Biphasic phenotypic adaptation of sheeppox virus consequent to serial passaging in heterologous host cell type

Sheeppox virus (SPPV) is a highly host-restricted capripoxvirus, and its biological behavior in heterologous cells remains poorly defined. Although heterologous cell lines such as Vero cells are widely used for SPPV propagation and vaccine production, the effects of prolonged adaptation to these systems on viral replication and release dynamics have not been systematically examined. In this study, the SP3-Nur strain of SPPV (PV167793.1) was serially passaged 45 times in Vero cells. The parent virus (P0) was assessed in lamb testicular (LT) cells, while Vero-adapted passage levels (P15, P30, and P45) were evaluated in both Vero and LT cells to examine how passage levels affect viral replication and extracellular virus release. Replication kinetics were quantified at 0, 2, 4, and 6 days post-infection (DPI) using plaque-forming unit (PFU) assays and quantitative PCR (qPCR). In Vero cells, while virus production was increased from 2.54 log₁₀ PFU/ml (P15) to 5.73 log₁₀ PFU/ml (P30) at 6 DPI, accompanied by an increase in mean plaque area from 7,266 μm² to 42,309 μm², respectively, while at P45, reduced extracellular release (4.26 log₁₀ PFU/ml at 6 DPI) and smaller plaques (5,319 μm²) were observed. Plaque size differences between P15 and P30 and between P30 and P45 were statistically significant (p < 0.01). The qPCR data in Vero cells revealed that viral genome copies in supernatant declined from 5.73 to 4.72 log₁₀ copies/ml, whereas cell-associated viral genome copies remained stable at 5.45 to 5.13 log₁₀ copies/ml in passages P30 and P45, respectively, at 6 DPI, indicating impaired virus release rather than loss of intracellular replication. A similar trend was observed in LT cells. This study showed biphasic phenotypic adaptation in heterologous cells characterized by altered replication dynamics and impaired viral release. This study provides important insights into SPPV biology in Vero cells, having implications in its virulence, vaccine production, and the development of a suitable animal model.