Study On Immunogenicity Differences Of The Major Glycoproteins Between The Variant Strain And The Vaccine Strain Of Pseudorabies Virus

Pseudorabies(PR),also known as Aujeszky’s disease(AD),is an acute infectious disease caused by Pseudorabies virus(PRV),which is characterized by breeding disorders in breeding pigs and massive death of newborn piglets.In order to control the domestic PR epidemic,China introduced the gene-deletion vaccine Bartha-K61 from Hungary in 1979,and PR was well controlled.However,since the end of 2011,many pig farms immunized with the traditional vaccine Bartha-K61 in China experienced frequent PR outbreaks,which caused huge economic losses to the pig industry.Studies showed that it were variant strains of PRV caused PR outbreaks in pig farms,and the variant PRV showed stronger virulence and pathogenicity than the classical PRV.A systematic analysis of the genetic evolution of newly isolated PRV variant strains in China showed that the domestic PRV variant and the foreign PRV variant are in two branches of the genetic evolution,indicating that PRV may have two genotypes.Therefore,we hypothesized that the main protective antigen gene of the PRV variant strain may have mutations compared with the vaccine strain,resulting in the vaccine strain being unable to provide effectively protection to the pigs from the PRV variant strain infection.We conducted a bioinformatics analysis on the PRV variant JS-2012 strain isolated in our laboratory,and the results showed that,compared with the vaccine strain Bartha-K61,the main protective antigens g B,g C and g D amino acids sequence of the PRV variant JS-2012 strain had different degrees of variation.In order to explore the relationship between the variation of protective antigens g B,g C and g D and the phenomenon of Bartha-K61 immunity failure,we did the following work.1.Construction of the gene-substituted recombinant PRVs.Using the technology of CRISPR/Cas9 combined with HDR,g B-substituted recombinant strains BJB and JBJ,g C-substituted recombinant strains Bar-JSg C and JS-Barg C,g D-substituted recombinant strains Bar-JSg D and JS-Barg D,g B/g C/g D triple-substituted recombinant strains Bar-JSg Bg Cg D and JS-Barg Bg Cg D were successfully constructed based on the backbone strains Bartha-K61 or JS-2012-Δg E/g I,respectively.2.In vitro biological characteristics of the recombinant strains.The biological characteristics of the recombinant strains and its parent strains are similar in vitro.Among them,the replication ability of JS-Barg C on PK-15 cell line was significantly reduced compared with the parent strain JS-2012-Δ g E/g I.3.Cross-immunoprotection test in mice.The recombinant strains and it’s parent strains Bartha-K61 and JS-2012-Δg E/g I were prepared into the inactivated vaccines,respectively.The mice were immunized with the vaccines respectively and challenged with the viarant strain JS-2012.The results showed that the g B-substituted strains significantly changed the immunoprotective effect of Bartha-K61 or JS-2012-Δ g E/g I from JS-2012 attacks.The cross-immunoprotection effect of g C or g D-substituted recombinant strains from JS-2012 attack was not significant.However,the results of q PCR showed that the variations of g C and g D influenced the immunogenicity of Bartha-K61 to some extent.To sum up,in this study we used the technology of CRISPR/Cas9 combined with HDR,successfully constructed the g B,g C or g D single gene-substituted recombinant strains and the triple gene-substituted recombinant strains,respectively.The results of cross-immunoprotection test in mice showed that variations in g B contribute to immunogenic differences between PRV variant JS-2012 and Bartha-K61,which suggested that the poor-immunoprotection of the vaccine strain against variant strain JS-2012 was closely related to g B variations.Furthermore,the variations in g C and g D changed the immunogenicity of Bartha-K61 to some extent.This study provided a theoretical basis for revealing the variations between PRV strains and the immune escape mechanism.