NA V241I and N369K enhance the expression and activity of A(H1N1)pdm09 NA proteins.

<p>Surface expressed NA protein and enzymatic activity was assessed 20-transfection of 293T cells with New17 OR (A), or A/California/7/2009 oseltamivir sensitive (Cal7 OS) (B), NA expression plasmids containing a C-terminal V5 epitope tag followed by an IRES-GFP, and encoding the amino acid mutations indicated. NA expression was assessed by flow cytometry after staining with a fluorescently conjugated anti-V5 antibody and gating on the GFP positive (transfected) cells. Enzyme activity was assessed by the <i>in vitro</i> NA inhibition assay. All results represent the mean and standard error of three replicate transfections and are normalised to the mean fluorescent intensity/NA activity of cells transfected with the New17 OR (A) or Cal7 OS (B) wild type NA plasmid. NA activity results for each group were compared to the relative NA activity of New17 OR, for groups shown in A, and Cal7 OS for groups shown in B, using a two-tailed <i>t</i> test. *  = <i>P</i>≤0.05, ** = <i>P</i>≤0.01, ns = <i>P</i>&gt;0.05.</p

The H275Y mutation does not compromise the fitness of HNE2011 A(H1N1)pdm09 viruses.

<p>Donor ferrets were infected with pure populations or virus mixtures of A/Newcastle/17/2011 oseltamivir resistant (New17 OR) and A/Newcastle/163/2011 oseltamivir sensitive (New163 OS). Daily nasal washes from donor and 1^st and 2^nd recipient ferrets were assayed to measure the viral replication and transmission kinetics of each virus mixture/pure population and to assess the relative proportions of each virus within mixtures. (A) The infectious virus titre in each nasal wash was determined by titration on MDCK cells. (B) The relative proportions of New17 OR virus encoding NA 275Y (black bars) and New163 OS virus encoding NA 275H (white bars) in each nasal wash were determined by pyrosequencing. (A) Virus in donor ferrets (grey), 1st recipient ferrets (black lines solid squares), 2nd recipient ferrets (black lines, white triangles).</p

Addition of NA V241I and N369K enhances the fitness of earlier H275Y A(H1N1)pdm09 viruses.

<p>Donor ferrets were infected with pure populations or virus mixtures of reverse genetics derived A/Perth/261/2009 oseltamivir resistant (rgPerth261 OR) and rgPerth261 V241I, N369K OR. Daily nasal washes from donor and 1st and 2nd recipient ferrets were assayed to measure the viral replication and transmission kinetics of each virus mixture/pure population and to assess the relative proportions of each virus within mixtures. (A) The infectious virus titre in each nasal wash was determined by titration on MDCK cells. (B) The relative proportions of virus encoding NA 241I, 369K (black bars) and NA 241V, 369N (white bars) in each nasal wash were determined by pyrosequencing. (A) Virus in donor ferrets (grey), 1st recipients (black lines solid squares), 2nd recipients (black lines, white triangles).</p

Removal of NA V241I and N369K decreases the fitness of recent H275Y A(H1N1)pdm09 viruses.

<p>Donor ferrets were infected with pure populations or virus mixtures of reverse genetics derived New17 OR (rgNew17 OR) and rgNew17 I241V, K369N OR. Daily nasal washes from donor, and naive 1st and 2nd recipient ferrets were assayed to measure the viral replication and transmission kinetics of each virus mixture/pure population and to assess the relative proportions of each virus in mixtures. (A) The infectious virus titre in each nasal wash was determined by titration on MDCK cells. (B) The relative proportions of virus encoding NA 241I, 369K (black bars) and NA 241V, 369N (white bars) in each nasal wash were determined by pyrosequencing. (A) Virus in donor ferrets (grey), 1st recipient ferrets (black lines solid squares), 2nd recipient ferrets (black lines, white triangles).</p

Relative within-host and transmission fitness of virus pairs used in ferret experiments.

<p>a, OS  =  Oseltamivir sensitive.</p><p>b, OR  =  Oseltamivir resistant due to the NA H275Y mutation.</p><p>c, Relative within-host fitness: values &gt;1 indicate advantage for virus B.</p><p>d, Relative transmission fitness: Values &gt;1 indicate advantage for virus B.</p><p>e, No significant difference is observed when the confidence intervals cross 1.</p