人柯萨奇病毒A组6型（Coxsackievirus A6，CVA6）属于小核糖核酸（RNA）病毒科肠道病毒属，是引起手足口病（Hand foot and mouth Disease ,HFMD）另一主要病原体之一。近几年，由CVA6引起的HFMD已在多个国家和地区广泛暴发和流行，成为一个重要的医学关注对象。CVA6除感染婴幼儿之外，也可感染免疫功能正常的成年人。在其流行季节，易与其他肠道病毒混合感染形成新型变异毒株。此外，也能通过基因重组的形式形成新型变异毒株。目前，针对CVA6尚无有效的抗病毒药物或疫苗。因此，建立合适的动物模型对于有关CVA6的基础研究、抗病毒药物研究及疫苗研发具有重要意义。

本论文的研究为两部分。第一部分，建立CVA6病毒的细胞培养体系与核酸检测方法。首先对RD细胞进行培养，建立细胞培养体系，用于病毒培养、扩增；采用微量滴定法测定病毒的感染性滴度；纯化含有T7启动子的VP1基因片段，加“A”尾，连接T载体，感受态细胞转化，提质粒并线性化，经体外转录获得RNA标准品，利用标准品建立标准曲线，并对建立的方法进行灵敏性、特异性和重复性评价。结果 CVA6的感染性滴度为10^7.5CCID₅₀/ml；建立的CVA6 TaqMan探针实时荧光定量RT-PCR(real-time fluorescence quantitative RT-PCR，RT-qPCR)方法具有较高的灵敏性、特异性和重复性。结论 CVA6的感染性滴度可用于实验研究；建立CVA6 TaqMan 探针RT-qPCR方法可用于检测CVA6病毒核酸中的拷贝数。

研究与探讨CVA6感染动物模型的可行性，并通过建立的动物模型分析感染CVA6后的免疫学、病理学和病原学等变化，初步探究其致病机制。选用与人类较为接近的恒河猴婴猴（3-4月龄），经呼吸道和消化道两种感染方式，以感染滴度为10^6.5CCID₅₀/ml、感染剂量为100μl的CVA6病毒液感染恒河猴婴猴，构建CVA6感染动物模型。在恒河猴婴猴感染CVA6后逐日观察记录其临床表现，按时间点采集样品（血液、咽拭子、粪便），并对样品进行血常规、生化、细胞因子及中和抗体效价进行检测，通过CVA6 TaqMan 探针RT-qPCR方法检测模型感染CVA6后样品中的病毒血症情况及主要组织器官中的病毒载量变化；利用中和试验检测抗体效价；通过病理切片观察各主要组织器官的病理变化。结果 CVA6病毒可通过消化道（口腔）和呼吸道（鼻腔粘膜）感染方式引起恒河猴婴猴出现与人类HFMD相似的发热和唇部、前肢出现疱疹及脱皮等临床症状，血常规和生化检测均发生了相应改变；CVA6 TaqMan 探针RT-qPCR方法检测血液、咽拭子、粪便中出现了典型的病毒血症和排毒情况，各组织器官中均有病毒载量，其中以各肠段和淋巴结中病毒载量最为高；通过病理切片观察，在皮肤疱疹病理组织中观察到有炎性细胞浸润及中性粒细胞和淋巴细胞出现,同时在不同组织器官（肝、肺 、淋巴结、回肠等）有不同的病理改变；通过免疫组化方法在各组织中检测CVA6的抗原；同时，通过免疫学（补体、免疫球蛋白、细胞因子）检测，免疫学指标均有变化。结论 CVA6病毒可感染3-4月龄恒河猴，出现手足口病临床症状；相关免疫细胞、免疫蛋白、免疫因子均发生变化；肝、肺及淋巴结等多器官出现病理样的改变。以上结果表明，CVA6病毒可在恒河猴机体内复制、增殖及被清除，该动物模型反映了一个完整的病毒感染过程。此外，该动物模型在构建过程中的检测结果对于CVA6的致病机制研究和疫苗及抗病毒药物的研制也给予了一定的提示。

