Representation of the nucleotide and amino acid differences detected in the five ILTV genomes.

<p>The outermost, thick circle is the NCBI ILTV reference sequence (Serva strain, GenBank accession no. HQ630064), where rectangles represent the coding sequences (CDS) and the colour code indicates the transcription strand: cyan refers to CDS in the plus strand, orange stands for minus strand. The inner circles represent the five ILTV genomes sequenced in this study (A = 4787/80, B = 193435/07, C = 757/11, D = MSD CEO vaccine, E = Zoetis CEO vaccine): on each circle, the blue bars indicate silent nucleotide substitutions, while red ones highlight those causing amino acid substitutions. The yellow ribbons in the inner part of the figure connect the coding sequences that are repeated in the ILTV genome. IR: internal repeat; TR: terminal repeat. The plot was obtained with Circos [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149529#pone.0149529.ref044" target="_blank">44</a>].</p

Evolutionary relationships of taxa.

<p>The evolutionary history was inferred using the Maximum Likelihood method based on the Tamura-Nei model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149529#pone.0149529.ref028" target="_blank">28</a>] on four different genes: UL27 (A), UL36 (B), ICP4 (C), and sORF4/3 (D). ILTV genomes sequenced in this study are highlighted in red (field strains) and green (vaccine strains). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149529#pone.0149529.ref045" target="_blank">45</a>]. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149529#pone.0149529.ref027" target="_blank">27</a>].</p

Detailed information of the ILTV complete genomes published in GenBank (accessed 2015 November 5) used in the alignment and phylogenetic analyses.

<p>Detailed information of the ILTV complete genomes published in GenBank (accessed 2015 November 5) used in the alignment and phylogenetic analyses.</p

Pairwise comparison of the five ILTV strains with the NCBI ILTV reference sequence (Serva strain) (global identity percentage and number of identities over the genome length of the shortest of the two).

<p>Pairwise comparison of the five ILTV strains with the NCBI ILTV reference sequence (Serva strain) (global identity percentage and number of identities over the genome length of the shortest of the two).</p

Summary of the amino acid differences in coding regions of the five ILTV strains <i>vs</i> other ILTV complete genomes published in GenBank (accessed 2015 November 5).

<p>Summary of the amino acid differences in coding regions of the five ILTV strains <i>vs</i> other ILTV complete genomes published in GenBank (accessed 2015 November 5).</p

Precocious and supernumerary sensory neuron specification in <i>sox10</i>^<i>baz1</i> mutants.

<p>A,B) <i>neurod1</i> expression is seen in more cells (close-ups in left panels; arrowheads indicate a subset of <i>neurod1</i>^<i>+</i> cells) and extending more posteriorly (right panels; arrowhead marks posteriormost <i>neurod1</i>^<i>+</i> DRG) in <i>baz1</i> mutants compared with WT siblings at both 36 and 45 hpf. C) Counts of <i>neurod1</i>^<i>+</i> cells on one side of embryo at 36 and 45 hpf embryos (N = 11 for all conditions except 36 hpf <i>baz1</i>, where N = 13). <i>baz1</i> mutants significantly different to WT siblings (Student’s <i>t</i> test; ***, p<0.0001. In this and all subsequent images, embryos are shown in lateral view with dorsal to the top and anterior to the left, unless otherwise stated. Scale bar, 100 μm.</p

Medial pathway neural precursors undergo precocious and supernumerary differentiation into neurons in baz1 mutants.

<p>Confocal images of developing trunk DRGs of WT (A, D, G, J), <i>baz1</i> (B, E, H, K) and <i>m618</i> mutants (C, F, I, L) showing Elav1/Hu (red) and <i>sox10</i>:<i>GFP</i> (green) at each of 36 (A-C), 42 (D-F), 48 hpf (G-I) and 5 dpf (J-L). Arrowheads indicate subset of Elav1/Hu⁺ DRG sensory neurons. M-P) Counts (mean±s.d.) of trunk (Tr) and tail (Ta) and total (TOT) Elav1⁺ cells in DRGs of <i>baz1</i> (yellow) and <i>m618</i> (blue) mutants and their respective WT siblings. Significantly elevated numbers of neurons are indicated (two-tailed Student’s <i>t</i> test; **, p<0.01; ***, p<0.001). Note in panels J-L) that variable prominence of Elav1/Hu detection in spinal cord is an artefact of antibody penetration into CNS at this late stage. Scale bar, 50 μm.</p

deltaA and deltaD gene expression overlaps with neurog1 in the nascent DRGs.

<p>A-C) <i>deltaA</i> expression (red) clearly overlaps with <i>neurog1</i> (green) in the nascent DRG (arrows) at 30 hpf. D-F) At 38 hpf, <i>deltaA</i> expression is clearly seen in the DRGs, but weaker signals make it difficult to discern if expression is in the same cells as express <i>neurog1</i> or simply in other cells of the ganglia. G-I) <i>deltaD</i> expression (red) clearly overlaps with <i>neurog1</i> (green) in the nascent DRG (arrows) at 38 hpf. All main panels are confocal images of fluorescent dual-color <i>in situ</i> hybridisations in lateral view, with insets showing <i>y-z</i> planes (left) and <i>x-z</i> planes (above) for each. Insets in the bottom right of panels C, F and I show enlargements of the double-labeled cells indicated by the arrows. nc, notochord; sc, spinal cord.</p