中文摘要

目的：随着地球自然环境的退化和全球化进程的加快，人畜共患病毒病对人类 的影响越来越大。新冠疫情已经在全世界造成几十万人的死亡，据世卫组织的溯源 报告中指出，新冠病毒极有可能是起源于动物。及时准确的实验室检测对于监测新 发人畜共患病和疫情的防控起着至关重要的。树鼩作为在亲缘性上比啮齿类动物更 接近于人类的动物，尽管在许多疾病模型上的研究已经有了不错的进展，但在病毒 组学的研究方面还比较欠缺。树鼩目前存在遗传背景纯度较低的问题，所以对于树 鼩病毒组学的研究相对迫切且对于人类多种病毒在树鼩体内的研究具有指导价值。

方法：（一）将树鼩血清样品通过 0.22μm 低蛋白过滤器将血清中的细菌和细 胞杂质去掉，然后基于病毒衣壳蛋白耐受核酸酶的特性，利用 DNase I 核酸酶处理 宿主核酸后用非序列依 赖 性引物 对 处 理后的 树 鼩 血 清 样 品 进 行 随 机 PCR （Polymerase Chain Reaction）扩增，再基于随机扩增片段设计特异性引物进行 长片段扩增和基因步移扩增，获得完整基因组片段。（二）通过体外转录出病毒基因 组 RNA 构建 Realtime-PCR 标准品，结合在保守非结构蛋白基因 NS5B 上设计的探针 引物，制备病毒核酸检测系统；通过对非结构蛋白 NS3 蛋白表达与纯化，建立病毒 抗体检测系统；通过制备 NS3 蛋白的单克隆抗体与多克隆抗体的制备，建立病毒的 抗原检测系统。

结果：我们发现了一种尚未报道过的树鼩肝炎病毒，将其命名为 Tupaia Hepacivirus（THV），并且存在两种不同的病毒株。成功获取到了二型的树鼩肝炎病 毒（THV2）株的完整 ORF 序列信息，并构建了 THV2 病毒的相关进化树。并且构建了 THV2 病毒的检测系统：通过 Realtime-PCR 检测系统可以检测树鼩血清或组织中的 病毒基因组拷贝数；通过非结构蛋白 NS3 蛋白的表达，我们可以从树鼩血清中检测 到抗 THV2-NS3 抗体的存在；通过抗 NS3 蛋白单克隆抗体和多克隆抗体的制备，可 以从树鼩肝脏组织中检测到抗原的存在，也可以从 IFA（Immunofluorescence Assay） 中判断出病毒 NS3 蛋白在细胞中的表达情况。

结论：树鼩肝炎病毒 Tupaia Hepacivirus 是一种未报道过的动物性肝炎病毒， 具有较强的嗜肝性，并且可以引起树鼩的慢性感染和肝炎的发生。在相似度上更加 2 接近于从地松鼠中发现的啮齿类肝炎病毒，而从亲缘关系上来看与啮齿类肝炎病毒 和环尾浣熊类肝炎病毒是属于同一支。从同源性上看，THV 病毒与 HCV（Hepatitis C Virus）的氨基酸同源性接近 30%。我们希望发现的是一种在病毒的特性和感染机 制上更加接近于人感染丙型肝炎病毒的病毒，来尽可能改善 HCV 病毒缺乏动物模型 的现状。

ABSTRACT

Objective: The tupaia, as a representative of the climbing shrews, is considered to be a close relative of non-human primates. Due to its small size that are easy to breed, it has been used as animal models to study human diseases. Standard research on this experimental animal under the condition of artificial breeding were carried out. However, there is still a lack of information about the infection of natural pathogens in tupaia. It is known that tupaia is often used in the research of blood borne virus infection such as human hepatitis B virus and human hepatitis C virus. In order to eliminate the potential threat of zoonosis and the interference to animal model research, as well as find new pathogens, this research is planned to conduct preliminary research and exploration on the new pathogens in the blood of tupaia. 

Methods: (1) The tupaia serum samples were filtered through a 0.22 μm low protein filter to remove bacteria and cellular impurities in the serum. Based on the characteristics of virus capsid protein’s resistance to the nuclease, the host nucleic acid is treated with DNase I nuclease, extracting the possible genetic material of pathogens protected by viral structural proteins. Sequence alignment was carried out after SISPA (Sequence-Independent Single Primer Amplification) technology, and the possible candidate fragments of virus genome were selected. Then, specific primers were designed based on the candidate virus fragments for long fragment amplification and gene step-by-step amplification, then the coding fragment sequence of novel virus gene histone was obtained. (2) According to the genome sequence of the new virus, a relatively complete 4 detection system of virus nucleic acid, protein and immune system was established.

Results: In this study, a new type of tupaia hepatitis virus was found. After successfully obtaining the complete ORF sequence information of the virus, the virus was found to be homologous with human hepatitis C virus, and it was named as Tupaia Hepacivirus type II. Furthermore, the nucleic acid, protein and antibody detection system of THV2 virus was constructed: The virus genome in tupaia serum or tissue can be detected quantitatively by Realtime-PCR detection system, the existence of anti THV2 NS3 antibody in the serum of infected tupaia can be evaluated by expressing non-structural protein NS3 protein, what’s more, through the preparation of monoclonal antibody and polyclonal antibody against THV2 NS3 protein, the NS3 virus antigen in tupaia tissue can be detected quantitatively and qualitatively by immunological methods.

Conclusion: Tupaia Hepacivirus is a new type of animal hepatitis C virus, which has strong infectivity and hepatotropism. Although it is similar to rodent hepacivirus in homology, THV virus and human HCV (hepatitis C virus) are still have high similarities in protein coding and infection mechanism. The discovery of tupaia hepacivirus and the subsequent research were expected to provide a new animal model for the study of hepatitis C virus infection and vaccine development.