Abstract:

Objective To investigate the immunological characteristics and pathogenicity of China's first isolated mosquito borne viruses Nam Dinh virus (NDiV) on cultured cells and Kunming mouse. Methods The following methods were used:C6/36 cells for observation on cytopathic effect (CPE), plaque assay for determination of NDiV titer, virus inoculation methods for detection of NDiV virulence in 3 days old mice brain. In C6/36 cells, neutralization test was done using neutralizing antibodies from immunized Kunming mice. The BHK21 cells were inoculated with virus neutralizing antibodies of NDiV, one hour exposure followed by culturing for 7-9 d. Daily observation was made and the cytopathic effect was recorded. The TaqMan-MGB Probe Real-time PCR method was used to detect the nucleic acid of NDiV in C6/36 cells. Results C6/36 cells were infected after incubation with NDiV, CPE appeared since fifth days, the virus titer was 9.25×10⁶ PFU/ml, the LD₅₀ was 10^4.12/ml by Reed-Muench method. After NDiV virus infected mice, strong immunogenicity and higher rate of virus protection were showed in NDiV virus antiserum group, TCID₅₀ was less than 10^-1 while the control group TCID₅₀ for 10^-3.5. The neutralization index greater than 1 000. The virus nucleic acid detection results indicated positive 3 d after inoculation, virus nucleic acid showed significantly positive with growth of virus post inoculation upon virus nucleic acid increase. Conclusion The NDiV has certain pathogenicity and antigenicity against the exposed C6/36 cells and Kunming mouse.

Key words: Nam Dinh virus, Biological characteristics, Toxicity

摘要：

目的 探讨我国首次分离的蚊媒病毒南定病毒（Nam Dinh virus，NDiV）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NDiV对C6/36细胞的病变效应（CPE）；用空斑法测定NDiV滴度；采用3日龄小鼠脑内接种NDiV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NDiV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TaqMan-MGB Probe Real-time PCR方法对C6/36细胞中NDiV核酸进行检测，观察病毒增殖情况。结果 NDiV接种C6/36后，第5天出现明显的CPE，测得病毒滴度为9.25×10⁶ PFU/ml，利用Reed-Muench法计算NDiV对昆明小鼠半数致死量（LD₅₀）为10^4.12/ml，NDiV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（TCID₅₀）<10^-1，而对照组TCID₅₀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，NDiV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 NDiV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。

关键词: 南定病毒, 生物学特征, 毒力