Construction Of An Infectious Clone Of Pseudorabies Virus Strain ZJ Genome Maintained As A Bacterial Artificial Chromosome

Pseudorabies virus(PRV),or"Aujeszky"s virus, is a member of alpha herpesvirinae, Herpesviridae,which can cause pseudorabies disease in swine,bovine,sheep and wild animals. Pseudorabies is an acute infection diease and characterized by high fever, extreme itching (except swine) and encephalomyelitis.Especially, porcine pseudorabies has become one of the most economically important diseases in worldwide swine industry. PRV genome is a double-stranded DNA of approximately 143 kb and encodes 70-100 proteins.Like other alpha herpesvirus, PRV has some typical chatacteristics of alpha herpesvirus such as latent infection and neurotropism.The main method studying on the fuctional genes of herpesvirus and pathogenesis was to construct genetic-deleting mutants in the viral genome. It's difficult to obtain the genetic-deleting mutants in the traditional method of homologous recombination mutants, which can not delet the essential gene. So that infectious bacterial artificial chromosome (BAC) clone of pseudorabies virus (PRV) strain ZJ, which can carry out the modification of the virus genome in E.coli and improve efficiency of recombinantion was constructed to serve as foundation for further study on PRV genes functions and viral vector.In this study, a transfer plasmid pUL22-gfp-24-HA2, consisted of pHA2 (including the GFP tag and BAC-vector) flanked by two homologous arms of PRV strain ZJ UL22 and UL24 gene segments, was constructed. The recombinant virus (PRV-ZJ-HA2) was observed in fluorescence microscope and then isolated by plaque-forming assay after co-transfection of transfer plasmid and PRV genome into Vero cell. The circular genome of the recombinant virus was electroporated into E.coli DH10B and a PRV BAC clone was screened by Restriction Fragment Length Polymorphism (RLFP). The recombinant virus (recPRV-ZJ) were reconstituted after PRV BAC transfected into Vero cells.The reproduction feature of PRV, PRV-ZJ-HA2 and recPRV was investigated by a comparison of relative plaque size, growth curve and animal experiment, the results showed that the reproduction rate but not viral titers of PRV-HA2 was indistinguishable to that of parental virus PRV. It indicated that the 8.8kb-HA2 sequnce inserted in the TK gene didn't influence the viral reproduction rate, but deletion of TK gene affects the activation state of virus infection. The reproduction rate and viral titers of recPRV was indistinguishable to that of PRV-ZJ-HA2, the results showed that PRV-ZJ-HA2 can be stable replicated either in E.coli (eukaryotic) or Vero cell (prokaryotic). The PRV BAC system will be allow for quick and reliable manipulation of the viral genome in the functional research of the genes and in the development of PRV vector in vaccine.