目的：手足口病一直是我国婴幼儿群体内高发的传染病，尤其是在近些年EV-A71疫苗的成功上市并大量接种后，CVA10病毒越来越成为手足口病的主要流行株之一，因此针对CVA10疫苗开发的意义越来越重要，对于我国乃至世界的公共卫生具有重要的意义。本研究旨在通过对CVA10病毒的灭活进行研究，并建立免疫荧光检测方法辅助进行灭活验证，为CVA10疫苗的开发提供基础。

方法：使用甲醛和BPL两种灭活剂，探究不同浓度灭活剂和不同的灭活时间对病毒灭活的影响。同时将得到的灭活条件应用于病毒收获液和病毒原液中，探究该灭活条件是否仍适用。使用肠道病毒实验常用的RD细胞、Vero细胞和MA104细胞，确定CVA10的最敏感细胞。建立免疫荧光检测CVA10病毒颗粒的方法，确定一抗最佳工作浓度、病毒最佳孵育时间、可以检测到的病毒下限以及特异性，并将免疫荧光方法应用于灭活验证中。

结果：甲醛50μg/ml灭活24h、100μg/ml灭活18h、150μg/ml灭活12h、200μg/ml灭活6h；BPL 1：1000浓度灭活1h、1：2000浓度灭活6h、1：3000浓度灭活6h、1：4000浓度灭活6h，以上条件均可以将病毒完全灭活，且灭活验证符合要求。

CVA10病毒在RD细胞、Vero细胞和MA104细胞中均能良好生长，无论在一步生长曲线还是增殖动力学曲线中，CVA10病毒感染RD细胞测得的病毒感染性滴度均值最高，并且增殖最快。结果表明RD细胞为CVA10最敏感细胞，因此在灭活验证中使用该种细胞进行实验。

本研究建立了免疫荧光检测CVA10病毒的方法，最佳一抗工作浓度为1：100，该方法500 CCID₅₀/ml的CVA10病毒量与Vero细胞孵育18小时即可有少量的病毒颗粒；感染细胞在病毒感染24h后可以检测到的病毒量下限为50 CCID₅₀/ml；病毒感染4天后可以检测到的病毒量下限为0.5 CCID₅₀/ml，并且特异性良好，不会与其他型肠道病毒产生交叉反应。将免疫荧光方法与细胞盲传实验结合进行灭活验证的确认，提高了传统灭活验证方法检测的可靠性。

结论：甲醛和BPL均可作为CVA10病毒的灭活剂。在肠道病毒常用细胞中，RD细胞是CVA10的最敏感细胞，使用RD细胞进行灭活验证可提高灭活验证结果的可靠性。成功建立了免疫荧光检测CVA10病毒颗粒的方法，该方法可以检测到的病毒量下限为0.5 CCID₅₀/ml，并且特异性良好，不与其他型肠道病毒产生交叉反应，可以辅助进行灭活验证实验，提高灭活验证结果的可靠性。

Objective: Hand, foot and mouth disease has always been a high incidence of infectious diseases among infants and young children in our country. Especially after the successful marketing of EV-A71 vaccine and a large number of vaccination in recent years, CVA10 virus has increasingly become one of the main epidemic strains of hand, foot and mouth disease. Therefore, the significance of development of CVA10 vaccine is becoming more and more important and has important significance for the public health of our country and even the world. This study aims to provide a basis for the development of CVA10 vaccine by studying the inactivation of CVA10 virus and establishing an immunofluorescence detection method to assist the inactivation verification.

Methods: Formaldehyde and BPL were used to investigate the effects of different concentration of inactivators and different inactivation time on virus inactivation. At the same time, the obtained inactivation conditions were applied to the virus harvest solution and the virus stock solution to explore whether the inactivation conditions were still applicable. The most sensitive cells of CVA10 were identified using RD cells, Vero cells and MA104 cells commonly used by enteroviruses. A method of immunofluorescence detection of CVA10 virus particles was established to determine the optimal working concentration of primary antibody, the optimal incubation time of the virus, the lower limit of the virus that can be detected and the specificity of the virus, and the immunofluorescence method was applied in inactivation verification.

Results: Use formaldehyde to inactivated for 24h with 50μg/ml, inactivated for 18h with 100μg/ml, inactivated for 12h with 150μg/ml and inactivated for 6h with 200μg/ml. Use  BPL 1:1000 to inactivated for 1h, 1:2000 inactivated for 6h, 1:3000 inactivated for 6h, and 1:400 inactivated for 6h. All the above conditions could completely inactivate the virus, and the inactivation verification met the requirements.

CVA10 virus can grow well in RD cells, Vero cells and MA104 cells. In both one-step growth curve and proliferation kinetics curve, CVA10 virus infected RD cells had the highest mean viral titer and the fastest proliferation. The results showed that RD cells were the most sensitive cells of CVA10, so this kind of cells were used in the experiment for inactivation verification.

In this study, a method of immunofluorescence detection of CVA10 virus was established. The optimal working concentration of primary antibody was 1:100. In this method, 500 CCID₅₀/ml of CVA10 virus was incubated with Vero cells for 18 hours, and a small number of virus particles could be obtained. The lower limit of virus amount detected by infected cells 24h after infection was 50 CCID₅₀/ml. The lower limit of virus amount detected 4 days after infection was 0.5 CCID₅₀/ml, and the specificity was good, without cross-reaction with other enterovirus types. The inactivation verification was confirmed by combining immunofluorescence method with blind cell transmission test, which improved the reliability of the traditional inactivation verification method.

Conclusion: Formaldehyde and BPL can be used as inactivators of CVA10 virus. Among the commonly used cells of enterovirus, RD cells are the most sensitive cells of CVA10. Using RD cells for inactivation verification can improve the reliability of the inactivation verification results. The immunofluorescence detection method for CVA10 virus particles was successfully established. The lower limit of virus amount detected by this method is 0.5 CCID₅₀/ml, and the method has good specificity. It does not cross-react with other enterovirus types, which can assist in inactivation verification experiment and improve the reliability of inactivation verification results.