REPLICATION IN-VITRO AND IN-VIVO OF AN EQUINE INFECTIOUS-ANEMIA VIRUSMUTANT DEFICIENT IN DUTPASE ACTIVITY

As an important enzyme in DNA synthesis, dUTPase is present in a wide variety of organisms anal viruses and has been identified as a compone nt of the equine infectious anemia virus (EIAV) pol gene. The role of EIAV dUTPase, designated DU, in virus replication in vitro and in vivo was investigated with a recently described infectious molecular clone of EIAV. A deletion mutant that was deficient in dUTPase activity was constructed, and its replication kinetics was examined in fetal equin e kidney (FEK) cells and primary equine bone marrow macrophage (EBMM) cells. In FEK cells, which are permissive for EIAV replication, the mu tant virus replicated as well as the parental virus. In primary cultur es of EBMM cells, which are primary targets of EIAV infection in vivo, the DU mutant showed delayed replication kinetics and replicated to a lower extent than did the parental virus. As the multiplicity of infe ction decreased, the difference bet ween the parental and mutant virus es increased, such that at the lowest multiplicity of infection tested , there was over a 100-fold difference in virus production. The mutant virus was also much less cytopathic. The role of DU in replication in vivo was examined using a Shetland pony model of EIAV infection. Shet land ponies that were infected with the parental and mutant viruses sh owed transient virus RNA levels in plasma approximately 5 to 10 days p ostinfection. The peak virus levels in plasma (as measured by a quanti tative reverse transcriptase PCR assay) were 10- to 100-fold lower in the mutant virus-infected animals than in the animals infected with th e parental virus. However, ponies infected with the mutant virus mount ed similar antibody responses despite the marked differences in virus replication. These studies demonstrate that EIAV DU is important for t he efficient replication of the virus in macrophages in vitro and in v ivo and suggests that variations in the DU sequence could markedly aff ect the biological and pathogenic properties of EIAV.