THE AFRICAN SWINE FEVER VIRUS THYMIDINE KINASE GENE IS REQUIRED FOR EFFICIENT REPLICATION IN SWINE MACROPHAGES AND FOR VIRULENCE IN SWINE

African swine fever virus (ASFV) replicates in the cytoplasm of infect ed cells and contains genes encoding a number of enzymes needed for DN A synthesis, including a thymidine kinase (TK) gene. Recombinant TK ge ne deletion viruses were produced by using two highly pathogenic isola te's of ASFV through homologous recombination with an ASFV p72 promote r-beta-glucuronidase indicator cassette (p72GUS) flanked by ASFV seque nces targeting the TK region. Attempts to isolate double-crossover TK gene deletion mutants on swine macrophages failed, suggesting a growth deficiency of TK- ASFV on macrophages. Two pathogenic ASFV isolates, ASFV Malawi and ASFV Haiti, partially adapted to Vero cells, were used successfully to construct TK deletion viruses on Vero cells. The sele cted viruses grew well on Vero cells, but both mutants exhibited a gro wth defect on swine macrophages at low multiplicities of infection (MO I), yielding 0.1 to 1.0% of wild-type levels. At high MOI, the macroph age growth defect was not apparent. The Malawi TK deletion mutant, sho wed reduced virulence for swine, producing transient fevers, lower vir emia titers, and reduced mortality. In contrast, 100% mortality was ob served for swine inoculated with the TK+ revertant virus. Swine surviv ing TK- ASFV infection remained free of clinical signs of African swin e fever following subsequent challenge with the parental pathogenic AS FV. The data indicate that the TK gene of ASFV is important for growth in swine macrophages in vitro and is a virus virulence factor in swin e.