Role of permissive neuraminidase mutations in influenza A/Brisbane/59/2007-like (H1N1) viruses

Abstract

Neuraminidase (NA) mutations conferring resistance to NA inhibitors were believed to compromise influenza virus fitness. Unexpectedly, an oseltamivir-resistant A/Brisbane/59/2007 (Bris07)-like H1N1 H275Y NA variant emerged in 2007 and completely replaced the wild-type (WT) strain in 2008-2009. The NA of such variant contained additional NA changes (R222Q, V234M and D344N) that potentially counteracted the detrimental effect of the H275Y mutation on viral fitness. Here, we rescued a recombinant Bris07-like WT virus and 4 NA mutants/revertants (H275Y, H275Y/Q222R, H275Y/M234V and H275Y/N344D) and characterized them in vitro and in ferrets. A fluorometric-based NA assay was used to determine Vmax and Km values. Replicative capacities were evaluated by yield assays in ST6Gal1-MDCK cells. Recombinant NA proteins were expressed in 293T cells and surface NA activity was determined. Infectivity and contact transmission experiments were evaluated for the WT, H275Y and H275Y/Q222R recombinants in ferrets. The H275Y mutation did not significantly alter Km and Vmax values compared to WT. The H275Y/N344D mutant had a reduced affinity (Km of 50 vs 12 µM) whereas the H275Y/M234V mutant had a reduced activity (22 vs 28 U/sec). In contrast, the H275Y/Q222R mutant showed a significant decrease of both affinity (40 µM) and activity (7 U/sec). The WT, H275Y, H275Y/M234V and H275Y/N344D recombinants had comparable replicative capacities contrasting with H275Y/Q222R mutant whose viral titers were significantly reduced. All studied mutations reduced the cell surface NA activity compared to WT with the maximum reduction being obtained for the H275Y/Q222R mutant. Comparable infectivity and transmissibility were seen between the WT and the H275Y mutant in ferrets whereas the H275Y/Q222R mutant was associated with significantly lower lung viral titers. In conclusion, the Q222R reversion mutation compromised Bris07-like H1N1 virus in vitro and in vivo. Thus, the R222Q NA mutation present in the WT virus may have facilitated the emergence of NAI-resistant Bris07 variants.

Figure 1. Neuraminidase (NA) enzyme kinetics of recombinant A/Brisbane/59/2007-like (H1N1) viruses.

The rate of substrate conversion velocity (V₀) by NA enzymes from a standardized dose of 10⁶ PFU/ml of recombinant virus was determined. The fluorogenic substrate (MUNANA) was used at final concentrations of 0 to 3000 µM. Fluorescence was measured every 90 sec for 53 min at 37 °C using excitation and emission wavelengths of 355 and 460 nm, respectively. The data of one representative experiment performed in triplicate is shown.

Figure 2. Surface activity of recombinant A/Brisbane/59/2007-like (H1N1) neuraminidase proteins.

293T cells were transfected with pCAGGS-PA, -PB1, -PB2, and -NP plasmids in addition to plasmids expressing the WT or mutant A/Brisbane/59/2007-like neuraminidases (NA) proteins. At 24 h post-transfection, cells were treated with a non-lysing buffer and surface NA activity was measured by using the fluorogenic substrate (MUNANA). Percent surface NA activities were determined in triplicate experiments ± standard deviations. **P<0.01 and ***P<0.001 compared to the WT surface NA activity.

Figure 3. Replication kinetics of recombinant A/Brisbane/59/2007-like viruses in vitro.

Confluent ST6Gal1-MDCK cells were infected with recombinant viruses at a multiplicity of infection (MOI) of 0.001 PFU/cell. Supernatants were harvested at 12 h, 24 h, 36 h, 48 h and 60 h post-infection and titrated by standard plaque assays. The mean values for three experiments with standard deviations are presented. *P<0.05 and ***P<0.001 for differences in viral titers when compared to the recombinant WT virus.

Figure 4. Body temperatures of infected and contact ferrets.

Body temperatures were recorded by rectal thermometer during 10 days post-inoculation in groups of 4 index ferrets infected with 1.25×10⁵ PFU of recombinant A/Brisbane/59/2007-like wild-type (WT) virus as well as H275Y and H275Y/Q222R mutants (A) and in groups of 4 naïve ferrets that were placed in direct contact with index ferrets 24 h later (B).

Figure 5. Mean viral titers in nasal wash samples of infected and contact ferrets.

Mean viral titers ± standard deviations were determined in nasal washes by using standard plaque assays in groups of 4 index ferrets infected with 1.25×10⁵ PFU of recombinant A/Brisbane/59/2007-like wild-type (WT) virus as well as H275Y and H275Y/Q222R mutants (A) and in groups of 4 naïve ferrets that were placed in direct contact with index ferrets 24 h later (B).*P<0.05 and **P<0.01 for differences in viral titers when compared to the recombinant WT virus.