Results of assemblies and mappings of five Hong Kong swine vesicular disease virus (SVDV) isolates.

<p>First row shows the names of the five isolates, passage history, and harvest date. Second row shows the number of reads assigned to each isolate after they were separated using MID barcodes, and the number of these that had unique sequences (i.e. with duplicates removed), which were then kept for assembly/mapping. A number of reads is also lost during de-multiplexing, however this is even smaller than the total number of duplicates (not shown). Row three shows the mean, the standard deviation and the maximum of the read lengths of the unique reads. The fourth row shows results from <i>de novo</i> assembly, including number of reads mapped and minimum, maximum, mean, and standard deviation figures for the depth of coverage of the contig produced. The following five rows show results from iterative mapping against the five selected starting reference sequences (two SVDV sequences, a coxsackievirus B5, a coxsackievirus B3, and a coxsackievirus B6 sequence). The three isolates with successful <i>de novo</i> assemblies were iteratively mapped against one or two of these sequences for validation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097180#pone-0097180-g001" target="_blank">Figure 1</a>), whereas the remaining two isolates were iteratively mapped against all five starting references. For each of these two isolates the final mapping statistics can be seen to be virtually identical across all five starting references, clearly indicating that convergence of the iterative mapping process has been obtained. The final row shows the total number of ‘polymorphic’ sites obtained when the consensus sequence from assembly (if successful) and all of the mappings are multiple-aligned for each isolate (in parenthesis is shown whether any of the ‘polymorphisms’ are due to base calls where a non-redundant call in one sequence matches the category of a redundant call in the other).</p

Phylogenetic validation of samples.

<p>Using a maximum likelihood approach as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097180#s2" target="_blank">Methods</a> on the 1B-1C-1D genome region, corresponding to the outer capsid proteins VP2, VP3 and VP1, final validation was obtained for the samples. The five Hong Kong SVDVs isolated in the 1970s and which were sequenced in this study, are shown in red and all other SVD virus isolates are shown in pink. Coxsackievirus B5 isolates are shown in blue, and other <i>Enterovirus B</i> serotypes are shown in black. As expected from the previous literature, all CV-B5 together with all SVDV form a monophyletic cluster. This is supported with a bootstrap proportion of 100/100. Within this cluster all the SVD virus isolates, including those sequenced in this study, form a monophyletic cluster with bootstrap support of 96/100. Additionally, none of the five presently sequenced Hong Kong isolates show any obvious aberrations with regards to their position in the topology concerning either geographical information or branch lengths (vs. age). The tree is rooted on the branch leading from CV-B5 to all other CV-B sequences, and all branch labels show support in the form of bootstrap proportions out of one hundred. Support values for nodes with minor importance regarding the verification are not shown.</p

Decision diagram for validation of consensus sequences.

<p>The diagram illustrates the logic of the applied methodology for obtaining consensus sequences validated for further analysis. The process begins with <i>de novo</i> assembly as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097180#s2" target="_blank">methods</a> section. The starting point from there is the blue-outline box in the top left hand corner. Positive answers follow the green ‘YES’ arrows, negative ones follow the red ‘NO’ arrows, and grey arrows are followed in all cases. Termination in a red box should lead to thorough analysis of upstream sources of error, including everything from contaminated samples to late stage <i>in silico</i> problems. Arriving at the first green box means that the consensus sequence of assembly/mapping has been verified. Arriving at the final green box means that the sample sequence is fully validated, now also with regard to sample provenance. The sample sequence is now ready to be used for further scientific analysis.</p

SAT 1 Phylogenetic Tree.

<p>Markov Chain Monte Carlo phylogenetic tree generated using the combined SAT 1 data set including both chronologically sampled (CHR) data sets and temporal sample clusters (CLU). Temporal sample clusters are collapsed and coloured. Posterior probabilities are given for each node and the scale bar indicates a branch length corresponding to 25 years.</p

<i>dN/dS</i> ratios for all datasets.

<p>Single likelihood ancestor counting (SLAC) P < 0.1. Serotype sequences constituted 221 amino acids (SAT 1), 216 amino acids (SAT 2).</p><p><i>dN/dS</i> ratios for all datasets.</p

SAT 2 Phylogenetic Tree.

<p>Markov Chain Monte Carlo phylogenetic tree generated using the combined SAT 2 data set including both chronologically sampled (CHR) data sets and temporal sample clusters (CLU). Temporal sample clusters are collapsed and coloured. Posterior probabilities are given for each node and the scale bar indicates a branch length corresponding to 25 years.</p

Estimated time in which FMDV might have been introduced causing the different Bulgarian outbreaks.

<p>This illustration was built according to the date of sample collection, the virus and serological results of the collected samples and the age of the lesions of the animals with clinical signs. The date of sample collection is coloured in yellow in case of the virus-positive outbreaks, and in dark green in case of the seropositive-only outbreaks. The age of the lesions of the animals with clinical signs (if any), according to the National Veterinary surgeons involved in the outbreaks, is coloured in orange. The incubation time, estimated to be 14 days, is coloured in blue. In the case of the seropositive-only outbreaks, two different times were considered to explain the presence of antibody-positive/virus-negative samples, depending on whether clinical signs where unobserved or mild (hypothesis 1, H1) or whether the lesions had healed (H2). In case of H1, a minimum of 5 days post-infection was estimated (shaded with vertical stripes), whereas a minimum of 21 days post-infection for H2 (pale green). All estimated times were based on previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049650#pone.0049650-Alexandersen1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049650#pone.0049650-Cottam3" target="_blank">[9]</a>. Only genetic data can prove a link between waves one and two.</p

Nucleotide and amino acid substitutions occurring along the genome of the FMDV sequences.

<p><b>A</b>) Data for sequences from Bulgaria (8 genomes): graphs represent the distribution of total nucleotide (nt) (black line) and non-synonymous (red) substitutions across the different genomic regions of FMDV (shown below). The pie chart and the bar chart show percentage of nt substitutions for each region, and nt variability within the region, respectively. <b>B</b>) Similar analysis to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049650#pone-0049650-g002" target="_blank">figure 2A</a>) undertaken for the 11 FMDVs genomes from Turkey and Israel.</p

Summary of the FMDV outbreaks which occurred in Burgas, Bulgaria, 2011.

<p>W = Wild boar; C = Cattle; S = Sheep; G = Goat; P = Pig; B = Buffalo; U = Unobserved (FMDV-seropositive-only holding);</p>a<p>1a Seropositive free range pigs (lesions) and cattle; 1b Village with seropositive sheep, goats and pigs; 1c Virus positive Hereford cattle.</p>b<p>Partial sampling.</p>c<p>Clinical signs seen in sheep at culling.</p

Summary of full genome sequenced viruses generated in this study.

a<p>WRLFMD = World Laboratory Reference for Foot-and-Mouth Disease, The Pirbright Institute, United Kingdom; SAP = Foot-and-Mouth Disease Institute, Turkey; DTU = National Veterinary Institute, Denmark.</p>b<p>E = Epithelium; CC = Cell culture.</p>c<p>Sample nomenclature assigned by DTU.</p>d<p>Sample nomenclature assigned by SAP.</p