The molecular epidemiology of Foot-and-Mouth Disease virus serotypes A and O from 1998 to 2004 in Turkey

BACKGROUND: Foot-and-Mouth Disease (FMD) causes significant economic losses in Turkish livestock. We have analysed the genetic diversity of the 1D sequences, encoding the hypervariable surface protein VP1, of Turkish isolates of serotype A and O collected from 1998 to 2004 in order to obtain epidemiological and immunological information. RESULTS: The 1D coding region of 33 serotype O and 20 serotype A isolates, obtained from outbreaks of FMD between 1998 and 2004, was sequenced. For serotype A, we confirmed the occurrence of the two subtypes IRN99 and IRN96. These subtypes are most divergent within the region encoding the immuno-dominant GH-loop. Also a close relationship to Foot-and-Mouth Disease virus (FMDV) serotype A isolates obtained from outbreaks in Iraq and Iran were detected and a clustering of isolates collected during the same period of time were found. The analysis of the deduced amino-acid sequences of these subtypes revealed evidence of positive selection in one site and one deletion, both within the GH-loop region. By inferring the ancestral history of the positively selected codon, two potential precursors were found. Furthermore, the structural alignment of IRN99 and IRN96 revealed differences between the tertiary structures of these subtypes. The similarity plot of the serotype O isolates suggested a more homogeneous group than the serotype A isolates. However, phylogenetic analysis revealed two major groups, each further divided in subgroups, of which some only consisted of Turkish isolates. Positively selected sites and structural differences of the Turkish isolates analysed, were not found. CONCLUSION: The sequence and structural analysis of the IRN99 strains is indicative of positive selection suggesting an immunological advantage compared to IRN96. However, results of antigenic comparison reported elsewhere do not substantiate such a conclusion. There is evidence that IRN99 was introduced to Turkey, in all probability from Iran. Since, a member of the IRN96 lineage was included as a component of the FMDV vaccine produced since 2000, the outbreaks caused by IRN96 strains in 2004 could be due to incomplete vaccine coverage. The Turkish type O strains, all with a VP1 structure similar to the O1/Manisa/69 vaccine, appear in several sublineages. Whether these sublineages reflect multiple samplings from a limited number of outbreaks, or if they reflect cross-boundary introductions is not clear

Reconstruction of the Transmission History of RNA Virus Outbreaks Using Full Genome Sequences: Foot-and-Mouth Disease Virus in Bulgaria in 2011

<div><p>Improvements to sequencing protocols and the development of computational phylogenetics have opened up opportunities to study the rapid evolution of RNA viruses in real time. In practical terms, these results can be combined with field data in order to reconstruct spatiotemporal scenarios that describe the origin and transmission pathways of viruses during an epidemic. In the case of notifiable diseases, such as foot-and-mouth disease (FMD), these analyses provide important insights into the epidemiology of field outbreaks that can support disease control programmes. This study reconstructs the origin and transmission history of the FMD outbreaks which occurred during 2011 in Burgas Province, Bulgaria, a country that had been previously FMD-free-without-vaccination since 1996. Nineteen full genome sequences (FGS) of FMD virus (FMDV) were generated and analysed, including eight representative viruses from all of the virus-positive outbreaks of the disease in the country and 11 closely-related contemporary viruses from countries in the region where FMD is endemic (Turkey and Israel). All Bulgarian sequences shared a single putative common ancestor which was closely related to the index case identified in wild boar. The closest relative from outside of Bulgaria was a FMDV collected during 2010 in Bursa (Anatolia, Turkey). Within Bulgaria, two discrete genetic clusters were detected that corresponded to two episodes of outbreaks that occurred during January and March-April 2011. The number of nucleotide substitutions that were present between, and within, these separate clusters provided evidence that undetected FMDV infection had occurred. These conclusions are supported by laboratory data that subsequently identified three additional FMDV-infected livestock premises by serosurveillance, as well as a number of antibody positive wild boar on both sides of the border with Turkish Thrace. This study highlights how FGS analysis can be used as an effective on-the-spot tool to support and help direct epidemiological investigations of field outbreaks.</p> </div

Monoclonal Antibodies to Conformational Epitopes of the Surface Glycoprotein of Caprine Arthritis-Encephalitis Virus: Potential Application to Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detecting Antibodies in Goat Sera

Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79–63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide- N -glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with N -acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were <10% of that of HRP-conjugated MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection

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

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

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

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

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

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