MOLECULAR EPIDEMIOLOGY AND WHOLE-GENOME CHARACTERIZATION OF FOOT AND MOUTH DISEASE VIRUS CIRCULATING IN ENDEMIC CLINICAL OUTBREAKS ACROSS PAKISTAN

Abstract

Foot-and-mouth disease (FMD) remains a major constraint to livestock productivity in Pakistan, causing significant economic losses and hindering trade. This study aimed to characterize circulating FMD virus (FMDV) strains using integrated virological and molecular diagnostics, based on clinical samples collected from cattle and buffalo in endemic regions of Punjab and Sindh. A total of 52 suspected samples were analyzed by RT-PCR, antigen-capture ELISA (Ag-ELISA), and virus isolation in IB-RS-2 cell culture. RT-PCR detected FMDV RNA in 34 samples, with serotype O predominating (34/34), alongside sporadic signals for serotype A and unconfirmed SAT-1. Cell culture confirmed viable virus in 14 isolates, all of which were consistent with serotype O or A, while Ag-ELISA showed lower sensitivity due to field sample limitations. Sequencing of the VP1 region from four representative isolates revealed two distinct genetic clusters: JW53/JW58 (99.21% identity) aligned with the PanAsia-2ANT lineage of serotype O, and JW43/JW48 (99.30% identity) matched the A/ASIA/G-VII topotype of serotype A. Inter-cluster identities (52–55%) confirmed serotypic divergence. Phylogenetic analysis reinforced regional links with strains from India and Afghanistan, highlighting transboundary transmission risks. The findings underscore that FMD in Pakistan is driven by endemic serotypes O and A, with no verified circulation of SAT types. These results advocate for a national control strategy centered on bivalent (O+A) vaccines matched to local lineages, enhanced molecular surveillance, and regional cooperation to mitigate cross-border spread.