Study On The Expression Characterization Of Large Latency Transcripts(LLT) Of Pseudorabies Virus

Pseudorabies(PR)is an acute and febrile infectious disease caused by Pseudorabies virus(PRV)in many domestic animals and wild animals.Pigs are the primary hosts and reservoir of PRV.Pseudorabies can cause great economic loss to pig industry,especially the outbreaks of PRV variant in China since the end of 2011,which seriously hindered the healthy development of pig industry.Similar to other herpes viruses,PRV can develop latent infection in the host after acute infection.In latent infection,the viral genome does not replicate or express viral proteins,but only a large number of latency associated transcripts(LATs)are transcribed.It were thought that LAT transcription should play an important role in PRV latent infection.However,the effects of LATs on viral infection and replication were unclear.The transcripyional characteristics and regulatory mechanisms of LATs were not clear,too.LATs are resulted from splicing of large latency transcripts(LLT)molecules.In order to study the mechanism of PRV LLT promoter LAP1 and LAP2 in transcriptional regulation,we first constructed a series of donor plasmids,including pB-L1-R1(deleting LAP1),pB-L1-cHS4-R1(deleting LAP1 and inserting insulator cHS4),pB-L1-CMVp-R1(deleting LAP1 and inserting CMV promoter),and pB-L1-BGH-R3(deleting both LAP1 and LAP2).Then,the galK gene was used to replace the promoter region of PRV LLT by BAC/galK system.After positive and negative screening,the target genes of different plasmids were used to replace galK gene.After identification by PCR,different positive clones were screened out.The BAC DNA of each positive clone was extracted,and co-transfected with pcDNA3.1-cre plasmid into Vero cells.Six mutant viruses with deleted promoters(vJS-? LAP1 ?vJS-cHS4-?LAP1a?vJS-cHS4-?LAP1b?vJS-CMVp-?LAP1?vJS-?LAP1/P2 and vJS-cHS4-?LAP1/P2)were successfully rescued.Then,the in vitro biological characteristics of parental virus JS-2012 and the mutant virus were compared.The results showed that the plaque size and morphology of mutant viruses with LAP1 and LAP2 deletions were not significantly different from that of parental viruses JS-2012.The one-step growth curve showed that the replication dynamics of mutant viruses were generally identical to that of their parent virus.Further,we analyzed the expression characteristics of Latency-associated transcripts of each mutant viruses,and found that the deletion of LAP1/P2 did not affect the transcription of ORF1 and ORF2 genes in LLT region,indicating that the deletion of LLT promoter did not cause the complete shutoff of LLT transcription.Moreover,the deletion of promoters LAP1 and LAP2 could not shut off the LLT expression in virus-infected mice.However,the deletion of LLT promoter significantly reduced the transcription level of multiple microRNAs(miR-2-3',miR-5 and miR-9-3')in LLT intron,suggesting that LLT promoter regulates the transcription of microRNAs in LLT intron.However,the biological significance of LLT promoter regulating viral microRNA expression needs further study.In conclusion,a series of PRV strains with LAP1 and LAP2 mutations on the basis of PRV JS-2012 were constructed using BAC/galK system.Through in vitro and in vivo biological characteristicsanalysis of the mutant strains,it was found that LLT promoter mutations could not significantly affect the biological characteristics of the virus in vivo and in vitro,nor the expression of ORF1 and ORF2 genes located in LLT region.However,the deletion of LLT promoter can significantly down-regulate the transcription of multiple microRNAs in viral LLT region.