Study On Molecular Bases Of Antigenic Variation Of Pseudorabies Virus

Pseudorabies virus(PRV)is the causative agent of Pseudorabies(PR).The disease is lethal to young piglets,and characterized by growth retardation and reproductive disorder.In the past decades,Bartha-K61 vaccine was widely used in Chinese pig farms to control and eradicate PR and showed complete protection,and there had been no report about the resistance to the vaccine.However,since late 2011,a severe PR outbroke in many Bartha-K61 vaccinated pig herds,and causes significant economic losses.It has been confirmed that a newly emerged PRV variant is the causative agent and the commercial PRV vaccine Bartha-K61 provided poor protection against PRV variants.Phylogenetic analysis based on complete genomic sequence revealed that PRV-variant strains isolated in China,such as HeN1 or JS-2012,displayed marked sequence divergence compared to European and American strains.In view of the role glycoprotein B(gB)plays in stimulating strong humoral immune responses and the genetic difference between Bartha-K61 and JS-2012,we are interested in the relationships between gB variation and the failure of Bartha-K61 vaccine to provide complete protection against PRV variants.In the present study,we generated gB-interchanged recombinant viruses based on JS-2012-?gE/g I and Bartha-K61 to identify the role of gB in antigenic variation and provide a novel PRV vaccine strain development strategy by ultilizing CRISPR/Cas9.Bioinformatic analysis suggested that the main antigen of PRV variant might occured antigenic variation.gB sequence analysis indicated that there was marked diversity between Chinese and foreign typical PRV strains.This was consistent with previous phylogenetic analyses based on the whole genome,which revealed that Chinese PRV strains,particularly outbreak-associated strains,are evolutionarily divergent compared with European–American strains.And,we showed that multiple amino acid mutations and deletions were precisely located in the identified epitope region.Utilizing specific CRISPR/Cas9 system combining with homologous recombination,we constructed two recombinant viruses BJB(PRV Bartha-K61 with JS-2012gB)and JBJ(PRV JS-2012-?gE/g I with Bartha-K61gB)by interchanging the full-length gB gene between Bartha-K61 and JS-2012-?gE/g I.The two recombinant viruses showed similar characteristics in growth kinetics in vitro and similar pathogenicity in mice to their parental strains.It provides the platform for the followed investigation on antigenic difference of gB variant.GB-substitution between Bartha-K61 and JS-2012-?gE/g I remarkably altered the immunogenicity of inactivated-Bartha-K61 and inactivated-JS-2012-?gE/gI according to the results from immunization and PRV variant JS-2012 challenge assays in mice.The protective efficacy of BJB was significantly higher than Bartha-K61,and JBJ provided poor protection compared with JS-2012-?gE/gI.It suggested that the variations in gB significantly induced antigenic and immunogenic variation between Bartha-K61 and JS-2012-?gE/gI,and revealed that gB variation contributes to the phenomenon that Bartha-K61 failed to protect piglets from the lethal challenge of PRV variant.Additionly,Bioinformatic analysis revealed that there was multiple sequence difference between PRV variant and classicial PRV strains in glycoprotein C(gC).We constructed recombinant viruses based on Bartha-K61 and JS-2012-?gE/gI by utilizing specific CRISPR/Cas9 system combining with homologous recombination and designated as BJbcB(Bartha-K61 with JS-2012 gB and JS-2012 gC),Bar-Jc(Bartha-K61 with JS-2012-g C)and JBcJ(JS-2012-?gE/gI with Bartha-K61gC)respectively.It provided an excellent platform for the study on antigenic difference of gC variant.Overall,variations in glycoprotein B contribute to immunogenic difference between PRV variant JS-2012 and Bartha-K61.The results obtained from the present study have made significant contribution to better understanding the importance of gB in the biology of PRV and also provide a promising vaccine candidate by an efficient vaccine development strategy for the PR eradication worldwide.