血清4型禽腺病毒3'端135-bp自然缺失影响病毒致病性的研究

New emerging avian disease of hepatitis-hydropericardium syndrome (HHS, 30-100% mortality) broke out in chicken flocks in China since mid of 2015. The disease emerged in most areas covered the Northeast, East, Central and the Northwest of China. The HSP is usually caused by serotype 4 of FAdV. Previously, FAdV was thought as common existing virus in chicken flocks, now the virus varied and caused high mortality in birds. It is still not clear how the virus varied to possess high virulence. On the bases of epidemiology of FAdV infection in fowl flocks and sequence analysis of FAdV-4 genome, we analyzed the genetic properties and molecular marker between the high virulent viruses and the nonvirulent strains of FAdV. Three discontinuous deletion (135-bp sequence) were found in the 3’ end of the genome in high virulence isolates. Homologous recombination was conducted to construct recombinant viruses that were manipulated by artificial completion the deleted sequence from nonvirulent virus. The pathogenicity of the wild virulent virus and the chimeric virus were evaluated in SPF chickens by comprehensively considering the clinical score, mortality, mean death time, viremia, virus shedding, the hydropericardium and the liver damage, and the expression of TNF-α and IL-1β. These results will be helpful for elucidating the correlation between the 135-bp deletion and the pathogenicity of the virus and further to uncover the molecular mechanism. This study would induce the interest on the molecular features of virulent FAdV and provide suggestions for the control and elimination of fowl adenovirus infection.

自2015年以来，我国大部分地区鸡群广泛发生禽新发疫病——以发病急和高致死率（30-100%）为特征的肝炎-心包积水综合征，血清4型禽腺病毒是其主要致病因子。禽腺病毒从最初被认为是鸡群常在病毒到对鸡群产生高致病性，其毒力变化机制及遗传演化规律未见报道。本研究在对我国血清4型禽腺病毒基因组解析基础上，分析强弱毒株分子遗传规律及分子标记，发现强毒株基因组3’端存在135-bp不连续缺失。利用已建立的反向遗传操作系统，构建人工补全缺失序列的嵌合毒株，比较分析野生型强毒株和插入135-bp弱毒株序列嵌合毒株的致病性差异。主要从病毒对SPF鸡临床致病指数、致死率、平均死亡时间、病毒血症和排毒、致肝脏损伤差异、炎症相关因子表达水平等指标，评价两个毒株间的致病性差异，明确135-bp缺失序列是否与病毒致病性相关，探讨病毒毒力变化的分子机制，为FAdV疫病防控和净化提供理论依据。

肝炎-心包积水综合征（Hepatitis-hydropericardium syndrome, HHS）是由禽腺病毒血清4型（Fowl adenovirus 4, FAdV-4）引起的以肝炎、心包积水为特征的疾病。自2015年以来，HHS在我国大面积流行，给我国养禽业造成了严重的经济损失。. 为探讨影响病毒毒力的分子标记，本研究以FAdV-4强毒株HN/151025为研究对象，通过同源重组技术，缺失病毒的ORF17基因，构建重组病毒，评价ORF17基因对病毒毒力的影响。此外，经序列比对分析，发现我国流行的FAdV-4在基因组3’末端缺失1966-bp序列，为了探明该序列是否为强弱毒的分子标记，本研究采用同源重组技术构建插入ON1弱毒株的1966-bp序列，评价该自然缺失序列对病毒毒力的影响。将获得的重组病毒与亲本病毒人工感染SPF鸡，从发病率、死亡率、抗体变化、经口咽/泄殖腔排毒等方面评价重组病毒的致病性，并与亲本病毒进行比较。结果表明，构建的两株重组病毒均未改变病毒对SPF鸡的致病性，但是重组病毒的免疫原性发生了改变。因此得出结论，ORF17基因缺失以及在病毒基因组的35430位置插入弱毒株的1966-bp序列后形成的重组病毒，对SPF鸡的致病性无明显影响，病毒对肝脏组织的亲和性未发生改变，但是病毒刺激机体产生抗体的能力下降，说明上述基因位点的改变，使病毒的免疫原性发生了变化。