Isolation and molecular characterization of serotype O foot-and-mouth disease virus circulating in cattle in East Java, Indonesia

Foot-and-mouth disease (FMD) is a highly contagious transboundary viral disease of cloven-hoofed animals that causes substantial economic losses worldwide. The re-emergence of FMD in Indonesia in 2022 after more than three decades of disease-free status has made continuous molecular surveillance essential for monitoring circulating foot-and-mouth disease virus (FMDV) strains and supporting effective control strategies. This study aimed to isolate FMDV serotype O from clinically affected cattle in East Java and to molecularly characterize the circulating field strains. A total of 29 clinical samples were collected from cattle with typical FMD lesions in four affected regencies in East Java in 2025. Virus isolation was performed using BHK-21 cell cultures, and viral infectivity was determined by the 50% tissue culture infectious dose (TCID50) assay. Viral RNA extracted from culture-positive isolates was analyzed by serotype O-specific reverse transcription-polymerase chain reaction (RT-PCR) targeting the VP3 gene, followed by sequencing and phylogenetic analysis. Infectious FMDV was successfully isolated from 18 of 29 samples (62.1%), with viral titers ranging from 5.7×105 to 3.7×107 TCID50/mL. All cytopathic effect-positive isolates were confirmed as FMDV serotype O by VP3-specific RT-PCR. Phylogenetic analysis showed that the representative isolates clustered within the O/ME-SA topotype, belonged to the Ind-2001e lineage, and were closely related to viruses associated with Indonesia's 2022 FMD outbreak. These findings provide updated molecular epidemiological evidence that the Ind-2001e lineage continues to circulate in East Java, and they highlight the importance of sustained molecular surveillance to monitor viral evolution and to support evidence-based FMD control and vaccine strain selection.