Construction And Application Of A Full-Length Pseudorabies Virus Variant Strain JS-2012 Genome As An Infectious Bacterial Artificial Chromosome

Pseudorabies(PR),caused by pseudorabies virus(PRV),is an acute infectious disease characterized by high fever,depression,respiratory distress,anorexia,cough,shivering,diarrhea,and systemic neurological symptoms.PR was well cotrolled in China by vaccination.Since the late 2011,however,outbreaks of PR occurred in many vaccinated pig farms in China and caused great economic losses to the swineindustry.Several research groups have confirmed that the outbreak of PR resulted from Pseudorabies virus variant.Compared with classical PRV,the variant virus appered mutations in many genes,and it virulence is obviously enhanced.In this study,a PRV variant strain JS-2012 was used as the research object,and the operation platform of PRV infectious bacteria artificial chromosome(BAC)was successfully constructed.1.Construction of recombinant PRV containing BAC vectorAn EGFP expression cassette flanked by a loxp site in both sides was inserted into the down stream of gG coding region of a pseudorabies virus variant strain PRV JS-2012 by homologous recombination,generating a recombinant virus rJS2012-gG/EGFP.After transfected with the pcDNA3.1-cre which expresses Cre recombinase,BHK-21 cells were infected with rJS2012-gG/EGFP.A recombinant virus rJS2012-gG/loxp carrying only one loxp site downstream of gG coding region was obtained.Then,the genome of rJS2012-gG/loxp was co-transfected with pcDNA3.1-cre and pBeloBAC11-EGFP into BHK-21 cells,generating a recombinant virus rJS2012-BAC.rJS2012-BAC carried the BAC vector sequences in its genome and grew slightly slower than its parent strain JS-2012 in vitro.2.Obtaining of PRV infectious BAC clones and the growth properties studying of rescue virusThe circular genome of recombinant virus rJS2012-BAC was extracted by Hirt method and transformed into E.coli DH10 B.The BAC clone pJS-2012 BAC containing the whole genome of PRV was screened out by PCR and RFLP analysis.The rescue virus was obtained after the pJS-2012 BAC was transfected into Vero cells.Vero cells were co-transfected with pJS-2012 BAC and pcDNA3.1-cre to generate the rescue virus vPRVJS-2012,in which the BAC vetor was removed.The viriological properties of parental virus PRV JS-2012,the rescued virus vPRVJS-2012 BAC and vPRVJS-2012 were analyzed by the plaque experiment and one step growth curve test in vitro,and the mouse pathogenicity experiment in vivo.The results showed that vPRVJS-2012 BAC had a slower rate of replication in vitro compared with parental virus,whereas the growth features of vPRVJS-2012 was virtually identical to that of parental PRV JS-2012 in Vero cells.Furthemore,the plaque areas of the vPRVJS-2012 were similar to that formed by parental virus in Vero cells and vPRVJS-2012 had the same pathogenicity to mice as JS-2012.It was indicated that vPRVJS-2012 have the same biological characteristics as parental viruses in vivo and in vitro.3.Establishment and preliminary application of PRV infection BAC clone operation platformIn this study,pJS-2012 BAC was transformed into E.coli strain SW102,and the positive clones SW102-PRVBAC containing pJS-2012 BAC were screened by PCR and RFLP analysis.SW102-PRVBAC was transformed with a DNA segment containing the galk gene flanked by specific 50-bp PRV sequences,generating the positive clone SW102-galk.The partial gE/gI was replaced by galk gene in SW102-galk.A gE/gI-deleted transfer vector was constructed and transformed into SW102-galk.A galk-negative clone,SW102-DgE/gI was screened out by galk negative screening and was identified by PCR and RFLP analysis.The plasmid pPRV-DgE/gI BAC was purified from SW102-DgE/gI and transfected into Vero cells,generating the virus vPRV-DgE/gI BAC.The virus vPRV-DgE/gI,in which the BAC vector was removed,also rescued from Vero cells by co-transfection with pPRV-DgE/gI BAC and pcDNA3.1-cre.Sequence analysis showed that gI and gE gene were partly deleted in vPRV-DgE/gI.The result of IFA displayed gE proteins were absent in Vero cells infected with vPRV-DgE/gI.The plaque areas of the vPRV-DgE/gI were significantly smaller than that formed by parental virus in Vero cells.One step growth curve experiments showed that vPRV-DgE/gI grew slightly slower than its parent strain which mean the intercellular diffusion capacityof vPRV-DgE/gI decreased.In conclusion,we constructed a full-length infectious clone pJS-2012 BAC of PRV variant JS-2012 by using cre/loxp syetem.Virus derived from the pJS-2012 BAC clone had the same biological characteristics as parental viruse in vivo and in vitro.PRV Infectious Bacterial Artificial Chromosome Operation Platform was successfully constructed by using pJS-2012 BAC and galk positive and negative screening system.This platform makes it possible to carry out efficiently and accurately genetic operations,such as deletion,substitution and mutation,for any gene of PRV.The platform will be a powerful tool to study the mechanism of enhanced virulence,antigenic variation,latent infection and immune escape mechanism of PRV vatiant.