鸭源基因Ⅶ型NDV对绍鸭、番鸭致病性差异的免疫学机制分析

Present research related to interaction mechanism between Newcastle disease virus and its hosts mainly focus on pathogen profile, it is seldom to illustrate this question based on host natural immunity, which acts important role in resistance to NDV infection. In our previous experiments, a NDV strain (NDV/Md/CH/GD/1/05) isolated from dead ducks was classified into genotype Ⅶbelonging to Class I, and its pathogenicity was evaluated on Shaoxing ducks and Muscovy ducks. Experimental results showed that Md strain could induce 100% morbidity and high mortality in Muscovy ducks, in contrast to the asymptomatic in Shaoxing ducks. The results indicated that Md strain had an obviously pathogenicity differentia to Shaoxing ducks and Muscovy ducks, in other words, there is diversity on duck breeds susceptible to NDV infection. To analysis the mechanism related to pathogenicity differentia, we select the host nature immune response as analysis objective to illustrate this mechanism. 8 kinds of immune-related genes correlation on antivirus nature immune were selected as analysis objective, they are some important cytokines, including IL-1, IL-2, IL-12, IFN-α, IFN-β, IFN-γ, and host cell pattern recognition receptors, such as Toll-like receptor 7(TLR7) and RIG-I receptor. Using real-time quantitative PCR, we examined the dynamic expression level of immune-related genes response to NDV duplication. On the other hands, we also expressed RIG-I gene in DF-I cell, and examined the dynamic expression level of other cytokines in response to Md strain infection. By analysis the relations: (1) immune-related genes dynamic expression level related to NDV- Md pathogenicity; (2) pattern recognition receptors expression level and other cytokine response; (3) immune-related genes related to duck breeds susceptibility to NDV infection. We hope that these data may provide insights into the molecular mechanism of NDV interaction with its hosts.

新城疫病毒与宿主间相互作用机制的研究，目前多侧重于病原角度，而在宿主抗NDV感染的天然免疫机制方面，迄今未见系统的研究报道。鉴于此，本项目基于前期试验中发现的一株鸭源基因Ⅶ型NDV对番鸭、绍鸭致病性差异较大这一现象，以与抗NDV感染密切相关的免疫因子为切入点，从宿主角度分析致病性差异的免疫学机制。研究以番鸭和绍鸭为感染模型，对细胞因子（IL-1、2、12;IFA-α、β、γ）和细胞模式识别受体（TLR7、RIG-I）mRNA动态表达水平、病毒复制水平进行定量检测；同时将鸭RIG-I基因在DF-I细胞中表达，定量检测病毒感染后细胞因子表达、病毒复制水平。通过分析免疫因子表达与病毒致病性、细胞模式识别受体与细胞因子调控，以及免疫因子与鸭品系对NDV易感性之间的关系，探讨NDV对番鸭、绍鸭致病性差异的免疫学机制。研究结果可为NDV与宿主间相互作用、病毒致病机制相关研究提供基础数据。

作为新城疫病毒（NDV）重要宿主，鸭在NDV进化、传播中有重要作用。以本项目为依托，选择不同禽源、基因型、毒力NDV，人工接种番鸭、绍鸭、金定鸭，力图从宿主、基因型、毒力方面分析NDV对几种鸭致病性差异。在此基础上，测定感染过程中宿主的主要免疫因子表达和变化情况，分析NDV致病性与鸭天然免疫的关系。.实验结果表明，NDV毒株对鸭致病性、病毒复制特点差异较大。疫苗株LaSota对番鸭、绍鸭、金定鸭无致病性，可刺激产生低水平血清抗体，组织中病毒分离率低，水平传播能力弱。ClassⅠ株对番鸭、金定鸭无致病性，接种鸭抗体水平显著高于LaSota，表明其在鸭体具有较高复制能力，这一数据可以部分解释ClassⅠ型NDV在我国禽群中近年来分离率高于疫苗株和流行强毒株这一现象。F48E9株对番鸭、绍鸭致病性和水平传播能力均较强。.Md株（鸭源）、WB363（野鸟）和HLJ01/06（鸡）同属于ClassⅡ基因Ⅶ型，属于近几十年来世界范围内流行的基因毒株。3株病毒基因组序列同源性在95%以上，对鸭致病性表现出基于鸭品系、病毒宿主源方面的差异。Md株对15日龄番鸭具有高致病性，对15日龄绍鸭未表现出致病性，WB363则对番鸭和绍鸭均未表现出致病性。鸭对致病性NDV具有较强日龄抵抗力，Md株对40日龄番鸭致病性低于15日龄番鸭；Md株对35日龄金定鸭致病性较低，WB363株则对2种鸭均未表现出致病性。鸡源HLJ01/06株对40日龄番鸭致病性高于35日龄金定鸭，但其致病性弱于Md株。.NDV的这种致病性差异目前在野鸭方面有报道，本项目属于首次将各种类型NDV代表株对番鸭进行致病性差异研究，这种差异也是分析NDV致病性与鸭天然免疫关系的基础。基因Ⅶ型NDV对番鸭、绍鸭、金定鸭致病性差异的各个免疫学指标正在测定中，未全部完成。目前只有20日龄绍鸭接种Md株后，主要细胞因子IL-2、IL-18、IL-4、IL-6、IFN-γ的检测变化情况。结果表明，IL-2在接种后48h的10种组织中均显著升高；IL-18、IL-4、IFN-γ在接种后48h的肺脏、肾脏中显著升高。