-6 and KC. Such increased levels of IL-6 were also observed in lymph nodes of ferrets infected with the mutant strain. Furthermore, the H274Y mutant strain was transmitted to ferrets. In conclusion, viral fitness of the H274Y pH1N1 isolate is not substantially altered and has the potential to induce severe disease and to disseminate. During the 2009 pandemic of the novel A/H1N1 (pH1N1) virus, the World Health Organization recommended oseltamivir as first-line agent for treatment of patients with severe infections leading to hospitalization and for those with underlying diseases predisposing to pulmonary complications. Oseltamivir-resistant isolates started to emerge at the end of June 2009 with now more than 100 strains reported worldwide including a few outbreaks where transmission of resistant viruses may have occurred. We characterized the fitness of a pair of oseltamivir-susceptible and oseltamivir-resistant strains emerging from the same familial cluster and that differed by only a single change (H274Y) in the neuraminidase protein. We found that the drug-resistant (mutant) virus was at least as virulent as the drug-susceptible (wild-type) virus in mice and ferrets. Based on these data, we believe that the H274Y pH1N1 mutant strain has the potential to disseminate in the population and to eventually replace the susceptible strain, a phenomenon that has been already observed with seasonal A/Brisbane/59/2007-like (H1N1) viruses.