Neuraminidase inhibitor-resistant recombinant A/Vietnam/1203/04 (H5N1) influenza viruses retain their replication efficiency and pathogenicity in vitro and in vivo

Abstract

Effective antiviral drugs are essential for early control of an influenza pandemic. It is therefore crucial to evaluate the possible threat posed by neuraminidase (NA) inhibitor-resistant influenza viruses with pandemic potential. Four NA mutations (E119G, H274Y, R292K, and N294S) that have been reported to confer resistance to NA inhibitors were each introduced into recombinant A/Vietnam/1203/04 (VN1203) H5N1 influenza virus. For comparison, the same mutations were introduced into recombinant A/Puerto Rico/8/34 (PR8) H1N1 influenza virus. The E119G and R292K mutations significantly compromised viral growth in vitro, but the H274Y and N294S mutations were stably maintained in VN1203 and PR8 viruses. In both backgrounds, the H274Y and N294S mutations conferred resistance to oseltamivir carboxylate (50% inhibitory concentration [IC(50)] increases, >250-fold and >20-fold, respectively), and the N294S mutation reduced susceptibility to zanamivir (IC(50) increase, >3.0-fold). Although the H274Y and N294S mutations did not compromise the replication efficiency of VN1203 or PR8 viruses in vitro, these mutations slightly reduced the lethality of PR8 virus in mice. However, the VN1203 virus carrying either the H274Y or N294S mutation exhibited lethality similar to that of the wild-type VN1203 virus. The different enzyme kinetic parameters (V(max) and K(m)) of avian-like VN1203 NA and human-like PR8 NA suggest that resistance-associated NA mutations can cause different levels of functional loss in NA glycoproteins of the same subtype. Our results suggest that NA inhibitor-resistant H5N1 variants may retain the high pathogenicity of the wild-type virus in mammalian species. Patients receiving NA inhibitors for H5N1 influenza virus infection should be closely monitored for the emergence of resistant variants.

FIG. 1.

Replication kinetics of the recombinant viruses in vitro. (A and B) Multistep growth curves for the recombinant VN1203, VN1203-H274Y, and VN1203-N294S viruses in MDCK (A) and MDCK-SIAT1 (B) cells. (C and D) Multistep growth curves for the recombinant PR8, PR8-H274Y, and PR8-N294S viruses in MDCK (C) and MDCK-SIAT1 (D) cells. Confluent cells were infected with recombinant viruses at an MOI of 0.0001 PFU/cell. The virus yield was titrated in MDCK cells at 12, 24, 36, 48, and 60 h postinfection. Each data point represents viral yield (log₁₀ PFU ± SD/ml) from three independent experiments. *, P < 0.05 compared to value for wild-type virus.

FIG. 2.

Lethality of the recombinant viruses in a BALB/c mouse model. (A) MLD₅₀ s of the recombinant viruses (expressed in PFU ± SD). (B and C) Survival of BALB/c mice inoculated with 10 PFU of recombinant VN1203, VN1203-H274Y, or VN1203-N294S virus (B) or 100 PFU of recombinant PR8, PR8-H274Y, or PR8-N294S virus (C). *, P < 0.05 compared to value for wild-type virus.

FIG. 3.

Virus titers in BALB/c mouse tissue at 1, 3, and 6 days after inoculation (d.p.i.). (A) Titers (log₁₀ EID₅₀ /ml) in blood, brain, and lungs after inoculation with 10 PFU of recombinant VN1203, VN1203-H274Y, or VN1203-N294S virus. (B) Titers (log₁₀ EID₅₀ /ml) in lungs after inoculation with 100 PFU of recombinant PR8, PR8-H274Y, or PR8-N294S virus. Each data point represents the value in an inoculated mouse, and the lines represent the mean values from three mice.

FIG. 4.

NA enzyme kinetics of the recombinant viruses, showing the substrate conversion velocity (V₀ ) of NA as a function of substrate concentration. (A) Recombinant VN1203, VN1203-H274Y, VN1203-N294S, and PR8 viruses; (B) recombinant PR8 viruses. The fluorogenic MUNANA substrate was used at final concentrations of 0 to 3333 μM. All recombinant viruses were standardized to an equivalent virus dose of 10^7.5 PFU/ml. The reaction was conducted at 37°C, and fluorescence was measured every 92 s for 45 min using excitation and emission wavelengths of 355 and 460 nm, respectively.