牛流行热病毒感染白纹伊蚊细胞免疫调控相关基因的差异表达分析

Differential expression of immune-regulatory genes in Aedes albopictus cells infected by bovine ephemeral fever virus

为了阐明牛流行热病毒(bovine ephemeral fever virus,BEFV)在媒介蚊形成持续感染过程中免疫相关基因的调控作用,利用白纹伊蚊幼蚊细胞C6/36从牛体传代抗凝血分离BEFV JT02L株,用geNorm和NormFinder软件分析感染细胞看家基因的mRNA表达水平稳定性,筛选获得最稳定表达的内参基因,并用荧光定量RT-PCR进行相对定量分析感染细胞Toll样受体通路、免疫缺陷通路(IMD)以及Janus激酶/信号转导与转录激活子通路(JAK-STAT)相关基因的mRNA水平。结果显示,利用C6/36细胞分离获得可稳定传代BEFV株,经微量免疫荧光试验测定其毒价为1×10^(-5.27)TCID_(50)/mL,在C6/36细胞复制48~72 h时病毒载量达到最高水平。BEFV感染C6/36细胞,核糖体蛋白s17基因为最稳定内参基因,Toll样受体通路分子转录因子rel1、抗菌肽attacin B、defa、diptericin、lysc以及丝氨酸蛋白酶clipB27基因mRNA水平显著上调,IMD通路转录因子2 mRNA水平上调较弱,但可通过上调其下游的细胞因子vago基因表达平刺激JAK-STAT信号通路抗病毒基因vir-1上调表达。本试验阐明了BEFV感染蚊媒细胞过程中调控性基因的差异表达情况,为深入研究BEFV的媒介传播奠定了基础。

In order to elucidate the regulatory role of immune-related genes in vector mosquitoes persistently infected by bovine ephemeral fever virus(BEFV),BEFV strain JT02 L was isolated from anticoagulant blood collected from calves using Aedes albopictus larvae cell line C6/36.The mRNA stability of housekeeping genes in C6/36 cells infected by BEFV was analysed by geNorm and NormFinder softwares to determine the most stably expressed internal control gene.The relative mRNA levels of the genes related to Toll-like receptor pathway,immune deficiency pathway(IMD)and Janus kinase-signal transduction and activator of transcription(JAK-STAT)in BEFV-infected C6/36 cells were analysed by qRT-PCR.The results showed that the stably proliferated BEFV strain was obtained by virus isolation using C6/36 cells,and the viral titre reached to 1×10^(-5.27)TCID_(50)/mL.The viral load of BEFV in C6/36 cells reached the highest level at 48-72 hpi.In C6/36 cells infected with BEFV,ribosomal protein s17 gene was the most stably expressed internal reference gene,while the mRNA levels of several genes in Toll-like receptor pathway,including transcription factor Rel1,antimicrobial peptide attacin B,defa,diptericin,lysc and serine protease clipB27,were significantly up-regulated.The mRNA level of transcription factor Rel2 of IMD pathway was weakly up-regulated,but its downstream cytokine vago was upregulated,which led to the up-regulation of the antiviral gene vir-1 of JAK-STAT pathway.This study clarifies the differential expression of regulatory genes in BEFV infected mosquito cells,and lays a foundation for further research on the vector transmission of BEFV.