Cloning of equine herpesvirus type 1 438/77 strain genome as a bacterial artificial chromosome

Equine herpesvirus type 1 (EHV-1) is a major cause of respiratory and reproductive diseases in horses worldwide. The genome of EHV-1 strain 438/77 (isolated from an aborted equine fetus) was cloned as a bacterial artificial chromosome (BAC) in E. coli without any gene deletions. The mini-F plasmid sequence was inserted in the middle of ORF19 and 20 via homologous recombination following co-transfection of viral DNA and plasmid pE19_20/HA into RK13 cells. Circular viral DNA was extracted from RK13 cells infected with purified recombinant virus expressing green fluorescent protein (GFP) and electrophorated into E. coli DH10B cells. The clone harboring the BAC was screened and analyzed by PCR and RFLP. Reconstitution of the recombinant virus was achieved successfully by transfection of the BAC DNA into RK13 cells. The mini-F sequence in the reconstituted virus was subsequently removed by homologous recombination between virus DNA and plasmid pE1920XM, inducing a point mutation in the Xbal site in ORF19. Comparison of RFLP profiles of the rescued, recovered and the wild-type viral genome demonstrated that no unexpected changes occurred during mutagenesis. In vitro replication assays showed that BAC-reconstituted virus mutant growth kinetics and plaque formation morphology/size were indistinguishable to those measured for wild-type virus.