诱导型一氧化氮合酶与精氨酸酶调节猪圆环病毒2型复制的细胞分子机制

Porcine circovirus type 2 (PCV2) is one of the primary causative agent, causing great economical loss affecting the global swine industry today. Mcrophages play an important role in antiviral effects and immuneregulation, particularly through the activities of inducible nitric oxide synthase (iNOS) and arginases. However, to date, mechanisms of PCV2 replication regulated by iNOS and arginases remain unknown. Porcine alveolar macrophages cells (PAMs) serve as cell models for PCV2 replication in vitro, in addition, quantitative PCR, indirect immunofluorescence, Western blot, flow cytometry and confocal laser scanning microscope, etc, are used to investigate the effects of iNOS and arginases on PCV2 replication and associated mechanisms in the project. In this proposal, studies will be conducted as follows: Effects of iNOS/arginase activities on PCV2 replication; Effects of L-arginine metabolism regulated by iNOS/arginase on PCV2 replication; Effects of iNOS/arginase on regulation of apoptosis and cell autophagy induced by PCV2. Mechanisms of PCV2 replication regulated by iNOS and arginase are estimated at cellular and molecular levels as noted above, so pathogenesis of PCV2 is enriched. Moreover, these results will provide crucial scientific basis and new strategies for prevention and control of PCVAD.

猪圆环病毒2型（PCV2）是目前猪场危害最大的病原之一，对世界养猪业造成巨大的经济损失。巨噬细胞在抗病毒和免疫调节过程中发挥重要作用，其作用主要受控于诱导型一氧化氮合酶(iNOS)和精氨酸酶的活性，目前，巨噬细胞中iNOS和精氨酸酶调控PCV2复制的机理尚无报道。本项目以猪肺泡巨噬细胞（PAMs）作为PCV2体外复制的细胞模型，通过定量PCR、间接免疫荧光、Western blot、流式细胞术和激光共聚焦等技术研究iNOS与精氨酸酶对PCV2复制的影响及其相关机理，具体研究如下：iNOS/精氨酸酶的活性对PCV2体外复制的影响；iNOS/精氨酸酶调节L-精氨酸代谢对PCV2复制的影响；iNOS/精氨酸酶对PCV2诱导的细胞凋亡与自噬的调控。从细胞和分子水平揭示iNOS与精氨酸酶调控PCV2复制的相关机机制，丰富了PCV2的致病机理，为猪圆环病毒相关疾病的防控提供新的科学依据。

猪圆环病毒2型（PCV2）是猪圆环病毒病（PCVAD）的主要病原之一，对世界养猪业造成巨大的经济损失。一氧化氮（NO）作为一种重要的信号分子，能抑制多种 DNA、RNA 病毒复制和感染。NO对PCV2复制的抑制作用本课题组的前期工作已经证实，然而，NO抑制PCV2复制的机理尚未见报道。本课题研究内容包括：一氧化氮供体 GSNO 抑制 PCV2 复制的体内外研究；NO 自由基（NO• ）对猪圆环病毒 2 型复制的抑制作用研究；SNAP 产生的外源性 NO 抑制 PCV2 的体外复制和调节 NF-κB 活性的研究。结果表明：体内、体外试验证实NO抑制PCV2的复制，NO抑制PCV2的复制通过NO自由基形式发挥作用，初步阐明PCV2感染中NO调节NF-κB通路的机制。上述结果从细胞和分子水平揭示NO抑制PCV2复制的机理，为猪圆环病毒病的防控提供新的策略。