目的：通过构建肠道病毒混合感染（Co-infection）叙利亚金黄地鼠（Syrian golden hamster）和恒河猴（Rhesus monkey）婴猴的动物模型，初步研究人类肠道病毒的混合感染机制，为多价疫苗和抗病毒药物研发奠定基础。

方法：本研究分为三部分，第一部分：培养柯萨奇B组1型（Coxsackievirus group B type 1，CVB1）和柯萨奇B组2型（Coxsackievirus group B type 2，CVB2）病毒，构建CVB1和CVB2的实时荧光定量逆转录聚合酶链式反应（Quantitative Reverse Transcription Polymerase Chain Reaction，qRT-PCR）检测方法。第二部分：建立CVB1和CVB2感染叙利亚金黄地鼠的动物模型，以鼻腔滴注单独或混合病毒液的方式感染地鼠，监测记录D0-D30的临床症状、排毒情况、生理和免疫指标，并在感染后D7随机安乐死3只地鼠后，进行组织病理学检测。第三部分：建立CVB1和CVB2感染恒河猴婴猴的动物模型，以鼻腔滴注单独或混合病毒液的方式感染婴猴，监测记录D0-D30的临床症状、排毒情况、生理和免疫指标，D5采集各组婴猴血液进行转录组测序，并在感染后D10分别安乐死对照组和混合感染组症状最重的婴猴后，进行组织病理学检测。

结果：第一部分：完成了CVB1和CVB2病毒原液的制备和CVB2 RNA标准品及其标准曲线的构建，建立了CVB1和CVB2 qRT-PCR检测方法。第二部分：通过鼻腔滴注成功构建了单独感染（CVB1、CVB2）以及混合感染（CVB1和CVB2）叙利亚金黄地鼠模型，各实验组地鼠出现手、足、口部疱疹症状；白细胞（White blood cell，WBC）、中性粒细胞（Neutrophil，NEUT）、淋巴细胞（Lymphocyte，LYMPH）增高；咽拭子、粪便、血液和组织中均能检测到CVB1和CVB2的病毒核酸；免疫学指标IL-1β、IL-6、IL-17、IFN-γ和TNF-α均表现出先升后降的趋势，IL-4则表现出先降后升的趋势，并且能检测到CVB1和CVB2的中和抗体；心脏和脑等组织出现病理损伤，值得注意的是，CVB2组的脑部病理损伤更为明显。第三部分：通过鼻腔滴注成功构建了单独感染（CVB1、CVB2）以及混合感染（CVB1和CVB2）恒河猴婴猴模型，各实验组婴猴均出现手、足、口部疱疹症状，混合感染组婴猴出现精神萎靡、食欲不振和腹泻等临床症状；WBC、NEUT、LYMPH降低；咽拭子、鼻拭子、粪便和血液中均能检测到CVB1和CVB2的病毒核酸；免疫学指标IL-1β、IL-6和TNF-α均表现出先升后降的趋势、IL-17和IFN-γ表现出逐渐上升的趋势、IL-4则表现出先降后升的趋势，此外，CD3+ T细胞 、CD4+ T细胞和NK细胞的数量减少，CD4+/CD8+的比值降低，CD8+ T细胞和B细胞的数量变化趋势相对平缓；混合感染组的主要内脏和神经组织中，均能检测到CVB1和CVB2的病毒核酸，并且均有不同程度的炎症病理变化，尤其是脑干、肠道和淋巴结等组织出现较为严重的病理损伤表现；混合感染组的血液转录组功能富集于干扰病毒的复制与代谢，诱发免疫细胞动态紊乱与炎症反应，以及导致线粒体功能障碍与氧化应激。

结论：本研究通过鼻腔滴注成功构建了CVB1和CVB2单独和混合感染叙利亚金黄地鼠和恒河猴婴猴的动物模型，研究发现：（1）叙利亚金黄地鼠和恒河猴婴猴均可以应用于肠道病毒感染模型的构建，叙利亚金黄地鼠可用于病毒性心肌炎、病毒性脑膜炎的模型研究，恒河猴婴猴模型能模拟人类手足口病的临床症状、免疫应答及病毒性脑干脑炎的病理进程。（2）CVB1表现出心脏嗜性和相对复制优势，CVB2表现出神经嗜性，初步提示CVB1在一定程度上可以影响CVB2的复制与排毒，二者的混合感染可能通过干扰代谢-免疫网络等途径，加重动物的炎症反应、氧化应激和免疫病理损伤。（3）不同型别肠道病毒的混合感染机制与毒株型别本身的性质和特征有一定相关性。

Objective：By constructing an animal model of enterovirus co-infection of syrian golden hamsters and rhesus monkeys, the co-infection mechanism of human enterovirus was initially studied, laying the foundation for the development of multivalent vaccines and antiviral drugs.

