鸭坦布苏病毒real time rt-pcr监测方法的建立及跨种间传播的初步研究-establishment of real time rt - pcr monitoring method for duck tembusu virus and preliminary study on its cross-species transmission.docx

进行检测，分析 DTMUV 对小鼠的抗体依赖增强作用，最后并对该试验现象进行验证。结果显示，DTMUV 以颅内接种方式感染小鼠时候，病毒可在小鼠的组织脏器中 进行增值，尤其以脑组织中病毒含量最高，病毒核酸最高可达 3000~4000 copies/mg；脾脏中病毒含量仅次于脑，肝脏中病毒含量最低约 800~600 copies/mg。感染小鼠还 表现为体重降低、采食废绝，脑部有轻微出血等症状，同时该病毒感染后小鼠还产生 高水平的中和抗体，其中 105TCID50 剂量组中 IgG 及 IgG2a 浓度可达 100 μg/mL 以上，而 104TCID50 及 103TCID50 剂量组显著低于 105TCID50 剂量组。另外，DTMUV 对小鼠 有明显的抗体依赖增强作用，二次感染后 4 天小鼠表现出严重的发病症状，并可在其组织中检测到约 350~1000 copies/mg 的病毒核酸，特异性 IgG 含量也极显著高于二次感染前水平。研究结果表明 DTMUV 具有一定的跨物种传播引起哺乳动物发病的能力，并且 该病毒可对哺乳动物产生抗体依赖增强作用。然而 DTMUV 是否具有潜在的跨种间传播感染人类的可能性尚需进一步的研究一旦该病毒在宿主细胞中找到合适的受体， 或该病毒在自然进化的过程中某些关键位点的氨基酸发生了特定的变异，很有可能会 导致病毒突破种属间传播的屏障引起人类的感染。关键词：鸭坦布苏病毒，Real-Time RT-PCR，跨物种传播，致病性AbstractA kind of acute infectious disease broke out successively in partial duck industries in some provinces of southeastern China since April 2010, mainly caused a sharp drop in egg production. The incidence was about 60%~100%. Once suffering from the disease after about 4~5 days, ducks led to decrease in egg production rapidly from the range of 80%~85% to 10%~30%, even didn’t lay eggs any more. There were some typical pathological changes in the infected ducks such as visible shrunken oaries, follicular bleeding, degradation and white tip shape necrosis of liver and so on. Originally the disease occurred in Jiangsu and Zhejiang provinces, soon continued in most duck industries in Hebei, Shandong, Anhui, Fujian, Guangdong province and so on. Besause of that, there was huge economic loss in duck industries in China. The viruses which caused the disease were isolated and identified as Duck Tembusu Viruses (DTMUV) by gene sequencing and virus cultures.This study aimed at establishing a rapid, sensitive and specific Real-Time RT-PCR diagnostic method for DTMUV. First of all, according to the the E gene sequence(GenBank login number：JQ957513)of DTMUV AH-10 strains, the specific primers weredesigned. Then the E gene was RT-PCR, and cloned into pBSK vector with a T7 promotor .The plasmid confirmed by sequencing was as a template,