狂犬病病毒和犬瘟热病毒疫苗株混合感染Vero细胞的实验研究

Experimental research on co-infection of rabies virus and canine distemper virus in Vero cells

目的研究狂犬病病毒(RV)及犬瘟热病毒(CDV)共同感染Vero细胞及混合感染对产毒量的影响,比较病毒检测方法的敏感性。方法将单独培养的RV、CDV及RV-CDV混合培养物进行连续10倍稀释后同步接种Vero细胞,37℃5%CO2培养5d,分别以直接免疫荧光(DFA)、RT-PCR及荧光定量RT-PCR方法检测病毒的半数细胞感染量,并对各病毒检测方法进行比较。结果RV和CDV可同时感染Vero细胞,并在其中增殖。CDV单独感染Vero细胞的TCID50为10-5.8/0.05ml,RV和CDV混合感染Vero细胞的TCID50为10-5.5/0.05ml。DFA、RT-PCR和荧光定量RT-PCR阳性的RV-CDV感染最大稀释度分别为10-6、10-5和10-6。结论混合培养对RV和CDV的感染滴度及产毒量影响很小。免疫荧光灶检测与RT-PCR及荧光定量RT-PCR方法检测的敏感性相当。犬瘟热-狂犬病毒联合疫苗的病毒滴度检测可不经中和其中的一个病毒直接将联合疫苗接种Vero细胞,然后分别进行免疫荧光检测。

Objective To investigate the co-infection of rabies virus（RV） and canine distemper virus（CDV） in Vero cells and the influence of virus titer caused by co-infection, compare the sensitivity of different virus detection methods. Methods After the RV and CDV which was cultured alone and coinfected in Vero cells were diluted, the virus was inoculated Vero cells and cultured under 37 ℃ 5%CO2 atmosphere. After 5 d, the virus titer was detected by direct immunofluorescent assay（DFA）, RT-PCR and real-time RT PCR, the sensitivity of three virus detecion methods were also compared. Results The RV and CDV could infect and proliferate in Vero cells at the same time, TCID50 of CDV cultured alone was 10^-5.8/0.05 ml, TCID50 of co-infection was 10^-5.5/0.05 ml. The maximum dilution of positive infection detected by DFA, RT-PCR and real-time RT-PCR was 10^-6 , 10^-5 and 10^-6. Conclusion Co infection had little influence on virus titer. The sensibility of DFA, RT-PCR and real-time RT-PCR was equivalent. The virus titer detection of mixed vaccine including RV and CDV could be tested by DFA after the mixed vaccine was inoculated in Vero cells directly without neutralization of one virus.