Evolutionary and Ecological Drivers Shape the Emergence and Extinction of Foot-and-Mouth Disease Virus Lineages.

Livestock farming across the world is constantly threatened by the evolutionary turnover of foot-and-mouth disease virus (FMDV) strains in endemic systems, the underlying dynamics of which remain to be elucidated. Here, we map the eco-evolutionary landscape of cocirculating FMDV lineages within an important endemic virus pool encompassing Western, Central, and parts of Southern Asia, reconstructing the evolutionary history and spatial dynamics over the last 20 years that shape the current epidemiological situation. We demonstrate that new FMDV variants periodically emerge from Southern Asia, precipitating waves of virus incursions that systematically travel in a westerly direction. We evidence how metapopulation dynamics drive the emergence and extinction of spatially structured virus populations, and how transmission in different host species regulates the evolutionary space of virus serotypes. Our work provides the first integrative framework that defines coevolutionary signatures of FMDV in regional contexts to help understand the complex interplay between virus phenotypes, host characteristics, and key epidemiological determinants of transmission that drive FMDV evolution in endemic settings.

Chitosan-based systems for intranasal immunization against foot-and-mouth disease.

Inactivated conventional vaccines against foot-and-mouth disease (FMD) are used routinely in endemic countries and are effective against clinical disease. Increased systemic IgG levels can be obtained with these vaccines whereas local response at the mucosal sites where the virus primarily enters the organism and replicates remains very limited. The aim of this study was to develop a safe, non-invasive and antigen compatible system for mucosal delivery of the FMD antigen which induces both the systemic and local immunity. Gel formulations were prepared using different types of chitosan at different concentrations and were incorporated with the whole inactivated FMD virion. The immune responses in guinea pigs were determined following intranasal administration. Chitosan-based FMD vaccine formulations have been shown to induce FMD antigen-specific serum IgG and nasal IgA levels, the latter response being significantly stronger as compared to that obtained following subcutaneous administration of the FMD antigen in Freund's incomplete adjuvant. Our results suggest that intranasal immunization with inactivated FMD virion delivered in the presence of chitosan is very promising, inducing both systemic and local immune responses.

Development of a Multiplex PCR Assay for Rapid Differentiation of Fowlpox and Pigeonpox Viruses.

The aim of this study was to develop a multiplex PCR assay capable of rapidly differentiating two major Avipoxvirus (APV) species, Fowlpox virus (FWPV) and Pigeonpox virus (PGPV), which cause disease in bird species. Despite the importance of a rapid differentiation assay, no such assay exists that can differentiate the APV species without sequencing. To achieve this, species-specific target DNA fragments were selected from the fpv122 gene of FWPV and the HM89_gp120 gene of PGPV, which are unique to each genome. Nine samples collected from unvaccinated chickens, pigeons, and a turkey with typical pox lesions were genetically identified as FWPV and PGPV. The designed primers and target DNA fragments were validated using in silico analyses with the nucleotide Basic Local Alignment Search Tool. The multiplex PCR assay consisted of species-specific primers and previously described PanAPV primers (genus-specific) and was able to differentiate FWPV and PGPV, consistent with the phylogenetic outputs. This study represents the first successful differentiation of FWPV and PGPV genomes using a conventional multiplex PCR test. This assay has the potential to facilitate the rapid diagnosis and control of APV infections.

Desarrollo de un ensayo de PCR múltiple para la diferenciación rápida de los virus de la viruela aviar y la viruela de paloma. El objetivo de este estudio fue desarrollar un ensayo de PCR múltiple capaz de diferenciar rápidamente dos especies principales de Avipoxvirus (APV) (viruela del pollo), el Fowlpox virus (FWPV) y el Pigeonpox virus (PGPV), (viruela de la gallina), que causan enfermedades en especies de aves. A pesar de la importancia de un ensayo de diferenciación rápida, no existe ningún ensayo que pueda diferenciar las especies de APV sin secuenciación. Para lograr esto, se seleccionaron fragmentos blanco de ADN específicos de especie del gene fpv122 de FWPV y el gene HM89_gp120 de Pigeonpox virus, que son únicos para cada genoma. Nueve muestras recolectadas de pollos, palomas y un pavo que no fueron vacunados con lesiones típicas de la viruela se identificaron genéticamente como FWPV y PGPV. Los iniciadores diseñados y los fragmentos de ADN blanco se validaron mediante análisis in silico mediante la herramienta de búsqueda de alineación local básica de nucleótidos (BLAST). El ensayo de PCR múltiple consistió en iniciadores específicos de especie y cebadores PanAPV previamente descritos (específicos de género) y fue capaz de diferenciar entre Fowlpox virus y Pigeonpox virus, de acuerdo con los resultados filogenéticos. Este estudio representa la primera diferenciación exitosa de los genomas de Fowlpox virus y Pigeonpox virus utilizando una prueba de PCR múltiple convencional. Este ensayo tiene el potencial de facilitar el diagnóstico rápido y el control de las infecciones por Avipoxvirus.

Evidence for multiple recombination events within foot-and-mouth disease viruses circulating in West Eurasia.

Phylogenetic studies on foot-and-mouth disease viruses (FMDVs) circulating in the West Eurasian region have largely focused on the genomic sequences encoding the structural proteins that determine the serotype. The present study has compared near-complete genome sequences of FMDVs representative of the viruses that circulate in this region. The near-complete genome sequences (ca. 7,600 nt) were generated from multiple overlapping RT-PCR products. These amplicons were from FMDVs belonging to serotypes O, A and Asia-1, including members of the O-PanAsia-II and the A-Iran05 lineages, and of Group-II and Group-VII (Sindh-08) within serotype Asia-1, which are currently predominant and widespread in West Eurasia. These new sequences were analysed together with other sequences obtained from GenBank. Comparison of different regions of the FMDVs genomes revealed evidence for multiple, inter-serotypic, recombination events between FMDVs belonging to the serotypes O, A and Asia-1. It is concluded from the present study that dramatic changes in virus sequences can occur in the field through recombination between different FMDV genomes. These analyses provide information about the ancestry of the serotype O, A and Asia-1 FMDVs that are currently circulating within the West Eurasian region.

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.

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.