| Pseudorabies is an acute infectious disease caused by pseudorabies virus in a variety of domestic animals and wild animals with fever,itching(except pigs),encephalomyelitis,respiratory and nervous system disorders as the main symptoms.Pigs are the natural hosts of PRV,as well as the long-term storage host and detoxification of this disease.Pseudorabies can establish latent infection in pigs that survive infection.Due to changes in the external environment or stress on the body,the pseudorabies virus in the latent infection state can be activated and released into the environment,thus infecting other animals,resulting in the long-term existence of the pseudorabies virus in pigs,which is difficult to eradicate.During latent infection,PRV does not replicate the viral genome,nor does it express the viral protein,but only a large number of virus latency-associated transcripts(LATs).It is unclear how the LATs gene is expressed during the latent infection period of PRV,but two promoters,LAP1 and LAP2 have been reported.Therefore,in this study,the pseudorabies BAC cloning and galk negative screening techniques were used to successfully construct a batch of viruses with two promoter deletions:JS-US and JS-RS,both LAP1/2 deleted;LAP1/2 deleted and added of transcription termination sequence BGH mutant virus JS-UBS;LAP1/2 deleted and added of insulator cHS4 and transcription termination sequence BGH for mutant viruses JS-U1 CBS and JS-U2 CBS.Camparing the in vitro biological characteristics of the mutant virus with that of the parental virus JS-2012,the results showed that the mutant viruses JS-US?JS-RS and JS-U1 CBS were not significantly different from the parental virus in terms of the size and morphology of the plaques or the characteristics of replication kinetics,but the replication speed of the mutant virus JS-UBS and JS-U2 CBS was much slower than that of the parental virus.Subsequently,we successfully constructed a simple and rapid RT-q PCR detection method for LLT transcription,which can avoid the influence of the presence of viral genome on gene transcription detection.The expression characteristics of LLT and EP0 were studied by RT-qPCR.The results showed that the LLT and EP0 could be detected at 2h after infection,and the expression level was highest at 8h,and then gradually decreased,and the expression level was significantly diferent in nerve cells and nonnerve cells.In both non-nerve and nerve cells,the expression level of LLT molecule was decreased after mutant virus infected cells compared with the parental virus.However,the expression of virus in nerve cells was higher than that in non-nerve cells,and that in Vero cells was higher than that in PK-15 cells.The second promoter,LAP2,can affect the expression of the LLT,and the absence of its results in a decrease in the expression of LLT.The pre-LAT promoter repeat sequence affects the expression of the LLT in nerve cells,and the loss also leads to a significant decrease in the expression of LLT.TK gene is the main virulence gene of PRV,and the deletion of TK gene will lead to the greatly reduced virulence of PRV,and the TK gene deletion virus cannot cause the death of mice.In order to establish an animal model of PRV latent infection,the PRV TK gene-deleted strain was inoculated into rats in this study.However,when the TK gene-deleted strains were inoculated with nasal dorps andintramuscular injections,the rats died.It was found that the virus content in the nerve tissue was always the highest by these two methods,and the viral load in the tissue was proportional to the dose.And the viral load in the tissuse challenged with the JS2012-?TK was significantly reduced compared with the parental virus.This shows that the TK gene deletion can reduce the pathogenicity of PRV in rats,but the TK gene deletion PRV is still lethal in rats. |
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