Impact of potential permissive neuraminidase mutations on viral fitness of the H275Y oseltamivir-resistant influenza A(H1N1)pdm09 virus in vitro, in mice and in ferrets

Abstract

Neuraminidase (NA) mutations conferring resistance to NA inhibitors (NAIs) generally compromise the fitness of influenza viruses. The only NAI-resistant virus that widely spread in the population, the A/Brisbane/59/2007 (H1N1) strain, contained permissive mutations that restored the detrimental effect caused by the H275Y change. Computational analysis predicted other permissive NA mutations for A(H1N1)pdm09 viruses. Here, we investigated the effect of T289M and N369K mutations on the viral fitness of the A(H1N1)pdm09 H275Y variant. Recombinant wild-type (WT) A(H1N1)pdm09 and the H275Y, H275Y/T289M, H275Y/N369K, and H275Y/V241I/N369K (a natural variant) NA mutants were generated by reverse genetics. Replication kinetics were performed by using ST6GalI-MDCK cells. Virulence was assessed in C57BL/6 mice, and contact transmission was evaluated in ferrets. The H275Y mutation significantly reduced viral titers during the first 12 to 36 h postinfection (p.i.) in vitro. Nevertheless, the WT and H275Y viruses induced comparable mortality rates, weight loss, and lung titers in mice. The T289M mutation eliminated the detrimental effect caused by the H275Y change in vitro while causing greater weight loss and mortality in mice, with significantly higher lung viral titers on days 3 and 6 p.i. than with the H275Y mutant. In index ferrets, the WT, H275Y, H275Y/T289M, and H275Y/V241I/N369K recombinants induced comparable fever, weight loss, and nasal wash viral titers. All tested viruses were transmitted at comparable rates in contact ferrets, with the H275Y/V241I/N369K recombinant demonstrating higher nasal wash viral titers than the H275Y mutant. Permissive mutations may enhance the fitness of A(H1N1)pdm09 H275Y viruses in vitro and in vivo. The emergence of such variants should be carefully monitored.

FIG 1

Surface activity of recombinant A(H1N1)pdm09 NA proteins. 293T cells were transfected with plasmids expressing the WT or mutant A(H1N1)pdm09-like NAs. At 24 h posttransfection, cells were treated with a nonlysing buffer, and surface NA activity was measured by using a fluorogenic substrate (MUNANA). Percent surface NA activities are shown compared to the WT from triplicate experiments ± standard deviations. *, P < 0.05; ***, P < 0.001 (compared to the recombinant H275Y virus).

FIG 2

In vitro replicative capacities of recombinant wild-type (WT) and NA mutant influenza A(H1N1)pdm09 viruses. Viral titers were determined at the indicated time points from supernatants of MDCK-α2,6 cells infected at a multiplicity of infection (MOI) of 0.001. Mean viral titers ± standard deviations from triplicate experiments were determined by using standard plaque assays. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to the recombinant H275Y virus).

FIG 3

Mean body weight loss of mice infected intranasally with 10⁵ PFU of recombinant WT or NA mutant influenza A(H1N1)pdm09 viruses. Percent body weight losses were determined daily up to day 12 postinoculation. *, P < 0.05; **, P < 0.01 (compared to the recombinant H275Y virus).

FIG 4

Mean viral titers ± standard deviations in nasal wash specimens of index ferrets infected with 5 × 10⁴ PFU of recombinant WT and NA mutant A(H1N1)pdm09 viruses (A) and in samples of their direct-contact animals (B). Viral titers were determined at the indicated days postinoculation by using standard plaque assays. *, P < 0.05 compared to the recombinant H275Y virus.