A Recombineering Platform Of Construction And Modification Of Infectious Clone Of Pseudorabies Virus

Pseudorabies virus(PRV)is the causative agent of pseudorabies or Aujezsky’s disease,which is widely spread worldwide and has caused great economic losses to the pig industry.Infection with PRV causes neurological disorders,respiratory distress,weight loss,death of piglets,and abortion.Although efforts to eradicate PRV in the United States and Europe have shown remarkable improvement,it remains an unresolved concern in several countries.Pseudorabies virus belongs to the family Herpesviridae and has a linear double-stranded DNA molecule with a long unique region(UL),a short unique region(US),a terminal repeat sequence(TRS),and an internal repeat sequence(IRS).Pseudorabies virus has become one of the biological models for the investigation of herpesvirus biology.Since the creation of the first herpesvirus bacterial artificial chromosome(BAC)recombinant mouse cytomegalovirus(MCMV),mutagenesis using the BAC technique has proven to be a valuable tool for studying herpesvirus pathogenesis and other biological properties.However,the use of traditional homologous recombination methods is not only time-consuming but also prone to accumulate genetic mutations during successive virus transmissions.Additional steps are required to eliminate the BAC vector in order to make the recombinant virus phenotype consistent with the wild virus.In this study,we introduced the ExoCET method to the construction of herpesvirus recombinant vectors using pseudorabies virus as an example.Placement of the BAC vector at the terminus of the linear virus genome can be excised from the viral genome by restriction nuclease for delivery into mammalian cells,followed by rapid rescue of the virus genetically identical to the original viral strain.The use of Redap recombination technology combined with site-specific recombination system to modify the viral vector genome opens up a new platform for extremely simplified herpesvirus vector construction and genome modification.Construction of PRV infectious clone(pBeloBACll-cm-DCDl)and deletion of gEgI,TK virus vectorThe PRV DCD-1 viral genomic DNA was cloned directly into the pBeloBAC11cm vector by using the ExoCET method,and two PmeI restriction sites were incorporated into oligonucleotides that were used to linearize the recombinant viral vector by binding to sequences on both ends of the viral genomic DNA during gene recombination.The correctness of the infectious clone of pBeloBAC11-cm-DCD1 was verified by restriction enzyme digestion and sequencing.The linearized viral vector pBeloBAC11-cm-DCD1 was transfected into Vero cells and cytopathic effects were observed 2 to 4 days after transfection,indicating successful rescue of the obtained recombinant virus.The growth kinetic curves revealed that the recombinant viruses converged with those of wild virus DCD-1.To reduce virulence,a recombinant viral vector pBeloBAC1l-cm-DCD1-ΔgEgIΔTK with deletion of virulence genes gEgI and TK was constructed by Redαβ-mediated recombination binding site-specific recombination of linear loop recombination.gEgI fragment was replaced with Flp-specific sites at both ends by reference to the deletion sequence of the corresponding gene in Bartha-K61 kan resistance gene with FLPspecific sites at both ends,and the TK fragment was replaced by the lox66-genta-lox77 gene cassette.The obtained monoclones were double delineated on LB plates with different resistance and the excess resistance genes were eliminated by expression of Flp and Cre site-specific recombinase to obtain the attenuated viral recombinant vector pBeloBAC11-cm-DCD1-ΔgEgI-ΔTK.The recombinant vector was transfected into Vero cells and cytopathic effects were observed in 2-3 days.The titer measurements and growth curves of the rescued recombinant viruses at different time points showed no significant changes in growth kinetics compared to wild virus,demonstrating that Redαβ-mediated linear loop recombination and site-specific recombination of genes on deletion PRV viral sequences were feasible,that TK and gEgI genes were successfully deleted without impacting other sequences of the viral genome,and that the viral The replication kinetic profile was not significantly altered.The correctness of the recombinant viral vector was confirmed by multiple restriction enzyme digestion and gene sequencing.In vitro stability of the recombinant virus was confirmed by PCR amplification with primers specific for both ends of the substitution/insertion region and sequencing after 15 generations of virus transmission.Using fluorescent protein(FP)to identify expression sites on recombinant viral vectorsIn order to establish a stable platform for exogenous gene expression on PRV recombinant vector,we expressed the genes of fluorescent proteins mNeonGreen,mTaqBFP and mCherry after the endogenous promoters of genes TK,gG and gI of PRV DCD-1,respectively.After vector expression and successful rescue,the effect of exogenous gene expression could be judged by live cell imaging data.Three independent fluorescent virus clones were constructed at three positions(TK,gG,gI),named pBeloBAC11-cm-DCD 1-ΔTK-mNeonGreen,pBeloBAC11-cm-DCD1-ΔgGmTaqBFP,and pBeloBAC11-cm-DCD1-ΔgI-mCherry.These three independent clones were subsequently transfected into Vero cells to rescue the recombinant virus.Experiments showed that all three clones produced phospho spots on PK-15 cells,and the correct fluorescent signal was observed in confocal analysis.To further investigate the differences in promoter expression properties in the PRV clones,we inserted the exogenous promoters CMV and CAG at the TK gene position and added the mNeonGreen fluorescent protein gene after the promoter,respectively.Upon viral rescue,confocal analysis showed that both promoters could fluoresce efficiently,indicating that at the TK position,the CMV and CAG promoters could efficiently initiate the expression of the exogenous genes.Verification of exogenous gene expression in recombinant virusesThe IL18 and IFN-γ immune factor was inserted at the gG gene position in order to enhance the immune response of the host during vaccine immunization.To analyze expression of IL18 and γ in gG locus,PK-15 cells were infected with the recombinant PRV DCD-1-ΔgG-IL18-γ or parental PRV DCD-1 and harvested after 12 h post infection.RNA in cell lysate were analyzed by qRT-PCR analysis.The qRT-PCR showed that IL18 and γ attained both high levels of transcription.For TK locus,PRRSV antigen genes GP3,GP5 and M at the TK region were designed to insert into TK region to make a PRV-PRRSV diphasic vaccine.To initiate the expression of the antigen genes,we adopted the endogenous promoter and two exogenous promoters,CMV and CAG,to express the antigen genes.By qRT-PCR analysis,PRRSV antigen genes were able to be transcribed at a high level with a slightly higher intensity of the CAG promoter than the other two,as shown in Fig.4C,which was consistent with the fluorescent gene expression pattern.Protein immunobinding analysis,using HA-tag and Flag-tag antibodies,of infected cell protein harvested at 12 h post infection(hpi)showed visible accumulation of inserted antigenic proteins y and M at the gG and TK locus respectively.