Methylation Analysis Of Pseudorabies Virus EPO During Latent Infection

Pseudorabies virus (PRV), a member of neurotrophic alphaherpesvirus canestablish latent infection in host, and its reservoir is pigs. This circulation mechanismof latency-activation determine the persistence of PRV in pigs, thus preventing theestablishment of latent infection and eliminating the latent virus in host play a crucialrole in controlling and eradicating of Pseudorabies (PR). However, the mechanism ofPRV latent infection PRV is unclear currently. DNA methylation plays a vital role inthe transcriptional regulation of viral genome and immune evasion. The role of PRVDNA methylation in the establishment and maintenance of latent infection isunknown.PR has re-emerged with devastating impact in Henan since early2012.Toinvestigate the reason of this outbreak and provide a reliable materials for thepost-study test, four strains of PRV were isolated from PRV suspected tissues fromdifferent areas of Henan from March2012to June2013through passages in Vero cell.The gE, TK and gD genes of PRV isolates, were amplified by polymerase chainreaction, cloned, sequenced, and analyzed. Sequence analysis showed that the gE、TK and gD genes of four PRV isolates from Henan had specific nucleotides andamino acids which were differ from the reference PRV strains; Phylogenetic analysisrevealed that four PRV isolates formed a separate subpopulation and kept a nearestrelationship with2013PRV isolates. And four PRV isolates were especially closelyrelated to other strains from China, but differ genetically from European and SouthAmerica strains. These results demonstrate that four strains of isolates are new varientPRV.Isolation and identification of PRV isolates was used to replicate theestablishment and maintenance of PRV latent infection in its host. Simultaneously, thetranscription of PRV IE180、EP0、LAT or gD gene was detected on DNA and RNAlevels. The results showed that the transcription of LAT gene was persistent, yet thetranscription of IE180、 EP0or gD genes was closed.These demonstrate that a pigmodel of PRV latent infection is established successfully. This also provides reliableexperimental materials and datas for further study on the mechanism of PRV and alpha herpes virus latent infection.To confirm the distribution of DNA methylation in the promoter of EP0gene, themethylation status in the promoter of EP0gene during latent infection was detectedusing bisulfite polymerase chain reaction basing on the prediction of the CpG islandsand related transcription factors. The result showed that only sporadic individualmethylation site existed in the promoter of EP0gene, however, these scatteredmethylation sites were not part of "GC" box sensitive transcription factor binding sites.This primarily suggests that methylation in the promoter of EP0gene during latentinfection is not likely to play an important regulatory role in the expression of gene,which provides a foundation of the research on the relationship of DNA methylationand the mechanism of PRV latent infection.