Estimates of evolutionary pairwise distances A. over the Austrian strains (including the SMB₁ plasma isolate) and their nine closest relatives, B. five major groups (clades), and C. six minor groups (clusters) among clade 2d, all defined according to the clustering in the phylogenetic tree (Fig 1).

<p>The average numbers of substitutions per site between nucleotide and amino acid sequences are indicated below and above the diagonal, respectively. Estimation of evolutionary distances was conducted in MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126381#pone.0126381.ref019" target="_blank">19</a>] using the MCL and p-distance algorithms for nucleotide and amino acid sequences, respectively. Group 2d-1 contains the Central/Southern European viruses including Austrian strains, and group 2d-5 consists of the Eastern European WNVs.</p><p>Abbreviations used: At-bd = strain Blood donor/Vienna/2014Austria (KP109691), SMB₁ = first passage of the suckling mouse brain isolate from the Austrian blood donor’s plasma, Cz 329 = strain Cz 13–329 (KM203861), Cz 479 = strain Cz 13–479 (KM203862), At-Cx = strain Cx pipiens/Vienna/2014Austria (KP109692), It AN2 = isolate Italy/2011/AN-2 (JN858070), It 32.1 = strain Italy/2013/Rovigo/32.1 (KF588365), It 33.2 = strain Italy/2013/Rovigo/33.2 (KF647249), It 34.1 = strain Italy/2013/Padova/34.1 (KF647251), Cz 104 = s train CZ 13–104 (KM203860), Cz 502 = strain Cz 13–502 (KM203863), At-gh = WNV strain Austria/2008_goshawk (KF179640).</p><p>Estimates of evolutionary pairwise distances A. over the Austrian strains (including the SMB₁ plasma isolate) and their nine closest relatives, B. five major groups (clades), and C. six minor groups (clusters) among clade 2d, all defined according to the clustering in the phylogenetic tree (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126381#pone.0126381.g001" target="_blank">Fig 1</a>).</p

Phylogenetic tree of 36 West Nile virus lineage 2 full length polyprotein-coding nucleotide sequences.

<p>The sequences determined in this study are marked with a red diamond (Viennese blood donor-derived WNV) and a black diamond (Viennese <i>Culex pipiens</i>-derived WNV), respectively. The Austrian goshawk-derived sequence from 2008 is marked with a contoured diamond. Five major clades and six clusters among clade 2d are indicated. Group 2d-1 contains the Central/Southern European viruses including Austrian strains, and group 2d-5 consists of the Eastern European WNVs. The phylogenetic tree was constructed using the NJ method with MCL algorithm of MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126381#pone.0126381.ref019" target="_blank">19</a>] with 1,000-fold bootstrap analysis. GenBank accession numbers, strain names, and (if known) species, countries of origins and years of isolations are indicated at the branches. Supporting bootstrap values >80% (the percentage of replicates in the bootstrap analysis) are displayed next to the nodes. The horizontal scale bar indicates genetic distances (here 2% nucleotide sequence divergence).</p

Mosquitoes collected in the city of Vienna between 28 August and 10 September 2014.

<p>The two WNV-positive samples are marked with (+). Abbreviations used: BFH, Baumgarten Cemetery; FH Ottakring, Ottakring Cemetery; <i>An</i>., <i>Anopheles</i>; <i>Cs</i>., <i>Culiseta</i>; <i>Cx</i>., <i>Culex</i>; A, adults; E, egg rafts; L, larvae; P, pupae; MEM, minimal essential medium.</p><p>Mosquitoes collected in the city of Vienna between 28 August and 10 September 2014.</p