Functional Studies Of LncRNA LEULR Encoded By Pseudorabies Virus

Pseudorabies virus(PRV)can establish life-long latency and reactivation in the trigeminal ganglion of swines,causing Pseudorabies(PR)in swines and causing serious economic losses to the swine industry.Since 2011,Chinese swine farms have successively broken out pseudorabies,which has been proved to be a new mutant strain.Long noncoding RNA(lnc RNA)is the most common and most versatile type of RNA without the ability to encode proteins,with a length of more than 200 nt.Lnc RNA can promote a variety of biological functions,including cell structural integrity,cell cycle,reprogramming,and apoptosis.Viruses have evolved various strategies to evade host defenses,and lnc RNAs have proven to play an important role in them.LEULR is a sort of lnc RNA located at 3-254 bp in the PRV genome.In order to study the functions of LEULR in the life cycle of PRV,the research contents of this topic are as follows:1.The homology analysis of LEULR among different strainsDownload the LEULR sequence of domestic and foreign classic and variant PRV strains on Genebank for homology analysis and phylogenetic tree analysis.And it is found that LEULR is highly conserved among different strains.2.LEULR transcription level in PK15 cellsIn order to further study the expression of LEULR,this study tested the LEULR transcription level of PRV Ea strain infecting PK-15 cells at different time points.The results showed that LEULR began to be expressed in small amounts afterPRV infection,and was expressed in large amounts at 9 h after infection.The expression abundance reached the highest at 24 h,and then began to gradually decrease.3.Construction of LEULR deletion strain and back-mutant strainIn order to study the function of LEULR,the BAC platform and homologous recombination methods were used to construct rPRV?LEULR and rPRV?LEULR R.And it was proved that LEULR sequence was successfully deleted and reverted at the PRV genome level.4.The influence of LEULR on the replication of PRVOne-step growth curve of WT PRV Ea,rPRV?LEULR,rPRV?LEULR R were detected by infecting with PK15 cells at 5 MOI;Multi-step growth curve of WT PRV Ea,rPRV?LEULR,rPRV?LEULR R were detected by infecting with PK15 cells at 0.01MOI.The plaque size of the three viruses was also tested.The results found that LEULR had no effect on the one-step proliferation curve,multi-step proliferation curve and plaque size of PRV,showing that LEULR is not necessary forPRV replication.5.The effect of LEULR on the virulence of PRVWT PRV Ea,rPRV?LEULR,rPRV?LEULR R,PK15 cell lysate(control)were injected into BLAB/c mice at a rate of 10~4 PFU/mouse.The symptoms and death of the mice were observed every day.It was found that LEULR did not affect the neurological symptoms and lethality of PRV in mice.6.The expression of LEULR in PRV latency and reactivation trigeminal ganglia modelThe total RNA was extracted from the porcine trigeminal ganglion samples with Mock,PRV latent,and PRV reactivated.The expression of LEULR was detected by RT-q PCR method.It was found that LEULR was expressed in PRV latent samples,but was not expressed in reactivated samples,suggesting that LEULRA may be involved in the PRV latent process.In conclusion,this study found that LEULR is highly conserved among different strains.Its expression abundance reached the highest when PRV infected PK-15 cells for24 h.LEULR deletion strains and back-mutant strains were successfully constructed in this project,and it was found that LEULR does not affect the replication and virulence of PRV.However,the expression of LEULR was detected in the PRV latent porcine trigeminal ganglion samples,suggesting that LEULR may be involved in the latent process of PRV.