Oseltamivir–Resistant Pandemic H1N1/2009 Influenza Virus Possesses Lower Transmissibility and Fitness in Ferrets

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Abstract
The neuraminidase (NA) inhibitor oseltamivir offers an important immediate option for the control of influenza, and its clinical use has increased substantially during the recent H1N1 pandemic. In view of the high prevalence of oseltamivir-resistant seasonal H1N1 influenza viruses in 2007–2008, there is an urgent need to characterize the transmissibility and fitness of oseltamivir-resistant H1N1/2009 viruses, although resistant variants have been isolated at a low rate. Here we studied the transmissibility of a closely matched pair of pandemic H1N1/2009 clinical isolates, one oseltamivir-sensitive and one resistant, in the ferret model. The resistant H275Y mutant was derived from a patient on oseltamivir prophylaxis and was the first oseltamivir-resistant isolate of the pandemic virus. Full genome sequencing revealed that the pair of viruses differed only at NA amino acid position 275. We found that the oseltamivir-resistant H1N1/2009 virus was not transmitted efficiently in ferrets via respiratory droplets (0/2), while it retained efficient transmission via direct contact (2/2). The sensitive H1N1/2009 virus was efficiently transmitted via both routes (2/2 and 1/2, respectively). The wild-type H1N1/2009 and the resistant mutant appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. We compared viral fitness within the host by co-infecting a ferret with oseltamivir-sensitive and -resistant H1N1/2009 viruses and found that the resistant virus showed less growth capability (fitness). The NA of the resistant virus showed reduced substrate-binding affinity and catalytic activity in vitro and delayed initial growth in MDCK and MDCK-SIAT1 cells. These findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and characterization of possible evolving viral proteins during the pandemic. Most of the currently circulating pandemic H1N1/2009 (“swine”) influenza viruses are susceptible to the anti-influenza drug oseltamivir. Many countries have stockpiled oseltamivir for pandemic preparedness, and to date only a small proportion of the H1N1/2009 viruses isolated have been oseltamivir-resistant. However, if these viruses can be readily transmitted, oseltamivir resistance may spread. We evaluated the transmissibility of a pair of pandemic H1N1/2009 influenza viruses in ferrets. One virus was oseltamivir-sensitive and the other carried the oseltamivir resistance-associated H275Y NA mutation. We also investigated the viruses' susceptibility to NA inhibitors (the drug class to which oseltamivir belongs), their NA enzyme kinetics, and their replication efficiency in cultured cells. Under identical conditions, the resistant H1N1/2009 virus was not transmitted by respiratory droplets but was efficiently transmitted by direct contact, while the sensitive H1N1/2009 virus was efficiently transmitted by both routes.