Oseltamivir-resistant pandemic H1N1/2009 influenza virus possesses lower transmissibility and fitness in ferrets

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.

Figure 1. Plaque morphology and replication kinetics of two H1N1/2009 influenza viruses in MDCK and MDCK-SIAT1 cells.

The diameters of 20 randomly selected value plaques were measured in MDCK cells (A) and MDCK-SIAT1 cells (B). Values are mean (± SD) plaque diameter (mm). Single-cycle (C, D left panel) and multiple-cycle (C, D right panel) growth curves were obtained by using an MOI of ∼2 and ∼0.001 PFU/cell, respectively. Virus in the supernatant was titrated in MDCK or MDCK-SIAT1 cells and expressed as log₁₀TCID₅₀/ml at the indicated time post-infection. Each point represents the mean log₁₀TCID₅₀/ml ± SD from three experiments. * P<0.05 compared to value for wild-type viruses.

Figure 2. Transmissibility of the two H1N1/2009 influenza viruses among ferrets.

The virus titer (A, B left panel) and total number of inflammatory cells (A, B right panel) in the nasal wash samples from each donor ferret, direct-contact (DC contact) ferret, and respiratory droplet-contact (RD contact ) ferret. The arrow indicates the first day of exposure of contact ferrets.

Figure 3. Co-infection in a ferret with oseltamivir-sensitive and -resistant H1N1/2009 influenza viruses.

Virus titers and inflammatory cell counts in the nasal wash specimens of ferrets co-inoculated with oseltamivir-sensitive and -resistant H1N1/2009 viruses (A). The arrow indicates the first day of exposure of contact ferrets. The proportion of wild-type virus (C in SNP sequence) in the mixed virus population (C+T in SNP sequence) in nasal wash samples from the donor ferret and two direct-contact ferrets (B). Values are the mean ± SD from three independent measurements. * P<0.05 compared to value for day 0 p.i.