狂犬病病毒CVS-11攻毒小鼠动物模型的建立及初步应用

Establishment and preliminary application of rabies virus CVS-11 challenge model in mice

目的:建立狂犬病病毒CVS-11以肌肉注射或脑内注射方式攻毒的BALB/c小鼠动物模型。方法:将由BSR细胞传代繁殖的滴度为2.7×10^7 FFU/ml的CVS-11毒株进行10-1~10-7倍系列稀释,分别以肌肉或脑内注射的方式对4周龄的雌性BALB/c小鼠进行攻毒,观察小鼠发病死亡情况,并对所有攻毒小鼠的脑组织进行直接免疫荧光试验(direct fluorescent assay,DFA)及逆转录聚合酶链反应(RT-PCR)检测以确定小鼠死因。确定不同攻毒方式下小鼠的半数致死量(median lethal dose,LD 50),建立小鼠模型。根据已建立的小鼠模型,评估4种国内市售狂犬病疫苗对CVS-11暴露后小鼠的免疫保护效果。结果:BALB/c小鼠脑内攻毒后于第6~12天开始出现典型的神经症状并死亡,其LD 50为18.3/0.1 ml;肌肉注射攻毒的小鼠在第8~15天出现临床症状并死亡,LD 50为2.7×10^5/0.1 ml。DFA检测结果显示,所有死亡小鼠脑组织印片中均出现特异性黄绿色荧光,RT-PCR检测结果显示所有的扩增产物均出现大小约250 bp的明亮条带。以上结果提示狂犬病病毒感染是小鼠的致死原因。4种不同的市售狂犬病疫苗在无免疫球蛋白应用情况下的保护效果试验显示,接种其中1种疫苗暴露后小鼠的存活率为50%,接种其他3种疫苗小鼠的存活率为30%。以上结果表明,在无狂犬病被动免疫制剂使用的情况下,所选用的4种狂犬病疫苗对经CVS-11暴露后的小鼠提供了部分保护作用。结论:本研究建立了狂犬病病毒CVS-11不同攻毒方式下的BALB/c小鼠动物模型,为狂犬病及狂犬病疫苗的相关研究提供了一个技术平台。

Objective To establish a rabies virus CVS-11 challenge model in BALB/c mice through intramuscular or intracerebral injection.Methods The CVS-11 strain propagated in BSR cells with a titer of 2.7×10^7 FFU/ml was serially diluted 10-1-10-7 times to infect 4-week-old female mice through intramuscular or intracerebral injection.The morbidity and mortality of mice were observed after virus challenge.Moreover,brain tissues of all challenged mice were subjected to direct immunofluorescence assay(DFA)and reverse transcription polymerase chain reaction(RT-PCR)to analyze the cause of death.The median lethal doses(LD50)in mice under different challenge methods were determined.Mouse challenge models were established to evaluate the immunoprotective effects of four domestically available rabies vaccines on mice after CVS-11 exposure.Results BALB/c mice developed typical neurological symptoms and died 6-12 d after intracerebral challenge and the LD50 was 18.3/0.1 ml.The mice intramuscularly challenged with CVS-11 showed clinical symptoms on 8-15 d and the LD50 was 2.7×10^5/0.1 ml.DFA results showed that specific yellow-green fluorescence appeared in the brain tissue prints of all dead mice.RT-PCR results showed that all amplified products showed bright bands at about 250 bp.These results suggest that rabies virus infection was the cause of death in mice.The protective effect test results of four different rabies vaccines on the market without immunoglobulin application showed that the survival rate of mice after exposure to one of the vaccines was 50%,and the survival rates of mice immunized with the other three vaccines were all 30%.The above results indicated that the four rabies vaccines provided partial protection for mice exposed to CVS-11 without the use of rabies passive immunization preparations.Conclusions This study established rabies virus CVS-11 challenge models in BALB/c mice under different challenge methods and provided a technical platform for related research on rabies and rabies vaccines.