Neuraminidase inhibitor-resistant influenza viruses may differ substantially in fitness and transmissibility

Abstract

Mutations of the conserved residues of influenza virus neuraminidase (NA) that are associated with NA inhibitor (NAI) resistance decrease the sialidase activity and/or stability of the NA, thus compromising viral fitness. In fact, clinically derived NAI-resistant variants with different NA mutations have shown different transmissibilities in ferrets (M. L. Herlocher, R. Truscon, S. Elias, H. Yen, N. A. Roberts, S. E. Ohmit, and A. S. Monto, J. Infect. Dis. 190:1627-1630, 2004). Molecular characterization of mutant viruses that have a homogeneous genetic background is required to determine the effect of single mutations at conserved NA residues. We generated recombinant viruses containing either the wild-type NA (RG WT virus) or a single amino acid change at NA residue 119 (RG E119V-NA virus) or 292 (RG R292K-NA virus) in the A/Wuhan/359/95 (H3N2) influenza virus background by reverse genetics. Both mutants showed decreased sensitivity to oseltamivir carboxylate, and the RG R292K-NA virus showed cross-resistance to zanamivir. We also observed differences between the two mutants in NA enzymatic activity and thermostability. The R292K mutation caused greater reduction of sialidase activity and thermostability than the E119V mutation. The NA defect caused by the R292K mutation was associated with compromised growth and transmissibility, whereas the growth and transmissibility of the RG E119V-NA virus were comparable to those of RG WT virus. Our results suggest that NAI-resistant influenza virus variants may differ substantially in fitness and transmissibility, depending on different levels of NA functional loss.

FIG. 1.

NA enzymatic activity (A) and thermostability (B) of RG WT, RG E119V-NA, and RG R292K-NA A/Wuhan/359/95 influenza viruses. MUNANA was used as the substrate at a final concentration of 100 μM. The thermostability of NA in the recombinant viruses was determined after 15 min of incubation at the indicated temperatures. Residual NA enzymatic activity was assayed and is presented as a percentage of the NA activity at 4°C. Datum points represent the mean ± SD from three experiments.

FIG. 2.

Plaque morphology of recombinant A/Wuhan/359/95 influenza viruses in MDCK cells. MDCK cells infected with the RG WT (A) and RG E119V-NA (B) viruses were incubated in the absence of C. perfringens NA in the agar overlay, while MDCK cells infected with RG R292K-NA virus were incubated in the absence (C) and in the presence (D) of 2 mU/ml C. perfringens NA. After 3 days at 37°C, the cell monolayers were stained and the diameters of 10 randomly selected plaques were measured. Values in parentheses are mean plaque diameters (millimeters) ± SD.

FIG. 3.

Replication kinetics of recombinant A/Wuhan/359/95 influenza viruses in MDCK cells. (A) Single-step growth curve. Cells were infected with RG WT, RG E119V-NA, or RG R292K-NA virus at an MOI of ∼2.2 PFU/cell. (B) Multistep growth curve. Cells were infected with the recombinant viruses at an MOI of 0.01 PFU/cell. Virus in the supernatant was titrated (log₁₀ PFU/ml) at the indicated times postinfection. Each datum point represents the mean ± SD from three experiments.

FIG. 4.

Replication kinetics of recombinant A/Wuhan/359/95 influenza viruses in MDCK-SIAT1 cells. (A, B) Single-step growth curves. Cells were infected with RG WT, RG E119V-NA, or RG R292K-NA virus at an MOI of ∼2.2 PFU/cell in the absence (A) or presence (B) of 2 mU/ml of C. perfringens NA in the medium. (C, D) Multistep growth curves. Cells were infected with the recombinant viruses at an MOI of 0.01 PFU/cell in the absence (C) or presence (D) of 2 mU/ml of C. perfringens NA in the medium. Virus in the supernatant was titrated (log₁₀ PFU/ml) at the indicated times postinfection. Each datum point represents the mean ± SD from three experiments. RG R292K-NA virus reverted to WT-NA (R292-NA) during multistep replication in MDCK-SIAT1 cells. Revertant (K292→R) NA was observed in at least one of the three experiments at 36, 48, and 60 h postinfection without supplementary C. perfringens NA (E, 60 h postinfection). This reversion caused the large SD seen for the RG R292K-NA virus in the absence of C. perfringens NA (C). In the presence of C. perfringens NA, plaques were homogeneous in size (F, 60 h postinfection).

FIG. 5.

Transmissibility of recombinant A/Wuhan/359/95 influenza viruses in a ferret contact model. Panels show virus titers (log₁₀ TCID₅₀/ml) in the nasal washes of ferrets infected with RG WT virus (A), RG E119V-NA virus (B), or RG R292K-NA virus (C). All donor ferrets (solid lines) were inoculated intranasally with 1,000 TCID₅₀ of recombinant virus in 0.5 ml sterile PBS. At 2 days p.i. (arrow), each donor ferret was moved into housing with two naive recipient ferrets (broken lines). Nasal washes were collected daily for 14 days, and virus was titrated in MDCK cells.