基因Ⅲ型新城疫病毒感染鸡外周血单个核细胞后的靶细胞分析  

Target Cells Analysis of GenotypeⅢNewcastle Disease Virus in Chicken PBMC

作　　者：孙一帆 陆小龙 王晓泉[1,2] 胡顺林 刘秀梵[1,2] 刘晓文 SUN Yifan;LU Xiaolong;WANG Xiaoquan;HU Shunlin;LIU Xiufan;LIU Xiaowen(Animal Infectious Disease Laboratory,College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院动物传染病实验室,扬州225009 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《病毒学报》2022年第1期168-174,共7页Chinese Journal of Virology

基　　金：国家自然科学基金面上项目(项目号:31873021),题目:新城疫病毒Ⅰ系苗(Mukteswar)的进化及其毒力增强的分子机制。

摘　　要：新城疫(ND)是鸡新城疫强毒株引起的一种鸡的烈性传染病,目前疫苗接种是防治该病的主要手段。临床上曾分离到与中等毒力疫苗株Mukteswar高度同源的强毒株JS/7/05/Ch,其通过静脉注射后毒力显著增强。为了探究JS/7/05/Ch经血液途径毒力增强的机制,本研究选用鸡外周血单个核细胞(PBMC)作为研究模型,分析基因Ⅲ型NDV在PBMC中的靶细胞。细胞分选结果表明病毒感染可诱导PBMC中单核细胞的增殖。实验组病毒感染后单核细胞相对占比上升,感染后1d Mukteswar组(Muk组)单核细胞占比16.3%,JS/7/05/Ch组(JS组)为13.21%,而对照组单核细胞仅占比3.18%。荧光定量PCR测定分选后各细胞中的病毒载量,感染后3d JS组单核细胞中的病毒含量与感染后1d相比极显著性增加(P<0.01)。感染后1d病毒以感染淋巴细胞为主,而在感染后3d单核细胞的相对病毒含量均超过淋巴细胞占据主导地位,Muk组和JS组分别为54.2%和60.2%。综上所述,基因Ⅲ型NDV在PBMC中的靶细胞是单核巨噬细胞。这为进一步研究这对基因Ⅲ型模式病毒毒力差异的机制奠定了一定基础。Newcastle disease(ND),caused by virulent Newcastle disease virus(NDV),is a highly contagious disease in chickens.Currently,vaccination is the main way to prevent and control ND prevalence worldwide.In recent years,emerging velogenic NDV strain JS/7/05/Ch is isolated from chicken flocks and its virulence is significantly enhanced via intravenous injection as compared to a highly homologous mesogenic vaccine strain Mukteswar.In order to explore the mechanism by which intravenous administration enhances the virulence of JS/7/05/Ch,peripheral blood mononuclear cells(PBMC)isolated from NDV-infected or-uninfected SPF chickens were used as a research model to analyze target cells of genotypeⅢNDV in blood.Flow cytometry sorting revealed that viral infection induced the differentiation of monocytes in PBMC,as the results showed that 16.3%and 13.21%monocytes were identified in PBMC isolated from Mukteswar-and JS/7/05/Ch-infected chickens at 1 day post infection(dpi)respectively,while only 3.18%in uninfected-chickens.In order to identify target cells of genotypeⅢNDV in PBMC,we employed quantitative real-time reverse transcriptionPCR(RT-qPCR)to measure viral load in the sorted cells and the results showed that copies of NDV in monocytes isolated from JS/7/05/Ch-infected chickens at 3 dpi were significantly upregulated than those at 1 dpi(P<0.01).Furthermore,RT-qPCR results showed that viruses mainly infected lymphocytes at 1 dpi,while the relative viral load in monocytes exceeded 50%at 3 dpi.In summary,we demonstrated that monocytes/macrophages are the target cells of genotypeⅢNDV in chicken PBMC.These data could provide a valuable basis for further investigation on the mechanism of virulence differences between JS/7/05/Ch and Mukteswar.

关 键 词：新城疫病毒(NDV) 外周血单个核细胞 单核细胞 流式细胞分析 荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]