Human Coxsackievirus group A type 6 (Coxsackievirus A6, CVA6) belongs to the enterovirus genus of the small ribonucleic acid (RNA) virus family and is one of the pathogens that cause hand foot and mouth disease (HFMD). In recent years, HFMD caused by CVA6 has been widely outbreaked and epidemicd in many countries and regions, and has become an important object of medical attention. In addition to infecting infants,CVA6 can also infect adults with normal immune function. In epidemic season, it’s easy to mix with other enteroviruses to form a new variant strain. In addition, new mutant strains can also be formed in the form of gene  recombination. Currently, there is no effective antiviral drugs or vaccines against CVA6. Therefore, the establishment of an appropriate animal models is of great significance for basic research, antiviral drug research and vaccine development related to CVA6.

The research of this paper is divided into two parts. In the first part, the cell culture system and nucleic acid detection method of CVA6 virus were established. Firstly, RD cells were cultured to establish a cell culture system for virus culture and amplification.The infected titer of virus was determined by microtitration. After purification of VP1 gene fragments containing T7 promoter, adding“A”tail, connecting T vector, transformation of receptive cells, plasmid extraction and linearization,RNA standard was obtained through in vitro transcription. Standard curve was established using the standard, and specificity, sensitivity and repeatability evaluation were conducted for the establishment method. Results The infective titer of CVA6 was 10^7.5CCID₅₀/ml; the established CVA6 TaqMan probe real-time fluorescence quantitative RT-PCR（RT-qPCR）method had high sensitivity, specificity and repeatability. Conclusion The infective titer of CVA6 can be used for experimental study; The CVA6 TaqMan probe RT-qPCR method was established to detect the copy number of CVA6 virus nucleic acid.

In the second part,the feasibility of animal model of CVA6 infection was studied and discussed, and the changes of immunology, pathology and etiology after CVA6 infection were analyzed through the established animal model, so as to preliminary explore the pathogenic mechanism. A CVA6 infected animal model was established by infecting rhesus macaque infants (aged 3-4 months)with 100μl does at 10^6.5 CCID₅₀/ml though two infection models of respiratory tract and digestive tract. Daily observe and record the clinical manifestations of rhesus monkey infant monkeys infected with CVA6, according to the time points collect samples (blood, throat swabs, fecal samples),and the samples of blood routine, biochemical, cytokine and neutralizing antibody titer test, though CVA6 TaqMan probe RT-qPCR method to detect the viremia in the sample after the model is infected with CVA6 and the viral load changes in the main tissues and organs;neutralization test was use to detect antibody titer; pathological changes of the main tissues and organs were observed through pathological sections. Results The CVA6 virus could induce clinical symptoms such as fever, mucosal herpes, herpes on the hands and feet, and peeling similar to HFMD in rhesus monkey infants through the infection of digestive tract (oral) and respiratory tract (nasal mucosa).Typical vireaemia and detoxification were detected in blood, throat swabs and fecal samples by CVA6 TaqMan probe RT-qPCR. Viral load was found in all tissues and organs,with the highest viral load in each intestinal segment and lymph node; Pathological examination showe inflammatory cell infiltration, neutrophils and lymphocytes were observed in the pathological tissues of skin herpes, different pathological changes in different tissues and organs (liver, lung, lymph node, ileum,etc).Immunohistochemical method was used to detect CVA6 antigen in various tissues. At the same time, through the immunological (complement, immunoglobulin, cytokine) detection, immunological indicators have changed. Conclusion CVA6 virus can infect rhesus macaques at 3-4 months old, presenting clinical symptoms of hand, foot and mouth disease.The related immune cells, immune proteins and immune factors all changed, and pathological changes in many organs, such as liver, lung and lymph nodes. These results indicated that CVA6 virus could replicate, proliferate and be cleared in the rhesus monkey, and the animal model reflects a complete process of viral infection. In addition, the detection results of this animal model provide some hints for the study of pathogenic mechanism of CVA6 and the development of vaccines and antiviral drugs.