| Pseudorabies is an acute infectious disease caused by PRV,which cause huge economic losses to China's pig industry.As a member of the Herpesviridae,PRV can establish a lifelong latent infection in natural host without any clinical symptoms.Once under stress,PRV may be reactivated and cause new infections.Therefore,PRV latent infection has become a major obstacle to the prevention and eradication of Pseudorabies.Although there have been many studies on the latent infection of herpesvirus,there are few reports on the mechanism of the latency reactivation of PRV using natural host pigs as models.In this study,a latency reactivation model of PRV infection was established using natural host piglets.On the basis of this model,the transcriptional sequence of typical viral genes was studied during the process of PRV latency reactivation.The changes of some cytokines were detected during PRV latency reactivation.iTRAQ technology was used to screen the phosphorylated host proteins in the process of latency reactivation,and bioinformatics was applied to analyze the differentially expressed phosphorylated proteins,so as to further verify the influence of differentially expressed proteins on PRV replication.Specific researches on this subject are as follows: 1 The latency reactivation model of PRV infection in natural host pigs was successfully established.PRV negative piglets were selected for testing,and eye and nose swabs were collected weekly for virus titer determination.The results showed that the virus particles could not be detected in the eye and nose swabs after 21 days of challenge,indicating that the piglets infected with PRV entered the latent infection stage.At the same time,whole blood of piglets was collected weekly,serum was separated,ELISA and antibody neutralization test was conducted,and The results showed that PRV neutralizing antibodies persisted when PRV was in latent infection stage.After 8 weeks of challenge,Reactivation with dexamethasone treatment for 3 h,all piglets were slaughtered,and trigeminal ganglion(TG)of each group was collected and soaked in formalin for immunohistochemical test.The results showed that PRV was activated after dexamethasone treatment.At the same time,fluorescence quantitative PCR detection of the copy number of the virus genome in TG showed that PRV changes from latent infection to reactivation in piglet after dexamethasone treatment.To sum up,the PRV latency reactivation model constructed was successful in this paper.2 The expression of typical virus genes is disordered during reactivation from latency.To further confirm changes in PRV genes at the transcriptional level during reactivation from latency.Several PRV typical viral genes IE180,EP0,VP16 and g B were selected to conduct q PCR test.The results showed that LLT gene was detected in Group latency,which was composed of exons 1,2 and introns.Exon 1 and 2 overlap EP0 and IE180 genes,respectively.IE180,EP0,LLT-intron and VP16 were detected in group reactivation,but g B genes were not detected.This suggested that the reactivation of PRV in natural host cells does not follow the IE-E-L pattern of epithelial cell infection.3 Changes of cytokines concentration during reactivation from latency.In order to evaluate the changes of cytokines during PRV reactivation from latency,Luminex x MAP technology was used to detect IL-1beta,IL-6,IL-8,IFN-alpha and TNF-alpha.The results showed that IL-6 concentration increased significantly during the process of latency reactivation,indicating that IL-6 was involved in this process.The concentration of IL-1beta and IFN-alpha was not significantly different between the control group and the latency group,but decreased in the reactivation group;The concentrations of IL-8 and TNF-alpha in latency group was significantly higher than those in control group and reactivation group.4 Identification of phosphorylation protein in TG during reactivation from latency.In order to further understand the effect of host protein phosphorylation during PRV latency reactivation,iTRAQ was used to quantitatively detect the phosphorylated proteins in TG of the control group and the test group.A total of 1876 phosphorylated proteins and 4661 phosphorylated peptides were detected.According to the standards of P <0.05 and expression changes more than 1.2 times(increasing Ratio>1.2,decreasing Ratio<0.83),125 were up-regulated and 117 were down-regulated phosphorylated proteins were screened during reactivation from latency.5 Effect of secretory phosphorylated protein(sPP1)on PRV proliferation.According to GO and KEGG analysis,sPP1 was predicted to play a certain role in the latency reactivation of PRV.After si RNA interference with sPP1 gene and overexpression of sPP1 gene,the effect of sPP1 on PRV proliferation was detected.The results showed that si RNA interference with sPP1 gene could inhibit PRV proliferation and overexpression of sPP1 gene could promote PRV proliferation.In this study,a latency reactivation model of PRV infection was established using natural host piglets.On the basis of this model,the transcriptional sequence of typical viral genes was studied,and The results showed that the reactivation of PRV in natural host cells does not follow the IE-E-L pattern.some cytokines were detected and IL-6 was found to be involved in the regulation of PRV latency reactivation,while IL-8 and TNF-alpha were found to be involved in the regulation of PRV latency infection.iTRAQ technology was used to screen the phosphorylated host proteins in the process of latency reactivation,and bioinformatics was used to predict the up-regulated 125 and down-regulated 117 of the differentially expressed phosphorylated proteins during latency reactivation.The predicted differentially expressed of secretory phosphorylated protein(sPP1)may have a certain effect on PRV replication,and it has been confirmed that sPP1 participated in PRV replication during the process of latency reactivation.These results will provide a theoretical basis for further exploring the pathogenic mechanism of PRV latency reactivation. |
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