伪狂犬病病毒在小鼠原代神经细胞中潜伏感染模型的建立  

Establishment of an in vitro model of latent infection of pseudorabies virus in mouse primary neuronal cells

作　　者：向广韬 吴红霞[1] 王异民 周末 王亚林 王翌 尹航 仇华吉[1] 孙元[1] XIANG Guang-tao;WU Hong-xia;WANG Yi-min;ZHOU Mo;WANG Ya-lin;WANG Yi;YIN Hang;QIU Hua-Ji;SUN Yuan(State Key Laboratory for Animal Diease Control and Prevention,,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2024年第1期1-6,15,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金面上项目“伪狂犬病病毒变异株神经毒力增强的分子机制”(31570149)。

摘　　要：缺少PRV良好的潜伏感染模型已成为研究其潜伏感染机制的障碍。为建立PRV潜伏感染的体外细胞模型,本研究通过分离新生鼠背根神经节(DRG)获取神经细胞,并利用间接免疫荧光试验(IFA)对其进行鉴定,获得了DRG中的神经细胞。采用重组报告病毒r PRV-EGFP感染神经细胞,同时添加阿昔洛韦(ACV)抑制病毒复制以建立潜伏感染,并采用棋盘法对病毒感染剂量和ACV浓度进行优化,结果显示,感染复数(MOI)为0.001,ACV浓度为0.5 mmol/L,病毒能够在神经细胞中稳定建立潜伏感染。采用荧光定量RT-PCR、病毒滴度测定、药物刺激再激活的方法对潜伏感染模型鉴定,结果显示,潜伏感染时病毒不能复制,且不产生感染性病毒粒子;而经LY294002(PI3K抑制剂)诱导病毒再激活后,病毒大量复制并产生感染性病毒粒子。本研究首次利用小鼠原代神经细胞建立了PRV的潜伏感染模型,该模型的建立为PRV潜伏感染机制的研究奠定了基础。Studying latent Pseudorabies virus(PRV)infection has been limited by the lack of a suitable latent infection models.The aimed of this study is to establish an in vitro model for studying latent infection with PRV.Nerve cells were collected by isolating the dorsal root ganglion(DRG)of newborn mice,then indirect immunofluorescence(IFA)was used to identify that nerve cells in the DRG were successfully obtained.The nerve cells were infected by reporter recombinant virus(rPRV-EGFP),and Acyclovir(ACV)were used to inhibit viral replication to establish latent infection model.In addition,the conditions affecting latent infection,the multiplicity of infection(MOI)and the concentration of ACV were optimized.The results showed that with MOI 0.001 and 0.5mmol/L ACV,the PRV could stably establish latent infection on nerve cells.Then the latent infection was confirmed by genomic copies,virus titration and drug stimulation.The results showed that PRV could not replicate and no infectious virions were produced during latent infection in nerve cells.However,the PI3K inhibitor,LY294002,induced PRV reactivation and the production of large numbers of infectious virus particles.Taken together,this study established a PRV latent infection model in mouse primary nerve cells,which lays a foundation for further study on the latent infection of PRV.

关 键 词：伪狂犬病毒 小鼠背根神经节 潜伏感染 再激活 模型 

分 类 号：S852.65[农业科学—基础兽医学]