Study On The Role Of Porcine LncRNA In The Proliferation Of Japanese Encephalitis Virus And Its Signaling Pathway

At present,the biggest threat to pig industry is disease,especially some common zoonoses,which will seriously affect human health.Japanese encephalitis is one of them.Japanese encephalitis?referred to as JE?,also known as Japanese encephalitis,the pathogen is epidemic encephalitis virus,referred to as Japanese encephalitis virus?JEV?.JEV is an arbovirus,belonging to flaviridae flavivirus,which can cause high fever,headache,vomiting and other symptoms.People who get the severe cases such as fever in whole body,cerebral edema,spasmodic spasm,dyspnea and circulatory failure,may lead to death and most of them will have sequelae.JE is a zoonotic disease,which not only causes acute disease such as the central nervous systems,but also has a high mortality rate.It's mainly transmitted by Culex tritaeniorhynchus in animals.There are many modes of transmittion like birds-mosquitoes-birds,mosquitoes-bats-mosquitoes,pigs-mosquitoes-pigs and pigs-mosquitoes-humans.JEV will infect pigs,cause reproductive dysfunction in breeding pigs,which ultimately leads to huge economic losses in the pig industry.In addition,the viremia time is much longer.The viral load in pigs'blood is high and highly contagious,which is the main source of human infection with JE.During the summer and autumn,JE is one of the main epidemic diseases in China.It is a susceptible area all over the country except in Qinghai,Tibet and Xinjiang.The number of infected people in China can reach 25000per year,and the fatality rate can reach 10%.Moreover,15%of patients have different degrees of sequelae.The aim of this study was to initially explore the proliferation of Japanese encephalitis virus?JEV?infected with PK15 cells,screen the lnc RNAs associated with viral infections,and predict its subcellular localization and target genes.The expression of viral structural protein E was detected by immunofluorescence,the proliferation of virus in PK15 cells was detected by TCID₅₀0 method,and Real-time PCR was used to detect the expression level of lncRNA after viral infection.Subcellular localization of lncRNA was carried out in the NONCODE database,target gene prediction and signal pathway analysis were performed by starBase,NONCODE,KEGG and other databases.This study laid a foundation for further exploration of the effect of host cell lncRNA on viral proliferation.1.Differentially expressed lncRNA study of JEV infected PK15 cellsIn the early stage of the study,PK15 cells were infected by JEV,the expression of virus structural protein E in different time periods after JEV infected PK15 cells was detected by immunofluorescence assay.The results showed that the expression of virus structural protein E significantly increased 36 hours later.TCID₅₀0 also confirmed that virus titer significantly increased36 hours later.The samples were sent to Beijing boao biological Co.,Ltd.for transcriptome sequencing after the virus infected the cells.The results showed that,compared with the Mock control group,the JEV treatment group's expression of 452 lncRNAs increased,while the expression of 404 lncRNAs decreased.The expression of 2253 m RNA was increased and 1912mRNA was decreased.Thermal maps of genetic differences showed the samples were reproducible.The lnc RNA initially explored in this study may play an important role in the process of JEV replication and proliferation.2.Innate immune-related lnc RNA regulates JEV proliferation and related signaling pathway researchlncRNA NONSUST006715.1,the subsequent study named it lnc RNA B.After JEV infected PK15 cells,the antisense nucleic acid of lncRNA was designed to reduce the expression of lncRNA.The results showed when the expression of lncRNA B was knocked down,the expression of JEV significantly increased,suggesting that lncRNA could inhibit the proliferation of JEV.In the study,PK15 cells were treated with inhibitors through several common signaling pathways to quantitatively detect the expression of lncRNA B.The expression of NS3 protein was detected by western blot.The results showed that the expression of lncRNA B decreased after most inhibitors treated the PK15 cells.However,the expression of lncRNA B increased and the expression of JEV NS3 protein was significantly decreased after ZK811752?CCR1 inhibitor?treated PK15 cells.The above studies indicate that CCR1,acting as a key protein in the chemokine signaling pathway related to host immunity,inhibit the expression of lncRNA B,thereby promoting the proliferation of JEV.In the late stage of the study,the effect of lncRNA B with CCR1 knockdown was detected by fluorescence quantitative PCR.The result showed that the inhibition of CCR1 enhanced the expression of lncRNA B,while lncRNA B inhibited the proliferation of JEV.Besides,fluorescence quantitative PCR was used to determine whether CCR1 knockdown had a certain effect on the expression of lncRNA B transcription factors.The results showed that CCR1 knockdown enhanced the expression of lncRNA B transcription factor SP1 in the lncRNA B promoter region.In order to explore the interaction between proteins and genes,CHIP assay was used to detect the effect of CCR1 knockdown on lncRNA B transcription factor SP1 binding in lncRNA B promoter region.The results showed that CCR1 knockdown did affect the binding of SP1 in the lncRNA B promoter region.This study confirms that lncRNA B has the function of inhibiting JEV proliferation,and the virus inhibits the binding of transcription factor SP1 to the lnc RNA B promoter region by activating the CCR1 protein in the chemokine signaling pathway,thereby down-regulating the expression of lncRNA B and ultimately further promoting viral proliferation.