Method：This study is divided into three parts. The first part: Cultivating CVB1 and CVB2 viruses, and constructing qRT-PCR detection methods for CVB1 and CVB2. Part 2: Establish an animal model of CVB1 and CVB2 infecting the syrian golden hamsters, and infect the hamsters with nasal infusion alone or mixed viral fluid. Monitor and record the clinical symptoms, detoxification status, physiological and immune indicators of D0-D30, and perform histopathological testing after randomly euthanizing three hamsters in D7 after infection. Part 3: Establish an animal model of CVB1 and CVB2 infecting rhesus monkeys, infecting monkeys with nasal infusion alone or mixed virus fluid, monitor and record the clinical symptoms, detoxification status, physiological and immune indicators of D0-D30, and collect blood from each group of monkeys for transcriptome sequencing. After D10 euthanizes the most severe symptoms of monkeys in the control group and co-infection group after infection, histopathological tests were performed.

Result：Part 1: The preparation of CVB1 and CVB2 virus stock solution and the construction of CVB2 RNA standards and their standard curves were completed, and the detection methods of CVB1 and CVB2 qRT-PCR were established. Part 2: The syrian golden hamster model was successfully constructed through nasal infusion (CVB1, CVB2) and co-infection (CVB1 and CVB2). The hamsters in each experimental group showed herpes symptoms in hand, foot and oral areas; WBC, NEUT, and LYMPH increased; viral nucleic acids of CVB1 and CVB2 can be detected in the throat swabs, feces, blood and tissues; the immunologic indicators IL-1β, IL-6, IL-17, IFN-γ and TNF-α all show a trend of rising first and then falling, while IL-4 shows a trend of falling first and then rising, and neutralizing antibodies of CVB1 and CVB2 can be detected; pathological damage occurs in tissues such as the heart and brain. It is worth noting that the brain pathological damage in the CVB2 group is more obvious. Part 3: The rhesus monkey model were successfully constructed through nasal infusion. The monkeys of the experiment group all showed symptoms of hand, foot, and oral herpes, and the monkeys of the co-infection group had clinical symptoms such as mental depression, loss of appetite and diarrhea; WBC, NEUT, and LYMPH were reduced; viral nucleic acids of CVB1 and CVB2 can be detected in throat swabs, nasal swabs, feces and blood; the immunologic indicators IL-1β, IL-6 and TNF-α all showed a trend of rising first and then falling, IL-17 and IFN-γ showed a trend of gradual upward trend, and IL-4 showed a trend of In addition, the number of CD3+ T cells, CD4+ T cells and NK cells decreased, the ratio of CD4+/CD8+ decreased, and the number of CD8+ T cells and B cells changed relatively smoothly; the viral nucleic acids of CVB1 and CVB2 can be detected in the main visceral and neural tissues of the co-infected group, and there are different degrees of inflammatory pathological changes, especially the brainstem, intestinal and lymph nodes and other tissues have relatively serious pathological damage; the blood transcriptome function of the co-infected group is enriched to interfere with the replication and metabolism of the virus, inducing dynamic disorders and inflammatory responses of immune cells, and leading to mitochondrial dysfunction and oxidative stress.

Conclusion：This study successfully constructed an animal model of CVB1 and CVB2 infected with syrian golden hamsters and rhesus monkeys alone and co-infecting syrian golden hamsters and rhesus monkeys. The study found that: (1) Syrian golden hamsters and rhesus monkeys can be used to construct enterovirus infection models, and syrian golden hamsters can be used to study viral myocarditis and viral meningitis model. The rhesus monkey model can simulate the clinical symptoms, immune response and pathological process of viral brainstem encephalitis in human hand, foot and mouth disease. (2) CVB1 shows cardiac tropism and relative replication advantages, and CVB2 shows neurotropism, which initially suggests that CVB1 can affect CVB2's replication and detoxification to a certain extent. The co-infection of the two may aggravate the inflammatory response, oxidative stress and immune pathological damage in animals through interfering with the metabolism-immune network and other channels. (3) The co-infection mechanism of enteroviruses of different types is related to the nature and characteristics of the strain type itself.