Construction Of Four Kinds Of Structural Protein Gene Inactivated Mutant Of Pseudorabies Virus Variant And Primary Study Of Their Biological Analysis

Pseudorabies virus(PRV)can infect pigs and cause an acute infectious disease characterized by high fever,respiratory disorders,and encephalomyelitis,namely,Pseudorabies(PR).Naturally,PRV infects pigs by mouth or nasal cavity,and enters the central nervous system along the head nerve after replicating in the oropharyngeal cells;or establishes a latent infection in the trigeminal ganglia.Latent infection does not produce infectious virions,and only a small number of sections of PRV genomes are transcribed.When the animal's body suffers from stress or other diseases that result in decreased immunity,the virus is activated,proliferates actively,and egresses again.Since 2011,a new type of PRV variant strain has been outbreak in pig farms across China,its virulence and spread are significantly stronger than traditional strains,and even there are reports of human infected of it.Although antibodies produced by humoral immunity can neutralize extracellular viruses,viruses that infect cells rely on cellular immunity to eliminate them.Major histocompatibility complex-I(MHC-I)can be expressed in all nucleated cells,and it can present digested endogenous antigen peptides to the surface of infected cells for CD8+T Cells recognizing and activate Cytotoxic T lymphocytes(CTLs),exerting immune effects.Studies have shown that US3,UL49.5,and UL56 encoding products of PRV can all down-regulate the expression of MHC-I on the surface of infected cells;the homologous coding product of UL43 of PRV in Equine Herpesvirus-I(EHV-1)can inhibit the expression of MHC-I in infected cells.In order to explore the effect and mechanism of the products encoded by US3,UL43,UL49.5,and UL56 of PRV variant strains on the anti-cell immunity,this study used the Bacterial artificial chromosome(BAC)technology of PRV variant strain AH02LA strain,followed by two steps of Red recombination method to construct gene mutation inactivated strain,and the biological characteristics of the mutant-inactivated strains were analyzed.The effect of the products encoded by US3,UL43,UL49.5,and UL56 on the replication and proliferation of PRV in ST cells and the pathogenicity of PRV in mice were studied.This study will lay a foundation for further revealing the role of the above genes in the immune mechanism of PRV escape;also for finding new targets for the deletion of gene sites for construction of the PRV vaccine.1 Construction of gene mutation inactivated strainIn order to study the effect and mechanism of the products encoded by US3,UL43,UL49.5,and UL56 on PRV anti-cellular immunity,relying on the Bacterial artificial chromosome(BAC)technology,The point mutations of the start codons of UL43,UL49.5 and UL56 of BACPRV-G were inactivated,and 4 gene mutation recombinant HAC strains were constructed:BACPRV-G-US3m0t,BACPRV-G-UL43mut,BACPRV-G-UL49.5mut,BACPRV-G-UL56mut.Then,each gene mutation recombinant BAC strain and BACPRV-G were co-transfected with gE/gI gene sequences to ST cells to rescue the virus and replace the mini-F sequence in BAC to obtain recombinant viruses:PRV-US3mut,PRV-UL43mut,PRV-UL49.5mut,PRV-UL56mut and PRVrec.2 Biological characteristics of mutant inactivated strainsTo investigate the effects of US3,UL43,UL49.5,and UL56 inactivation on PRV proliferation in vitro,growth kinetics of the parental virus PRV AH02LA and PRV-US3mut,PRV-UL43mut,PRV-UL49.5mut,PRV-UL56mut,and PRVrec were tested on ST cells and growth curves were draw.The results showed that the products encoded by UL43 and UL56 had no effect on the proliferation of PRV;the products encoded by US3 and UL49.5 participated in the early proliferation stage after PRV infection of the cells.After inactivation of US3 and UL49.5,the early proliferation of the virus is slowed down,and US3 inactivation has no significant effect on the maximum toxic value achieved by the virus.The highest toxic value of PRV-UL49.5mut is 106.3TCID20/mL,compared with 107.63 TCID50/mL,the highest toxic value of the parental virus PRV AH02LA,indicating that the product encoded by UL49.5 participated in the replication and proliferation of PRV.The proliferative ability of the parental recovery virus PRVrec is basically the same as that of the parental virus,indicating that the construction of PRV into BAC will not have an irreversible effect on the growth performance of the virus in vitro.Genetic stability test results showed that PRV-US3mut,PRV-UL43mut,PRV-UL49.5mut,and PRV-UL56mut were continuously passaged in ST cells for 15 generations,and the start codons of their respective mutant genes remained in point mutation status,indicating 4 genetically inactivated strains have good genetic stability and is suitable for continuous passage on ST cells.The pathogenicity test results on mice showed that after diluting each gene mutation inactivated strainand the parental virus and the parental recovery virus from 10-2 to 10-5,the mice were challenged with 0.2mL.All PRV-US3mut challenged mice survived;the LD50 of PRV-UL43mut,PRV-UL49.5mut,and PRV-UL56mut on mice also significantly decreased compared with the parental virus PRV AH02LA.It shows that the products encoded by US3,UL43,UL49.5 and UL56 are all involved in the pathogenic effect of PRV in mice.The LD50 of PRVrec on mice had no significant difference between the parent strain,indicating that the construction of PRV into BAC will not have an irreversible effect on the pathogenicity of the virus in mice.