不同毒力犬瘟热病毒株调控Ⅰ型干扰素信号通路机制的比较研究

Canine distemper virus (CDV) infects a broad range of terrestrial carnivores and mainly cause high morbidity and mortality epidemics in dogs and fur-bearing animals, including mink, raccoon dog, and fox. Now, canine distemper (CD) is a very serious problem for dogs industry and fur-bearing animals industry in our country. Currently, attenuated CDV strains are routinely used worldwide as live vaccines against CDV infection in dogs, minks, and other carnivores. Vaccination of CD attenuated vaccines for dog is an efficient method to prevent CDV infection. However, it sometimes can cause apparent infection, even fatal infections, in minks and other carnivores. Virulent strains of morbilliviruses, including meals virus (MV) and CDV, use different mechanisms to inhibit type I interferon (IFN) signaling pathways, which contribute to virus to evade host innate immune response and help to infection and spread of virus. However, previous study showed that MV vaccine strains can induce the robust production of IFN-β in human myeloid dendritic cells. So far, type I interferon-regulatory mechanisms of CD attenuated vaccines are poorly understood. In this study, we will compare type I interferon-regulatory mechanisms of attenuated CDV vaccine strains with those of the wild-type CDV isolated and passage on Vero-SLAM cells, which contribute to clarify the question that vaccination of CD attenuated vaccines sometimes can cause apparent infection, even fatal infections, in minks and other carnivores. On its basis, we will modify the genome of CDV attenuated vaccine strains using site-directed mutagenesis to develop the safe and effective CD vaccine by reverse genetics, which protect fur-bearing animals from CDV infection.

犬瘟热病毒宿主范围广泛，主要引起犬和貂、貉、狐等肉食动物发病和死亡，是对我国养犬业和毛皮动物养殖业危害最大的疫病原之一。目前，弱毒疫苗是国内外使用最为广泛的犬瘟热疫苗，免疫犬可取得良好的免疫保护效果，但免疫接种水貂、貉、狐等皮毛动物，有时能引起症状明显的、甚至致死性的感染。犬瘟热病毒、麻疹病毒等麻疹病毒属成员强毒株感染宿主，可通过不同的分子机制阻断Ⅰ型干扰素信号转导途径，有助于病毒逃逸宿主天然免疫监视，从而有助于病毒感染和扩散。然而，对麻疹病毒疫苗株研究表明，其感染树状突细胞可诱导β-干扰素表达。现在，不同毒力犬瘟热弱毒疫苗株调控宿主Ⅰ型干扰素信号转导机制还不是十分清楚。本研究致力于比较不同毒力犬瘟热病毒在犬源细胞和貂源细胞中调控Ⅰ型干扰素信号转导机制，从而探寻犬瘟热弱毒疫苗免疫接种水貂引发疾病的原因，并在此基础上对犬瘟热弱毒疫苗株基因组修饰突变，从而构建更加安全、有效的犬瘟热疫苗。

犬瘟热病毒宿主范围广泛，主要引起犬和貂、貉、狐等肉食动物发病和死亡，是对我国养犬业和毛皮动物养殖业危害最大的疫病原之一。目前，弱毒疫苗是国内外使用最为广泛的犬瘟热疫苗，免疫犬可取得良好的免疫保护效果，但免疫接种水貂、貉、狐等皮毛动物，有时能引起症状明显的、甚至致死性的感染。犬瘟热病毒、麻疹病毒等麻疹病毒属成员强毒株感染宿主，可通过不同的分子机制阻断Ⅰ型干扰素信号转导途径，有助于病毒逃逸宿主天然免疫监视，从而有助于病毒感染和扩散。本研究以不同毒力犬瘟热病毒P、C、V蛋白为研究对象，对3种蛋白抑制宿主细胞Ⅰ型干扰素信号转导的能力及差异进行分析。研究证实，P和C蛋白仅有轻微地抑制Ⅰ型干扰素信号转导的能力，且各毒株之间无显著性差异；但是，V蛋白能明显地抑制Ⅰ型干扰素信号转导，且不同毒株之间存在显著地差异，分离株JL17、TM-CC和强毒株A75-17 V蛋白可显著地抑制宿主细胞Ⅰ型干扰素信号的转导，而疫苗株Onderstepoort V蛋白仅有轻微的抑制作用，然而疫苗株20/8也具有显著地抑制作用。结果表明，V蛋白是犬瘟热病毒抑制Ⅰ型干扰素信号转导主要因子之一。为了进一步研究V蛋白的作用机制，本研究构建了用于酵母双杂交的犬源细胞cDNA文库和诱饵质粒，3个毒株的缺失表达V和/或C蛋白的重组犬瘟热病毒和表达EGFP、且缺失表达V和/或C蛋白的重组犬瘟热病毒，建立了另外一株疫苗病毒Snyder Hill的反向遗传技术平台，分离、并正在建立分离株JL17的反向遗传技术平台，为进一步系统研究犬瘟热病毒致病机制研究和探索研制一种安全、有效的貂犬瘟热疫苗奠定了基础，将具有广阔的应用前景。