Construction Of UL3,UL46 And UL50 Mutants And Truncated UL43 Mutants Of Pseudorabies Virus And Primary Identifiation Of Biological Analysis

Pseudorabies virus（PRV）can infect many mammals except primates.Pigs are natural hosts of PRV which can evade the immune system of pigs and establish latent infection.PRV can lead to high mortality in piglets,respiratory and neurological symptoms in adult pigs and reproductive dysfunction in sows.In 2011,the emergence of PRV mutants caused huge losses to pig farms in China.The control of the novel PRV mutants has become an important problem to be solved in recent years.Many papers have shown that several proteins encoded by PRV can play important roles for immune suppression,including interference of Major histocompatibility complex-I（MHC-I）molecule presentation,interferon pathway,complement regulation pathway and so on.UL43 can suppress MHC-I presentation in Equine Herpesvirus I homologues.The intracellular region is suspected to be crucial signal transduction regions of p UL43,which may play a role in protein signal transduction.The function of p UL3 of PRV has not been reported till now;p UL46 of PRV can induce nuclear membrane rupture and accelerate the invasion and packaging process of virus particles;p UL50 of PRV,as a pyrophosphatase of PRV,can also antagonize the reaction ofα-interferon.In this study,based on the Bacterial artificial chromosome（BAC）of a virulent PRV isolates AH02LA strain,mutants of intracellular region truncated UL43mutants and deficient UL3,UL46 and UL50 mutants were generated by Red two-step recombination.And biological characteristics of these mutants were determined for future study on the role of these proteins and selection of novel gene deletion target of PRV vaccine development.1 Construction of UL3,UL46 and UL50 deficient PRV mutants and biological characteristics analysisBased on the Bacterial artificial chromosome（BAC）of a virulent PRV isolates AH02LA strain,deficient mutants of UL3,UL46 and UL50 of PRV were generated by deletion of start codon through Red two-step recombination,nominated BAC^PRV-UL3knock,BAC^{PRV-UL46knock},BAC^{PRV-UL50knock}.Recombinant virus were rescued by transfection of BAC mutants on ST cells and isolation with several round of plaque picking to obtain purified virus named PRV-UL3^knock,PRV-UL46^knock and PRV-UL50^knock.The growth kinetics,passage stability and pathogenicity of PRV-UL3^knock,PRV-UL46^knock,PRV-UL50^knock and PRV AH02LA parental strains were determined.Growth kinetics results showed that virus titers of PRV-UL3^knock,PRV-UL46^knock and PRV-UL50^knockreached the peak at 48～72hpi,which were 10^6.8TCID₅₀/m L,10^6.7TCID₅₀/m L,10^7.1TCID₅₀/m L respectively.The titers were slightly lower than that of PRV AH02LA parental strain(10^7.5TCID50/m L),but there was no siginificant difference.The results showed that p UL3,p UL46,p UL50 have no significant effect on virus replication in vitro.Progeny virus was not detected for PRV-UL46^knocktill 12 hours post infection,and the titer of PRV-UL46^knock at 24hpi(10^3.625TCID₅₀/m L)was significantly lower than that of PRV AH02LA parental strain(10^6.4TCID50/m L),indicating p UL46 may play an important role on early stage of replication of PRV.Stability was proved by serial passageing on ST cells for these mutants by sequencing,showing no recovering or mutation in the deletions sites.Animal tests show that LD₅₀ of PRV-UL3^knock,PRV-UL46^knock,PRV-UL50^knock were 10^4.44TCID₅₀,10^4.5TCID₅₀ and 10^4.05TCID₅₀,which were similar to PRV AH02LA parental strain(LD₅₀ was 10^4.91TCID₅₀),demostrating that p UL3,p UL46,p UL50 have no weakening effect on PRV pathogenicity to mice.2 Generation of intracellular region truncated UL43 mutants and biological characteristics analysisBased on the Bacterial artificial chromosome（BAC）of a virulent PRV isolates AH02LA strain,three mutants were constructed by deletion of UL43 intracellular region nucleotides of 286～291bp、571～585bp、850～858bp respectively.The resulting recombinant BAC were nominated as BAC^{PRV-UL43Δ1knock},BAC^{PRV-UL43Δ2knock} and BAC^{PRV-UL43Δ3knock}.Recombinant virus were rescued by transfection of BAC mutants on ST cells and isolation with several round of plaque picking to obtain purified virus named PRV-UL43^Δ1knock,PRV-UL43^Δ2knock,PRV-UL43^Δ3knock.The growth kinetics,passage stability and pathogenicity of PRV-UL43^Δ1knock,PRV-UL43^Δ2knock,PRV-UL43^Δ3knock and PRV AH02LA parental strains were determined.Growth kinetics results showed that at 12hpi,the titers of PRV-UL43^Δ1knock,PRV-UL43^Δ2knock,PRV-UL43^Δ3knock were 10^2.4TCID₅₀/m L,10^2.6TCID₅₀/m L,10^2.5 TCID₅₀/m L respectively,lower than that of PRV AH02LA parental strain(10^3.0TCID₅₀/m L).At 36hpi,the titers of three truncated UL43 strains were 10^6.5TCID₅₀/m L,10^6.0TCID₅₀/m L,10^6.48TCID₅₀/m L respectively,which were aslo lower than PRV AH02LA parental strain(10^7.3TCID₅₀/m L),demostrating that the intracellular region of UL43 is involved in the regulation of early and middle viral replication.The titers of three truncated strains reached the peak at 48～72hpi,which were 10^7.86TCID₅₀/m L,10^7.3TCID₅₀/m L,10^7.15TCID₅₀/m L respectively,similar to PRV AH02LA parental strain(10^7.5TCID₅₀/m L),showing that the intracellular region of UL43 has no significant effect on virus replication in vitro at this time point.These three truncated mutants were stable when were passaged on ST cells for10 times,no mutant happened in the deletion sites.Animal tests showed that LD₅₀ of PRV-UL43^Δ1knock,PRV-UL43^Δ2knock,PRV-UL43^Δ3knock were 10^3.83TCID₅₀,10^4.97TCID₅₀,10^4.73TCID₅₀ respectively,similar to that of PRV AH02LA parental strain(LD₅₀ was10^4.91TCID₅₀).indicating that the checked intracellualar regions of UL43 may have no weakening effect on PRV pathogenicity to mice.Through the study of UL3,UL46,UL50 mutants and truncated UL43 mutants,we can provide reference and theoretical basis for the development of new PRV vaccine.