伪狂犬病病毒感染猪肺泡巨噬细胞和小肠上皮细胞的转录组动态分析

Transcriptome dynamics of pseudorabies virus-infected porcine alveolar macrophages and small intestinal epithelial cells

为探究伪狂犬病病毒(pseudorbies virus,PRV)与猪肺泡巨噬细胞及小肠上皮细胞的相互作用,将PRV感染猪肺泡巨噬细胞株3D4/21及小肠上皮细胞株IPEC-J2,测定其感染后病毒滴度及0~48 h内各时间点病毒gE基因拷贝数,观察细胞病变(CPE)和免疫荧光,采用荧光定量PCR(RT-qPCR)、ELISA检测肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)和白介素-6(IL-6),对PRV感染后0、6、12、18 h样本进行转录组测序(RNA-seq)分析。结果显示:PRV感染3D4/21细胞及IPEC-J2细胞96 h时,病毒滴度分别为10^(-8.16)TCID_(50)/0.1 mL、10^(-7.76)TCID_(50)/0.1 mL,病毒gE基因拷贝数分别在18、24 h达到最大,CPE分别在10、12 h开始出现,病毒蛋白表达随感染时间延长逐渐增加;TNF-α、IL-6和IL-1β表达量在PRV感染后均逐渐升高;PRV感染3D4/21细胞及IPEC-J2细胞后,经RNA-seq联合分析,在0、6、12、18 h的4个时间点筛出2种细胞共有差异表达基因分别为8313、6999、6693、5714个,其中Ras关联域家族成员6(RASSF6)、锌指蛋白667(ZNF667)、激肽超家族蛋白3C(KIF3C)、早期应答基因3(IER3)显著变化基因可能与细胞炎症反应有关,且RT-qPCR验证与RNA-seq分析结果一致。基因本体论(GO)分析显示差异基因富集到细胞过程、生物调控、细胞代谢调节等;京都基因与基因组百科全书(KEGG)通路富集显示差异基因主要涉及癌症通路、丝裂原活化蛋白激酶(MAPK)信号通路、Toll样受体(TLRs)信号通路等,其中参与炎症的Toll样受体4(TLR4)-髓样分化因子88(MYD88)-核转录因子кB(NF-кB)通路验证结果与KEGG通路分析数据变化趋势总体一致。提示:PRV感染会影响3D4/21细胞及IPEC-J2细胞的炎症反应。本研究为进一步探索PRV的致病机制提供了参考。

To investigate the interaction of pseudorabies virus(PRV)with porcine alveolar macrophages and small intestinal epithelial cells,PRV was infected with porcine alveolar macrophage cell line(3D4/21)and small intestinal epithelial cell line(IPEC-J2);the viral titer and viral gE gene copy number were measured at each time point from 0-48 h after the infection;the cytopathic lesions(CPE)and im⁃munofluorescence were observed,and RT-qPCR,ELISA for tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-6(IL-6).Transcriptome sequencing(RNA-seq)analyses were performed on the samples at 0,6,12 and 18 h after the PRV infection.The results showed that,when PRV infected 3D4/21 cells and IPEC-J2 cells for 96 h,the viral titer was 10^(-8.16) TCID_(50)/0.1 mL and 10^(-7.76) TCID_(50)/0.1 mL,respectively;the viral gE gene copy number reached the maximum at 18 and 24 h,respectively;the CPE began to appear at 10 and 12 h,respectively;the viral protein expression increased gradually with the infection.The expression of the viral proteins gradually increased with the prolongation of infection time;the expression of TNF-α,IL-6 and IL-1βgradually increased after the PRV in⁃fection;3D4/21 cells and IPEC-J2 cells were analyzed by RNA-seq at four time points,namely,0,6,12 and 18 h of the PRV infection,and the differentially expressed genes were screened out at 8313,6999,6693,5714,among which the significantly altered genes of Ras off structural domain family 6(RASSF6),zinc finger protein 667(ZNF667),kinin superfamily protein 3C(KIF3C),and early-response gene 3(IER3)might be related to cellular inflammatory response,and the RT-qPCR validation was in agreement with the RNA-seq re⁃sults.The gene ontology(GO)analysis showed that the differential genes were enriched in cellular processes,bioregulation,and regulation of cellular metabolism,etc.the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment showed that the differential genes were mainly involved in the cancer pathway,the mitogen-activated protein kinase(MAPK)signaling pathway,and Toll-like receptors(TLRs)signaling pathway,etc.,among which,the significantly altered genes of the Toll-like receptor 4(TLR4)-myeloid differentiation factor 88(MYD88)-nuclear transcription factorкB(NF-кB)pathway involved in inflammation were in general agreement with the trend of the KEGG pathway analysis data.To conclude,PRV infection affected the inflammatory response of 3D4/21 cells and IPEC-J2 cells,and this study laid the foundation for further exploration of the pathogenic mechanism of PRV.