新城疫病毒P基因编辑频率与病毒复制及宿主免疫状态之间关系的研究

Paramyxovirus antagonize the host IFN system by mediating viral IFN-antagonistic proteins, V, W and so on, which are encoded by a process of viral phosphoprotein (P)-gene mRNA editing. And the expression levels of mRNA of V and W proteins of Nipah virus are dynamic variation during different time of virus infection. We found that the ability of IFN antagonism of NDV V protein is closely related to its virulence. Thus infer that the editing frequency of the P gene must be dynamic during Newcastle disease virus (NDV) infection in cells and hosts. In different situation of infection or different immune state of cells and hosts, virus may adjust the level of V/W by changing the P gene editing frequency to antagonize IFN and other immune factors and the viral infection and proliferation in cells and tissues. Total five NDV isolates with different virulence and genotypes will be used for infection in chicken cells, SPF chickens and different immune state chickens in this study. And the editing frequencies of its P gene, IFN and other immune cytokines expression levels, pathological changes of cells and tissues, viral titers and detoxifications will be analysized during different infection time. This study will clarify the relationships between the frequency variation of P gene mRNA editing and viral virulence, proliferation, immune state in the process of NDV infection in vitro and in vivo. Will eventually reveal the role of the variation of NDV P/V/W gene editing frequencies in affecting virus proliferation and the mechanisms of breaking the host immune defense, which would provide a new information to further clarify the NDV pathogenesis and immune mechanism.

副粘病毒通过P基因的mRNA编辑编码V、W等蛋白来拮抗宿主的IFN系统，且NiV V/W蛋白的mRNA水平随感染状态而实时变化。我们发现NDV V蛋白拮抗IFN的能力与毒力密切相关。因此推断，NDV在感染细胞和机体中P基因编辑频率也应动态变化，病毒可能在感染不同免疫状态的机体、细胞时，通过P基因编辑频率改变以调控V/W的水平来拮抗IFN等免疫因子与病毒在组织、细胞中的感染、增殖程度。本研究拟以5株不同毒力与基因型的NDV感染细胞、SPF鸡和免疫状态不同的鸡，测定不同时间点P基因编辑频率、IFN等免疫因子的量、细胞与组织病变程度、病毒滴度及排毒量，从体外和体内水平阐明NDV感染过程中P基因编辑频率的动态变化与病毒毒力、增殖能力和免疫状态间的关系，揭示NDV P/V/W基因编辑频率的变化在影响病毒增殖与突破机体免疫防御中发挥的作用，为进一步阐明NDV致病机理与免疫机制提供新的资料。

新城疫病毒P基因可以编码P、V和W 3种蛋白，其中V蛋白与病毒拮抗宿主干扰素相关，但并不清楚P、V、W三种基因编辑比例及与宿主免疫反应之间的关系。本项目围绕NDV感染后P、V、W编辑变化、宿主基因转录变化及与V蛋白相关的宿主蛋白功能展开研究。结果显示：F48E9和La Sota两种病毒感染DF-1细胞后，利用二代测序发现，不同时间点P基因编辑频率较为稳定，P、V及W mRNA表达分别为53.17%～59.73%，31.84％～37.37%和8.44％～10.48%。鸡胚感染NDV后测序发现，F48E9 P、V、W转录比为50.48%: 30.68%: 18.84%，La Sota为47.3%: 30.33%: 22.37%。NDV感染法氏囊测序发现，P、V、W转录比为48.6-54.54%: 29.24-32.63%: 16.22-18.94%，与细胞和鸡胚中测得结果相似。这些结果表明在NDV感染宿主时，P、V、W三种基因的比例相对稳定，受宿主因素影响较小。利用鸡胚感染模型研究NDV感染对IFN调控发现，F48E9感染可上调1190个基因，下调845个基因，La Sota感染可上调992个基因，下调612个基因，筛选与干扰素相关基因，F48E9所引起的差异基因明显多于La Sota引起的，表明NDV强弱毒对IFN诱导能力存在差异；将转录组与小RNA数据库关联分析，得到130个与免疫和炎症反应相关miRNA-mRNA pairs。对F48E9感染的不同时间点法氏囊测序分析发现，共有176个共同表达的差异基因，且随着感染时间，干扰素诱导基因的表达越来越少，而抑制宿主天然免疫反应的基因随之表达，有利于病毒的复制。对SOCS3深入研究发现，过表达可明显促进NDV增殖，这是通过抑制STAT1蛋白磷酸化，进而抑制干扰素刺激基因OASL和MX1表达来实现的。mRNA-miRNA关联分析发现，SOCS3是miR-455-5p作用靶点，NDV感染能够抑制gga-miR-455-5p表达而促进SOCS3表达。利用酵母双杂交技术筛选出22个与V蛋白互作的宿主蛋白，证实了V蛋白可负调控C1QC、TXNL1、CacyBP和hnRNPH1表达，参与ERK、Bcl-2/Bax、Caspase-3和细胞增殖等信号通路，利于NDV的增殖。这些研究扩充了对NDV病毒基因的研究，也为疫苗研究奠定了基础。