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目的探讨我国首次分离的蚊媒病毒南定病毒(Nam Dinh virus,NDi V)的生物学特征及对培养细胞和昆明小鼠的致病能力。方法连续9 d观察NDi V对C6/36细胞的病变效应(CPE);用空斑法测定NDi V滴度;采用3日龄小鼠脑内接种NDi V方法检测病毒毒力;中和实验以免疫后灭活的昆明小鼠血清为中和抗体,在C6/36细胞中接种NDi V和中和抗体,作用1 h后继续培养7~9 d,逐日观察并记录细胞病变情况;采用Taq Man-MGB Probe Real-timePCR方法对C6/36细胞中NDi V核酸进行检测,观察病毒增殖情况。结果 NDi V接种C6/36后,第5天出现明显的CPE,测得病毒滴度为9.25×10~6PFU/ml,利用Reed-Muench法计算NDi V对昆明小鼠半数致死量(LD_(50))为10~(4.12)/ml,NDi V感染小鼠后,具有较强的免疫原性,且攻毒保护率较高。病毒抗血清组半数组织培养感染剂量(TCID_(50))<10~(-1),而对照组TCID_(50)为10~(-3.5),中和指数>1 000,病毒核酸检测结果显示,接种3 d后,NDi V核酸呈明显阳性,病毒核酸量随病毒接种后时间的延长而增加。结论 NDi V对C6/36细胞和昆明小鼠具有一定的致病力和较强的抗原性。
Objective To investigate the biological characteristics of the first NDV isolated in China and its virulence to cultured cells and Kunming mice. Methods The pathogenic effect (CPE) of NDi V on C6 / 36 cells was observed for 9 days. NDi V titers were measured by plaque assay. The virulence of NDi V inoculated intranasally in 3-day-old mice was measured. Immunized Kunming mouse serum neutralizing antibody, inoculated with NDi V and neutralizing antibody in C6 / 36 cells, the role of 1 h and then cultured for 7 to 9 days, daily observation and recording of cytopathic effect; using Taq Man -MGB Probe Real-timePCR method to detect NDi V nucleic acid in C6 / 36 cells to observe the proliferation of the virus. Results Significant CPE appeared on day 5 after inoculation of C6 / 36 with NDi V, and the titer of virus was 9.25 × 10 ~ 6 PFU / ml. The NDe V was calculated by Reed-Muench method. )) Was 10 ~ (4.12) / ml, NDi V infected mice, with strong immunogenicity, and a high rate of attack protection. The virus titer (TCID50) was less than 10 ~ (-1) in virus antiserum, while TCID_ (50) in control group was 10 ~ (-3.5) After 3 days of inoculation, the NDi V nucleic acid was significantly positive and the amount of viral nucleic acid increased with the prolongation of virus inoculation time. Conclusion NDi V has certain virulence and antigenicity to C6 / 36 cells and Kunming mice.