Study On Genetic Variation Of Pseudorabies Virus And Function Analysis Of UL41 And UL24 Genes

Pseudorabies(PR),also known as Aujeszky's disease,is an acute disease characterized by reproductive disorders in sows and respiratory and neurological symptoms in piglets caused by pseudorabies virus(PRV).For a long time China's pig farms have adopted attenuated vaccines to prevent the disease,and the disease has also been effectively controlled.However,since 2011,a large number of pig farms vaccinated with Bartha-K61 vaccine in China have undergone PRV variant infection,resulting in increased sow abortion and piglet mortality,which has caused huge economic losses to pig industry.However,the genetic differences between the circulating strains and classical strains and then the characteristics and functions of virulence proteins of the PRV variant are still unclear.In this study,we performed genome-wide sequencing of a domestic classical PRV strain SC and a variant HLJ8,and obtained their complete genome sequence.Further,we obtained the genome sequences and gC gene sequences deposited in GenBank.Through genome comparison and phylogenetic analysis,we demonstrated that PRV strains from different geographical regions showed different evolutionary trends and in particular,the early strains and the current variant strains in China constituted new genotypes.In addition,through recombination and phylogenetic analysis,we first reported a recombination event that occurred between PRV strains and showed that SC is a recombinant strain of Chinese early strain and Bartha-vaccine-like strain.UL41 is a conserved protein of the ? herpesviruses.Studies on HSV-1 have shown that it has RNase activity and is an important regulatory protein.To study the function of PRV UL41 in the pathogenesis of the virus,homologous recombination technology was used to link the UL41 upstream and downstream sequences and the eGFP to a linearized pBluescript vector to generate a donor plasmid.Then the plasmid was co-transfected into Vero cells together with the PRV JS-2012 genome.The ?UL41 eGFP PRV recombinant virus was obtained;then donor plasmid only containing UL41 upstream and downstream sequences and another plasmid containing the UL41 ORF and an inframe HA tag sequence were simultaneously constructed,which respectively along with a CRISPR/Cas9 plasmid that cuts the eGFP sequence were cotransfected with the ?UL41 eGFP PRV genome into Vero.By screening via PCR obtained UL41 null and revertant PRV.UL41 null PRV produced fewer plaque sizes and lower virus titers in Vero cells than JS-2012,indicating that the replication and diffusion capacity of the mutant virus decreased;however,the titers on PK-15 and MDBK were comparable between strains,indicating that deletion of UL41 impaired replication in specific cells.Furthermore,the lack of UL41 reduced the lethality and neuroinvasion of the virus in mice.Luciferase reporter assay showed that UL41 vaccination activity of SC strain and JS-2012 strain was different,and UL41 of SC strain had more significant vhs activity.Further qRT-PCR showed that regardless of the absence of UL41,the virus could reduce the expression of host-related genes,suggesting that other viral factors may be able to participate in the early host shutoff.The UL24 homolog of PRV is very conserved in herpesviruses.Studies on several other ? herpesviruses have shown that UL24 is an important virulence determinant.However,no studies have been conducted on PRV UL24.Here the mRNA of UL24 gene can be detected at 8 h after infection,and PAA can inhibit its transcription,indicating that UL24 is a late gene of PRV.Further UL24 protein can be detected in viral infection,indicating that UL24 gene can encode protein.Heterologously expressed UL24 can be localized to the nucleus,suggesting that it may enter nucleus during viral infection.Using the CRISPR/Cas9 technique we constructed UL24 deleted,TK deleted,and UL24 and TK deleted PRVs.Further studies showed that TK or UL24-deleted strains could replicate on Vero cells,but the titers at late phase of infection of deleted strains were lower than wild type and the plaque sizes were also significantly smaller than wild type.In vivo studies showed that UL24 deleted strain exhibited reduced virulence in mice;after TK deletion,the virus is not pathogenic in mice.Further virus could not be detected in the nerve tissue infected with the TK deleted strain but a small amount of viral genomic DNA was detected,indicating that the virus can infect the trigeminal nerve but lose the ability to replicate.In addition,the expression of IFN-? in UL24 deleted virus-infected cells was significantly increased,indicating that UL24 protein can participate in inhibition of IFN-? expression.Luciferase reporter assays showed that UL24 protein regulated IFN-? expression through inhibition of cGAS-STING-activated NF-?B pathway.But mechanisms of UL24 regulation of IFN-? expression needs further research.In summary,the whole genome level sequencing and comparative study found that the partial sequence of the domestic classical strain SC genome is homologous to that of Bartha,indicating that SC is a potential recombinant strain of the domestic strain and the Bartha vaccine strain.By studying the characteristics of UL41 and UL24,it was found that deletion of UL41 reduced the PRV replication and infectivity both in vitro and in vivo,and that the UL41 protein of PRV variant had weaker vhs activity than the classical strain SC owing to amino acids mutation;While UL24 deletion can reduce the replication and infectivity of PRV both in vitro and in vivo,and further UL24 protein can inhibit the expression of IFN-? by cGAS-STING-activated NF-?B pathway.