Sequential Adaptation of Foot-and-Mouth Disease Virus Serotype O Field Isolates in ZZR-127 and BHK-21 Cell Lines

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals. Continuous genetic variation in circulating field strains complicates vaccine matching and highlights the need for efficient virus isolation for vaccine seed production. Nineteen ELISA-confirmed FMDV serotype O field isolates from the Veterinary Research Institute (VRI), Peshawar, were sequentially cultured in ZZR-127 and BHK-21 cell lines. Each cell line underwent three serial passages. Antigen detection in culture supernatants (antigen-capture ELISA, positivity threshold OD ≥ 0.1, set empirically) and qualitative cytopathic effect (CPE) were used as indicators of virus recovery and propagation; infectious virus was not titrated. RT-PCR, performed on BHK-21 supernatants only, confirmed the presence of FMDV genomic material. Passage-dependent changes and predictors of adaptation success were analyzed using linear mixed-effects models, linear regression, and logistic regression. On ZZR-127 cells, antigen was detectable at passage 3 (P3; operationally defined here as “adaptation success”) in 12/19 isolates (63.2%). A non-significant positive trend in ELISA optical density (OD) values was observed across ZZR-127 passages (β = +0.123, p = 0.069); because the inoculum volume was doubled from 0.5 mL (P1) to 1.0 mL (P2–P3), this trend cannot be attributed to adaptation. In an exploratory logistic regression, initial OD was inversely associated with P3 positivity (β = −2.3769, p = 0.036), but the estimate was imprecise (OR = 0.093, 95% CI 0.009–0.956; 12 adapted vs. 7 non-adapted isolates) and sensitive to the omission of single isolates; this is a hypothesis-generating observation only. Antigen was detectable in all 12 isolates (100%) by P3 on BHK-21 cells, but in only 6/12 (50%) at P1 and 8/12 (66.7%) at P2; the 100% figure therefore depends on the arbitrary choice of P3 as the endpoint. A model-based decline in OD across BHK-21 passages (β = −0.171, p = 0.033) reflected the inclusion of the ZZR-127 P3 inoculum reading as “passage 0”; when restricted to BHK-21 readings (P1–P3), OD increased with passage (β = +0.317 per passage), so no conclusion on attenuation or antigen yield can be drawn. BHK-21 supernatants from all 12 isolates were RT-PCR positive for the FMDV VP1 region; ZZR-127 supernatants were not tested. The sequential use of ZZR-127 followed by BHK-21 cells was examined descriptively. Because no concurrent BHK-21-only control, mock-infected control, infectivity assay (TCID50/plaque assay) or sequencing was included, no conclusion can be drawn on efficiency relative to direct BHK-21 isolation, on viral replication or adaptation, or on vaccine seed potential, and comparison with published BHK-21 isolation rates is indicative only. These preliminary data are hypothesis-generating and require validation in controlled experiments, including cross-neutralization (r1 values), antigen stability and in vivo protection studies.