Development of molecular tools for the characterisation of foot-and-mouth disease virus strains circulating in Southeast Asia

Foot-and-mouth disease is an acute and contagious vesicular disease affecting wild or domesticated cloven hoofed animals. The disease is caused by Foot-and-mouth disease virus (FMDV) of Aphthovirus genus within Picornaviridae family. FMDV exists as seven distinct serotypes; O, A, C, Asia 1, SAT (Southern Africa Territories) 1, SAT 2 and SAT 3, each with multiple subtypes which are not equally distributed around the world. In this study, VPI analysis of clinical samples cattle and pigs received from Southeast Asian countries namely Malaysia, Thailand, Lao PDR, Myanmar, Vietnam and Cambodia between 2000 to 2009 revealed seven FMDV lineages circulating in the region. The lineages were O/SEA/Mya-98, O/SEA/Cam-94, O/ME-SA/PanAsia, O/ME-SA/PanAsia-2, O/CATHAY, A/ASIA, and Asia 1. Some of these lineages were highly prevalent such as O/SEA/Mya98 and serotype A, while O/ME-SA/PanAsia-2 was unique to Malaysia within Southeast Asia. Phylogenetic analysis of VP1 showed two distinct patterns of genetic relationships between viruses from Malaysia and the other neighbouring countries. These data suggest that viruses of O/SEA/Mya-98 and serotype A lineage were repeatedly introduced into the country on a yearly basis. In contrast, viruses belonging to O/ME-SA/PanAsia-2 sublineage were maintained in the country to cause outbreaks in following continuous years. However, phylogenetic analysis cannot conclude the link between Malaysian isolates with those from neighbouring countries. Since Malaysia is a major beef and animal importer, the sources of these FMDVs were most likely introduced via imports of infected animals or contaminated animal products into Malaysia. In order to improve the resolution, by which the molecular epidemiology of FMDV in the region can be studied, RT-PCR protocols to amplify complete genome sequences of each lineage were developed. The protocols utilised a single set of universal primers for NSP region and specific primers sets for the capsid region. Complete genomes from representative isolates from the seven virus lineages were generated from Malaysian samples. Basic characteristic of genomes generated with this protocol were consistent with previous published sequences such as deletions at the 3A region for O/CATHAY and O/SEA/Cam-94 lineage. In addition, preliminary analysis of O/ME-SA/PanAsia-2 sequence showed numerous nucleotide substitutions observed at the 5’UTR and Lpro region of genome to that of O/ME-SA/PanAsia which indicated a possible event of recombination. The O/SEA/Mya-98 protocol was proven to be robust when it was utilised to generate two complete genome sequences of O/SEA/Mya-98 viruses from outbreaks in East Asian countries, in Hong Kong SAR and Republic of Korea that have occurred during 2010. Analysis of the Hong Kong genome sequence revealed a block deletion of 70 nucleotides located within the S-fragment. Subsequent analysis showed that this feature was shared with other viruses with related VP1 sequences. The methods developed in this study provide a basis for a more comprehensive understanding of the diversity and evolution of FMDV that will hopefully develop improved epidemiological and diagnostic tools that will contribute to the existing disease control programmes in the region.