戊型肝炎病毒兔分离株跨种系传播及致病性研究

The morbidity of Hepatitis E (HE), caused by hepatitis E virus (HEV), has increased dramatically in China over the last decade. HEV has been isolated from a variety of domestic and wild mammals, including pigs, boar and deer, and swine is the main reservoir of HEV. Recently HEV has been first isolated from rabbits in China by researchers including our group. The newly identified rabbit HEVs shared high nucleotide sequence identity (73%-79%) with HEV genotypes 1-4 and was most closely related to HEV genotype 3. Because of the zoonosis specificity of HEV genotype 3, the possibility of cross-species transmission of rabbit HEV is supposed. Rabbits are mainly domestic animals which has frequent contacts with humans, therefore, it is urgent to determine the possibility of cross-species transmission of rabbit HEV. Besides, new problems associated with HEV infection, such as chronicity and neurological symptoms, are emerging in recent years, so that further study for the pathogenicity of HEV is urgently needed..Our previous study supposed that 25 amino acid positions were involved in specifying the host range of HEV and/or determining the severity of hepatitis E disease. The present study will focus on the mechanism of host specificity and pathogenicity of rabbit HEV. To find out direct evidences of zoonotic transmission of rabbit HEV, the investigation of HEV antibody prevalence among persons who work with rabbits and patients with acute viral hepatitis will be carried out. Experimental infection of rabbits, cynomolgus monkeys and pigs with rabbit HEV will be also done to demonstrate the possibility of cross-species transmission of rabbit HEV. Meanwhile, infectious full-length cDNA clones of rabbit HEV will be constructed and the mechanisms of cross-species infection will be clarified using site-directed mutagenesis to mutate the 25 specific amino acid positions mentioned above. The results of this study will greatly contribute to the prevention of hepatitis E in our country.

由戊型肝炎病毒(HEV)引起的戊型肝炎(HE)近年来在我国发病呈迅速上升趋势。目前已从猪、野猪、鹿等动物中分离到HEV，但猪是HEV主要宿主。最近我国学者及本研究组首先从兔群分离到HEV，与已知4个型别HEV同源性高达73%-79%，但与3型最接近。3型HEV具有人畜共患特征，因此兔HEV也有可能跨种系传播。鉴于人与兔接触密切，兔HEV跨种系传播问题亟待明确。此外近年HE出现了慢性化、神经症状等新问题，亟待深入研究HEV致病性。.本课题组前期研究发现25个氨基酸位点与HEV宿主范围和致病性相关。本课题将围绕兔HEV跨种系传播及致病性深入研究并阐明其分子机制。拟采用与兔密切接触人群的流行病学调查、兔HEV实验感染兔、猴和猪，构建兔HEV感染性克隆并定点突变与宿主特异性相关位点等方法，揭示兔HEV跨种系传播的可能性并验证其宿主特异性和致病性的分子机制。本研究成果对我国HE防控有重要意义。

背景：.由戊型肝炎病毒(HEV)引起的戊型肝炎(HE)近年来在我国发病呈迅速上升趋势。目前已从猪、野猪、鹿等中分离到HEV，但猪是HEV主要宿主。最近我国学者及本研究组首先从兔群分离到HEV，与已知4个HEV型同源性高达73%-79%，但与3型最接近，目前被归为HEV-3。3型HEV具有人畜共患特性，因此兔HEV也有可能跨种系传播。鉴于人与兔接触密切，兔HEV跨种系传播问题亟待明确。此外近年HE出现了慢性化、神经症状等，亟待深入研究HEV致病性。本课题组前期已发现25个氨基酸位点与HEV宿主范围、致病性相关。本课题围绕兔HEV跨种系传播及致病性深入研究并阐明其分子机制。.内容：.采用与兔密切接触人群的流行病学调查、兔HEV感染兔、猴，构建兔HEV感染性克隆并定点突变与宿主特异性相关位点等方法，揭示兔HEV跨种系传播的可能性并验证其宿主特异性和致病性的分子机制。.重要结果、关键数据、科学意义：.（1）首次证实兔HEV可跨种系感染猕猴并致其出现典型急性戊肝，包括ALT升高、抗HEV抗体阳转、粪便排毒等，并从感染猴子粪便中分离到HEV RNA，其与注射毒株HEV同源性接近100%，说明兔HEV能成功感染猕猴并在体内复制，间接说明兔HEV有潜在感染人的可能性。.（2）首次发现兔感染兔HEV后出现了超过半年以上粪便排毒并伴有肝炎，提示兔HEV感染可致动物出现慢性化。此外，肝外组织如肾、小肠等均检测到了正、负链HEV RNA，说明HEV有肝外复制。同时，首次在HEV慢性感染兔中发现严重肾损伤，肾组织HEV正负链RNA检测和病理、免疫组化结果表明，肾损伤是因HEV在肾脏中直接复制所致。此结果首次在动物模型水平上发现了HEV引起肝外症状的原因。另外，首次发现孕兔感染兔HEV后出现了与孕妇感染HEV相同的不良反应（流产、死胎、孕兔高死亡率），并发现HEV可在胎盘复制，推测这些不良结局与其有关。首次应用兔模型进行疫苗评估，证实了2次10 μg的HEV 239疫苗可保护兔免受高载量HEV感染。这些结果使基础研究与临床研究有机结合起来，对深入阐述HEV致病特点及预防措施均有重要意义，也提示兔可用于HEV急、慢性感染、肝外复制、孕妇高病死率、疫苗评估研究的理想动物模型。.（3）成功构建了第一个兔HEV全基因组cDNA克隆，证实了其感染性。并通过基因敲除证明了其特有的31个氨基酸与其复制有关。