EVALUATION OF THE BIOLOGICAL ACTIVITY OF ORTHOPOXVIRUS STRAINS IN PRIMARY CHICKEN EMBRYO FIBROBLASTS

Understanding the biological features of the genus, Orthopoxvirus, including their replicative activity, is essential for their application in virology and vaccinology. Comparative analysis of biological activity in cell cultures makes it possible to evaluate the replicative potential of strains and the degree of their adaptation to the cellular substrate. The aim of the study was to standardize a method for obtaining primary chicken embryo fibroblasts (CEF) and to use them as an in vitro substrate for assessing the biological activity of various Orthopoxvirus strains, with an emphasis on cytopathic effect (CPE) and infectious activity. Vaccinia virus, cowpox virus, and camelpox virus were used in the study. Primary CEF were obtained from 9-11-day embryonated chicken eggs (ECE), the optimal seeding concentration for the formation of a monolayer substrate was determined, and the cultures were infected with the viruses under study; CPE and virus titer were assessed during sequential passages. The results showed that a seeding concentration of 5×105 cells/mL ensured the formation of a uniform CEF monolayer within 24 h. Upon infection, all three viruses induced similar CPE, characterized by cell rounding, loss of elongated morphology, and partial destruction of the monolayer, with the main difference being the timing of CPE onset. Vaccinia virus strains induced CPE within 24 h, whereas cowpox virus and camelpox virus induced CPE after 36 and 48 h, respectively. Both vaccinia virus strains exhibited high infectious activity and rapid adaptation (titers from 5.50 to 6.58±0.12 lg TCID₅₀/mL), whereas cowpox and camelpox viruses showed a lower and stable titer (titers from 5.50 to 6.58±0.12 lg TCID₅₀/mL), and delayed CPE development. The results emphasize the significance of CPE onset time and infectious titer level as key parameters for evaluating replicative capacity and strain adaptation, which is important for laboratory studies and the selection of vaccine candidates.