Susceptibility profiles of recombinant A/Brisbane/59/2007-like (H1N1) viruses against neuraminidase (NA) inhibitors as assessed by MUNANA NA inhibition assays.

a<p>Values indicated mean IC₅₀ values of three experiments ± standard deviations.</p><p>**<i>P<0.01</i> compared to the single H275Y mutant.</p

Neuraminidase enzymatic properties and plaque areas of recombinant A/Brisbane/59/2007-like (H1N1) viruses.

a<p>Values indicate mean <i>Km</i> and relative NA activity (<i>Vmax)</i> values of a representative experiment performed in triplicate ± standard deviations (SD).</p>b<p>Values indicate mean plaque area (N = 16) ± SD.</p><p>*P<0.05,</p><p>***P<0.001 compared to WT.</p

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

<p>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).*<i>P</i><0.05 and **<i>P</i><0.01 for differences in viral titers when compared to the recombinant WT virus.</p

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

<p>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. **<i>P</i><0.01 and ***<i>P</i><0.001 compared to the WT surface NA activity.</p

Replication kinetics of recombinant A/Brisbane/59/2007-like viruses <i>in vitro</i>.

<p>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. *<i>P</i><0.05 and ***<i>P</i><0.001 for differences in viral titers when compared to the recombinant WT virus.</p

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

<p>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.</p

Body temperatures of infected and contact ferrets.

<p>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).</p

Neuraminidase activity assay results.

a<p>Units of are “U/h” where U is dependent on the concentration of neuraminidase enzyme, which may differ between strains (despite the fact that the infecting virus titer, in PFU/mL, is fixed).</p

Infection parameters identified by experiment-model analysis.

a<p>Errors denoted by a range of values are 95% confidence intervals, taken from the extent of the light blue areas in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014767#pone-0014767-g006" target="_blank">Figures 6C and 6F</a> for the parallel plaque and yield experiment and from fits to 1000 bootstrap replicates for the single-cycle yield experiment.</p>b<p>Assuming a normal distribution of latent infection periods.</p>c<p>The “” indicates a value not calculated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014767#pone-0014767-g006" target="_blank">Figure 6F</a>.</p

Dependence of plaque velocity and viral titer growth rate on parameters.

<p><b>A</b>. Effect of a - to 10-fold variation of the parameters on the plaque velocity. <b>B</b>. Effect of a - to 10-fold variation of the parameters on the viral titer exponential growth rate (for the multiple-cycle viral yield experiment), as determined by Equation (3). <b>C</b>. Radial plaque velocity as a function of the infecting time and latent infection period (labeled contours have units of ). <b>D</b>. Viral titer growth rate as a function of the infecting time and latent infection period (labeled contours have units of ). When not varied, the following base values for each parameter were used: infectious lifespan, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014767#pone.0014767-Gaush1" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014767#pone.0014767-Takizawa1" target="_blank">[64]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014767#pone.0014767-Zhirnov1" target="_blank">[66]</a>; infecting time, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014767#pone.0014767-Baccam1" target="_blank">[34]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014767#pone.0014767-Beauchemin1" target="_blank">[35]</a>; latent infection period, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014767#pone.0014767-Gaush1" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014767#pone.0014767-Kaverin1" target="_blank">[67]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014767#pone.0014767-Min1" target="_blank">[69]</a>.</p