Spatiotemporal dynamics of the FMDV epidemic in Bulgarian 2011.

<p>The simplest option was considered for the selection of the gamma-random-relaxed-walk (RRW) continuous diffusion model. The isolate from Israel was excluded from this analysis,. Satellite imagery: GoogleEarth. Date accessed: 25 May 2012. Co-ordinates: 40°57′25.89″N, 28°27′28.38″E (A); 42°04′27.07″N, 27°39′47.60″E (B) 42°09′01.30″N, 27°09′42.18″E (C). <b>A</b>. FMDV spread from the North-West of Turkey throughout Bursa (Anatolia, Turkey) to Brugas (Bulgaria). The uncertainity on the location the virus is represented by transparent polygons (80% HPD). Turkish Thrace might have been infected before November 2010, which is plausible with the serological results in wild boar. <b>B</b>. The transmission infection pathways between the wild boar, outbreaks 1, 2, 3 and the second wave of outbreaks is not clarified. It might be explained by un-sampled notified sites/outbreaks or by a reservoir in wildlife (i.e. wild boar), both hypotheses are compatible with a genetically and spatiotemporally close FMDV replicating within a host. <b>C</b>. The genetic spatiotemporal reconstruction of the second wave of outbreaks linked them to each other, in agreement with epidemiological data, i.e. owners from animals in outbreak 6 had animals in the location of outbreak 4.</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

Statistical parsimony trees as implemented by TCS using the full genomes of 19 sequenced FMDVs.

<p><b>A</b>. Edited TCS tree in which putative virus ancestors (○), except those corresponding to nodes, were removed. The length of the branches is directly proportional to the number of nucleotide (nt) changes. The vertical axis represents a time scale which denotes the date when the viruses were collected. <b>B</b>. Detailed TCS tree showing the viruses corresponding to the Bulgarian outbreaks and their closest ancestor within the Middle East. Open circles and lines correspond to putative genetic intermediates separated by single nt changes. Putative common (red circle) and secondary ancestors for each wave are shaded (blue circle, first; green circle, second). Lines in bold correspond to non-synonymous changes. The square shows the number of nt versus non-synonymous changes. The specific amino-acid changes are indicated, as well as the viral proteins involved. Non-conservative amino-acid substitutions (according to GONNET matrix, as implemented in BioEdit software) are highlighted in bold.</p

Bayesian maximum-clade-credibility time-scaled phylogenetic tree (BEAST) generated using 19 sequenced FMDV full genomes.

<p>The analysis was undertaken using a GTR substitution model, relaxed clock, constant population size, sampling 30.000 trees from 30 million generations. Uncertainty for the date of each node (95% highest posterior density – HPD - intervals) is displayed in bars. Only node labels with posterior over 0.8 are indicated. Overall, a rate of nucleotide substitution of 9.05×10⁻³ (95% HPD: 6.99–11.11×10⁻³) per site per year was estimated.</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