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目的研究狂犬病不同固定毒株对动物的致病性和对细胞的感染性。方法以小鼠脑内和肌内途径接种比较不同毒株的致病性,研究并建立HEPES(4-羟乙基哌嗪乙磺酸)诱导的空斑形成技术测定病毒空斑形成单位(plaque-forming unit,PFU)和细胞病变感染技术测定病毒感染滴度(CCID50),并用以比较不同毒株的病毒滴度,实验同时以常规的直接免疫荧光法(direct immunofluorescent assay,dFA)做对照。结果不同固定毒株对10~12g小鼠的脑内致病力普遍很高,但肌内毒力普遍较低,脑腔感染致病力比肌内感染致病力高3.0~5.0lg LD50,CVS株相差最大为5.0lg LD50。用两种新的方法 (PFU和CCID50)测定不同毒株的病毒滴度与dFA法测定的结果比较,除个别株外无明显差异,如CVS-11、4aG、PV株用三种方法测定的滴度均在7.1~7.9lg。结论用dFA法测定病毒滴度的结果与小鼠脑内测定的结果有可比性,用以替代小鼠脑内法测定病毒滴度是可行的。两种细胞感染法测定的病毒滴度操作简便,无需贵重仪器和昂贵试剂,可以更广泛地应用于狂犬病病毒和疫苗发展的研究。
Objective To study the pathogenicity and cell infectivity of different fixed strains of rabies in animals. Methods The pathogenicity of different strains was inoculated intracerebrally and intramuscularly in mice. The plaque formation induced by HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) -forming unit (PFU) and cytopathic effect (CCI) technique were used to determine the virus titer (CCID50). The titer was compared with that of different strains. The control was also performed by the routine direct immunofluorescent assay (dFA). Results The results showed that the pathogenicity of 10 ~ 12g mice in different fixed strains was generally high, but the intramuscular toxicity was generally low. The pathogenicity of intracavity infection was 3.0 ~ 5.0lg higher than that of intramuscular infection. CVS strains vary up to 5.0lg LD50. Two new methods (PFU and CCID50) to determine the virus titer of different strains and dFA method compared to the results, except for some strains no significant differences, such as CVS-11, 4aG, PV strains determined by three methods Titers were 7.1 ~ 7.9lg. Conclusion The result of determination of virus titer by dFA method is comparable with that of mouse brain assay. It is feasible to determine the virus titer by intracerebral method in mice. The virus titers determined by both cell-based assays are easy to use and do not require expensive instrumentation and expensive reagents for broader research into rabies virus and vaccine development.