高致病性禽腺病毒4型反向遗传技术平台的建立与毒力相关基因鉴定

Hepatitis-hydropericardium syndrome (HHS) caused by a novel fowl aviadenovirus 4 (FAdV-4) genotype is a new emerging poultry infectious disease in China, causing significant losses to Chinese poultry industry. Our previous research found that, when compared with non-pathogenic FAdV-4 strains, the novel Chinese FAdV-4 genotype contains various genomic deletions which caused the loss of ORF19, ORF27 and ORF48, and multiple distinct amino-acid mutations in major structural genes, Hexon, Fiber1 and Fiber 2. Investigating the association between these variations and the enhanced virulence of the novel FAdV-4 genotype is very important for elucidation of the pathogenisis of FAdV-4. By taking advantage of the lambda Red recombinase system, a double- selection markers system consisting of the antibiotic resistance gene and the E. coli temperature-sensitive promoter PL-controlled E. coli kil gene, the reverse genetic technique platform for highly pathogenic FAdV-4 will be established in this proposal. A series of mutants of highly pathogenic FAdV-4 will be developed by using the reverse genetic technique platform. Differences in pathogenicity and virulence between mutant and parent highly pathogenic FAdV-4 , identification of virulence-associated genes and critical sites of virulence-determiants will be mainly focused on. The results will provide theoretical foundation for elucidating the pathogenisis of FAdV-4, developing new anti-FAdV-4 drugs and highly efficient vaccines for the control of HHS.

禽腺病毒4型（FAdV-4）新基因型导致的鸡肝炎-心包积液综合征（HHS）是危害我国养禽业的新发传染病。我们前期研究发现，与非致病性FAdV-4基因组序列相比，该新基因型基因组有多处缺失，缺失导致ORF19、27和48的消失，主要结构基因Hexon、Fiber1 和Fiber2均存在特定位点氨基酸替换。研究这些变异与该新基因型毒力增强的相关性对于阐明FAdV-4致病机理具有重要意义。我们将利用λ噬菌体Red重组酶技术、抗生素抗性基因与λ噬菌体温度敏感启动子PL控制的大肠杆菌素释放基因（kil）组成的双标记筛选系统，建立基于高致病性FAdV-4的反向遗传技术平台，利用此技术平台构建一系列突变毒株。重点研究突变株与亲本株对SPF易感鸡的致病性差异，鉴定FAdV-4毒力相关基因和基因中决定毒力的关键位点。项目将为解析FAdV-4的致病机理、研发新型抗腺病毒药物和控制HHS的高效疫苗提供理论基础。

2015年5月份以来，由禽腺病毒4型（FAdV-4）新基因型导致的鸡肝炎-心包积液综合征（HHS）在我国暴发流行，对我国养禽业造成严重经济损失。我们前期研究发现，与非致病性FAdV-4基因组序列相比，FAdV-4新基因型基因组有多处缺失，主要结构基因Hexon、Fiber1 和Fiber2均存在特定位点氨基酸替换。为了研究这些变异与FAdV-4新基因型毒力增强的相关性，本项目首先利用ExoCET（核酸外切酶结合RecET重组)技术构建了FAdV-4致病性毒株HNJZ和非致病性毒株ON1的全基因组感染性克隆，然后利用Redα/β重组技术和大肠杆菌自杀基因ccdB反向筛选技术，构建了penton、Fiber1、Fiber2、Hexon和1966bp缺失片段替换的突变株，并比较了各突变株和亲本毒株对3周龄SPF鸡的致病性差异。研究结果证明，FAdV-4的毒力不依赖于Fiber1、Penton和缺失的1966bp片段，但与Fiber2和Hexon密切相关。在此基础上，为了确定Hexon和Fiber2蛋白与FAdV-4毒力相关的关键氨基酸，本项目构建了FAdV-4致病性毒株HNJZ 的Hexon和Fiber2特定位点氨基酸突变株，并研究了这些突变毒株与亲本毒株HNJZ对SPF易感鸡的致病性差异，鉴定出Hexon的第188位氨基酸和Fiber2基因的第219位氨基酸是决定毒力的关键位点。本项目还研究了致病性FAdV-4毒株的感染是否激活鸡巨噬细胞中NLRP3炎症小体。研究结果显示，致病性FAdV-4激活鸡巨噬细胞中NLRP3炎症小体，进而诱导NLRP3依赖性IL-1β的分泌，最终导致机体组织的炎性损伤。项目研究结果将为解析FAdV-4致病机理奠定基础，项目建立的反向遗传技术平台将为基因功能研究和研发基因工程疫苗提供技术支撑。