禽腺病毒血清4型感染诱导内质网应激介导凋亡和自噬的机制研究

Hydropericardium syndrome caused by fowl adenovirus serotype 4 (FAdV-4) has been widespread in China with a high mortality rate. Acute viral hepatitis is an important cause of death in chicken. Many studies have shown that some viral infections can induce endoplasmic reticulum stress in cells. When the stress is excessive or persistent, apoptosis and autophagy will be activated, eventually leading to occurrence of diseases. However, there is no report that FAdV-4 infection can induce endoplasmic reticulum stress-mediated apoptosis and autophagy. Previous studies have found that FAdV-4 infection can cause irregular vacuoles swelling in the endoplasmic reticulum of hepatocyte and significantly increase the transcriptional levels of endoplasmic reticulum stress marker protein. It could induce apoptosis and autophagy as well. Therefore, the scientific problem of endoplasmic reticulum stress-mediated apoptosis and autophagy induced by FAdV-4 infection has been focused. Through an infected cell model and molecular biology techniques, we will explore the endoplasmic reticulum stress triggered by FAdV-4 infection and its effect on viral replication; the relationship among endoplasmic reticulum stress, apoptosis and autophagy; as well as the activation pathway. These studies will provide theoretical basis for elucidating the pathogenesis of acute viral hepatitis caused by FAdV-4 infection, and provide experimental basis and novel ideas for the development of antiviral drugs.

禽腺病毒血清4型（Fowl adenovirus serotype 4, FAdV-4）引起的鸡心包积液综合征在中国大面积爆发流行，死亡率极高，急性变质性肝炎是鸡只死亡的重要原因。研究表明某些病毒感染可诱导细胞发生内质网应激，当应激过大或持续不减弱时，凋亡和自噬会被激活，导致疾病发生。目前对于FAdV-4感染诱导内质网应激介导凋亡和自噬的研究未见报道。前期试验发现，FAdV-4感染会导致肝细胞内质网呈无规则的空泡状肿胀，引起内质网应激标志蛋白转录水平显著升高，还诱导细胞发生凋亡和自噬。围绕FAdV-4感染诱导内质网应激介导凋亡和自噬这一问题，拟通过感染细胞模型，利用分子生物学技术，研究FAdV-4感染后触发的内质网应激及对病毒复制的影响，探索内质网应激与凋亡和自噬的关系及激活通路，为阐明FAdV-4感染导致急性变质性肝炎的致病机制提供理论依据，也为开发靶向抗病毒药物提供实验依据和新的思路。

心包积液综合征感染引发的急性病毒性肝炎是FAdV-4直接作用的结果，也是机体死亡的重要原因之一，阐明病毒导致急性病毒性肝炎的原因，可为深入研究FAdV-4的致病机制奠定基础。许多研究表明，一些病毒感染可诱导内质网应激。当内质网压力超过或持续时，它会激活自噬，最终引发疾病的发作。然而，从未有报告表明FAdV-4感染可诱导内质网应激介导自噬。先前的研究已经确定FAdV-4感染在肝细胞中可以诱发自噬；然而这种激活的潜在机制仍然未知。本研究探讨了FAdV-4感染诱导内质网应激介导的自噬分子机制。结果发现，FAdV-4感染可以导致细胞中内质网发生扩张，呈现无规则的空泡状；内质网应激蛋白标志物葡萄糖调节蛋白78的表达增加，表明FAdV-4可以诱导内质网应激的发生，并激活未折叠蛋白反应(UPR中三条相关的途径，分别包括PKR样ER蛋白激酶、转录因子6和肌醇需要酶1）途径。使用含有内质网应激诱导剂（衣霉素Tu）或是抑制剂（牛磺脱氧胆酸TUDCA）处理LMH细胞，发现病毒复制也是受到抑制和促进作用。为了探索FAdV-4可以通过PERK/eIF2α或IRE1信号通路激活自噬，用小干扰RNA或特异性化学抑制剂处理，均可抑制FAdV-4复制并影响自噬的发生。另外，通过构建的四种主要结构蛋白，发现其均可以诱导LMH细胞发生内质网应激并促进自噬。总之，目前的研究结果揭示FAdV-4可以激活内质网应激及相关的3条UPR途径，也为FAdV-4诱导自噬发生机制提供了新的见解，并为开发抗病毒药物提供了实验基础和新思路。FAdV-4感染LMH 24h后，代谢组学发现，FAdV-4可以影响LMH细胞中的代谢网络。在葡萄糖或谷氨酰胺缺乏的培养基中，FAdV-4的产生显著减少。结果发现宿主细胞代谢在病毒复制中的重要性，研究FAdV-4-调控宿主细胞代谢的异同，有助于研发靶向药物，减少感染。