血清4型禽腺病毒ORF19、ORF27的天然缺失与病毒毒力增强的相关性研究

Since July 2015, a natural outbreak of Hydropericardium syndrome (HPS) occurred in Heilongjiang, Henan, Jilin et al provinces in China, and the disease rapidly spread to different areas of the country. The mortality was 50-80% in broilers and 50% in layers, respectively. HPS appeared not only in chickens but also in ducks with a mortality of 15%. Therefore, the HPS introduced serious economic losses to the poultry industry of China. Based on virus isolation and characterization, fowl adenovirus 4 (FAdV-4) infection was responsible for HPS. FAdV-4 single infection was able to induce 80% mortality in SPF chickens, and severe hydropericardium and inclusion body hepatitis, which were incidence with the clinical signs of the natural infected chickens. Complete genome assay showed that all 14 strains isolated from different areas and different kinds of hosts had the same natural deletion of a 1966 bp sequence. The natural deletion sequence included two open reading frames (ORF), ORF19 and ORF 27. The relationship between the natural deletion of ORF19 and ORF27 and the increasing viral virulence of fowl adenovirus 4 is not clear until now. Therefore, we want to construct infectious clones of recombinant natural deletion, ORF19 insert, ORF 27 insert, ORF19+ORF27 insert viruses based on bacterial artificial chromosome (BAC) technology, and evaluate the viral virulence variation of different recombinant strains in vitro and in vivo. Finally, a certain relationship between the natural deletion of ORF19 and ORF27 and the increasing viral virulence of fowl adenovirus 4 will be obtained, which is very important for revealing the molecular basis of FAdV-4 virulence evolution. Simultaneously, the relationship between the natural deletion and viral virulence was critical for the prevention and control of HPS.

2015年7月以来，心包积液综合征（HPS）在黑龙江、河南、吉林等地大规模流行，肉鸡死亡率达50～80%，蛋鸡达50%。此外，该病在鸭群中也有感染，死亡率达15%，给家禽养殖业造成严重经济损失。前期研究证明，该病由FAdV-4强毒株感染所致，其单独感染SPF鸡可导致80%的死亡率，并成功复制出严重心包积液和包涵体肝炎，与临床症状一致。全基因组测序发现，不同省份、宿主来源的14株临床分离株都在35413～37378位置出现1966 bp规律性缺失，该缺失包含ORF19、ORF27，它们与病毒毒力增强的相关性尚无报道。本研究拟利用细菌人工染色体技术（BAC），构建FAdV-4全基因组感染性BAC克隆，拯救ORF19、ORF27天然缺失、人工补全病毒，综合评价天然缺失对病毒毒力的影响，揭示天然缺失与毒力增强的相关性，对阐明FAdV-4毒力增强的分子基础并有针对性地开展HPS防控研究具有重要意义。

2015年7月以来，由新型FAdV-4强毒株感染所致的心包积液综合征（HPS）在我国大规模流行，给家禽养殖业造成严重经济损失。前期全基因组测序发现，我国流行毒株在35413～37378位置出现1966 bp规律性缺失，该缺失包含ORF19、ORF27，它们与病毒毒力增强的相关性尚无报道。本研究利用CRISPR/Cas9技术，首次建立了FAdV-4的反向遗传操作平台，成功拯救了ORF19、ORF27天然缺失、人工补全病毒，通过体内外综合评价，首次证明1966 bp的大片段缺失对病毒毒力增强没有影响。此外，本研究分离鉴定了第1株鸭源FAdV-4，并完成了全基因组测序，结果显示鸭源FAdV-4也存在1966 bp的天然缺失。诊断试剂方面，我们建立了禽腺病毒I群ELISA血清抗体检测方法，制备筛选了群特异性和型特异性的单克隆抗体，为抗原诊断方法的建立提供了工具。综上所述，本研究首次证明了我国FAdV-4流行毒株特有的ORF19、ORF27天然缺失对病毒毒力增强没有影响，建立的ELISA方法和制备的单克隆抗体对我国HPS的综合防控具有重要的意义。