| 摘要: |
| 【目的】分析1株血清4型禽腺病毒（FAdV-4）流行株的分子特征及其感染对细胞因子的影响。【方法】采集安徽省某禽类养殖场疑似心包积水 肝炎综合症的鸡肝脏组织样本，用鸡肝癌细胞（LMH）对病原进行分离，采用PCR、透射电镜（TEM）观察和间接免疫荧光试验（IFA）对分离的毒株进行鉴定。采用分段扩增后拼接的方法克隆分离毒株的全基因组，对其进行遗传进化分析和序列重组分析。基于柯赫法则，用分离毒株接种无特定病原体（SPF）鸡，检验其致病性。采用实时荧光定量PCR法，检测分离毒株对LMH细胞天然免疫相关细胞因子环鸟苷酸-腺苷酸（cGAS）、干扰素刺激因子（STING）、白介素-1β（IL-1β）、IL-6、IL-8、干扰素-α（IFN-α）、IFN-β、干扰素调节因子7（IRF7）、线粒体抗病毒信号蛋白（MAVS）、主要组织相容性复合体（MHC）Ⅰ-α、MHCⅡ-β等11种细胞因子的诱导作用。【结果】分离鉴定到1株FAdV，其在LMH细胞中培养未出现明显细胞病变效应；病毒粒子直径为70~90 nm，符合FAdV-4的结构特征；免疫荧光试验结果显示，在接种分离毒株的LMH细胞内可观察到亮红色荧光，而对照细胞没有荧光；将该毒株命名为FAdV-4-AH-F41。采用分段扩增后拼接的策略获得了FAdV-4-AH-F41全基因组序列（43 708 bp）；遗传进化分析和序列重组分析发现，FAdV-4-AH-F41与FAdV-4株核苷酸相似性为99.6%；存在3个重组事件，第1个重组事件发生在30 453-30 674 bp，第2个发生在36 884-36 988 bp，第3个发生在43 401-43 715 bp。致病性试验显示，FAdV-4-AH-F41是导致鸡心包积水-肝炎综合症的病原。实时荧光定量PCR检测结果显示，与对照（DMEM/F12处理）相比，用FAdV-4-AH-F41处理后LMH细胞中11种免疫因子的表达量水平均有提升，其中cGAS、IL-6、IRF7、MHCⅡ β差异达极显著水平（P<0.01），IL-1β、IFN-α、MAVS差异达显著水平（P<0.05），STING、IL-8、IFN-β、MHCⅠ-α差异不显著。【结论】成功分离1株重组血清4型禽腺病毒，感染LMH细胞会诱导强烈的天然免疫反应。 |
| 关键词: 禽腺病毒4型 鸡肝癌细胞 致病性 细胞因子 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金项目（31972642） |
|
| Molecular characterization of an endemic strain of avian adenovirus FAdV-4-AH-F41 and its induction of cytokines |
|
WEI Ningbo,LIU Qi,XU Liang,et al
|
| Abstract: |
| 【Objective】This study analyzed the molecular characteristics of a prevalent strain of serotype 4 avian adenovirus (FAdV-4) and the effect of its infection on cytokines.【Method】Liver tissue samples were collected from chickens with suspected pericardial effusion-hepatitis syndrome in a poultry farm in Anhui Province,and the pathogen was isolated using leghorn male hepatoma (LMH).The isolated strains were identified by PCR,transmission electron microscopy (TEM) observation and indirect immunofluorescence assay (IFA).The whole genome of the isolated virulent strain was cloned by segmental amplification followed by splicing,and genetic evolutionary analysis and sequence recombination analysis were performed.Based on Koch’s rule,the isolated strain was inoculated with specific pathogen free (SPF) chickens to test its pathogenicity.Real-time fluorescence quantitative PCR was used to detect the effects of the isolate on 11 natural immunity-related cytokines including cyclic guanosine-adenylate (cGAS),interferon-stimulating factor (STING),interleukin-1β (IL-1β),IL-6,IL-8,interferon-α (IFN-α),IFN-β,interferon regulatory factor 7 (IRF7),mitochondrial antiviral signaling protein (MAVS),major histocompatibility complex (MHC) Ⅰ-α and MHC Ⅱ-β.【Result】A strain of FAdV was isolated and identified,which showed no obvious cytopathic effect in LMH cells.The viral particles were about 70-90 nm in diameter,consistent with the structural features of FAdV 4.Immunofluorescence assays showed that bright red fluorescence could be observed in LMH cells inoculated with the isolated strain,while no fluorescence was observed in control cells.The strain was named FAdV-4-AH-F41 and its whole genome sequence (43 708 bp) was obtained by segmental amplification followed by splicing strategy.Genetic evolutionary analysis and sequence recombination analysis revealed that the identity between FAdV-4-AH-F41 and FAdV-4 strain was 99.6%.There were three recombination events,occurring at 30 453-30 674 bp,36 884-36 988 bp and 43 401-43 715 bp,respectively.The pathogenicity test showed that FAdV-4-AH-F41 was the causative agent of pericardial effusion-hepatitis syndrome in chickens.The real-time fluorescence quantitative PCR assay showed that the expression levels of 11 immune factors in LMH cells were elevated compared with the control (DMEM/F12),among which cGAS,IL-6,IRF7 and MHC Ⅱ-β differed at highly significant levels (P<0.01),IL-1β,IFN-α and MAVS differed at significant levels (P<0.05),while STING,IL-8,IFN-β and MHCⅠ-α had no significant differences.【Conclusion】A recombinant serotype 4 avian adenovirus strain was successfully isolated,and infection of LMH cells induced strong natural immune responses. |
| Key words: type 4 fowl adenovirus leghorn male hepatoma pathogenicity cytokines |
