狂犬病病毒CTNCEC25株的感染力和致病力特征分析

The Infectious and Pathogenic Characteristics of Rabies Virus Strain CTNCEC25

研究狂犬病病毒CTNCEC25株的增殖能力、病毒的感染力及致病力等表型特征,为将该毒株应用于疫苗生产提供基础信息。将CTNCEC25株及其母株CTN-1株分别接种非洲绿猴肾细胞(Vero细胞)、原代鸡胚成纤维细胞(Primary chicken embryo cell,CEC)和小鼠神经瘤母细胞(Mouse neuroblastoma N2acells,N2a),观察其细胞病变特性及其复制规律;以脑内注射的方式接种动物,观察其对乳鼠、小鼠、豚鼠及家兔等动物的致病力;同时还将CTNCEC25株在CEC上连续传20代,在乳鼠脑内传3代,观察其增殖能力、病毒感染力及致病力的稳定性。研究结果表明,与母株CTN-1株相比,CTNCEC25株在Vero细胞和N2a细胞上的增殖并没有显著差异,但在CEC上,CTNCEC25株具有明显优势,其72h滴度可达10^(7.5^7.6) FFU/mL,而CTN-1株滴度只有10^(5.8) FFU/mL;此外CTNCEC25株在N2a细胞、Vero细胞及CEC上均可产生明显的细胞病变。脑内致病力实验结果表明,CTNCEC25株虽然对1~3日龄的乳鼠仍然具有较强的脑内致病性,但对成年小鼠的脑内致病力大大减弱,对豚鼠和家兔不致病。经细胞和乳鼠脑内连续传代,CTNCEC25株在CEC上增殖稳定,其减弱的毒力并未回升,无弱毒返祖现象。综上所述,本研究初步证实CTNCEC25株是一株高度减毒、不易返祖的弱毒株,其在CEC上可稳定、快速增殖,病毒滴度高(>10^(7.0) FFU/mL),是一株安全性较高的狂犬病疫苗候选毒株。

To investigate the phenotypic characteristics of the strain of the rabies virus CTNCEC25, the strain of the China rabies virus CTN-1 adapted to primary chicken embryo cells （CECs）, Vero cells, and mouse neuroblastoma N2a cells was inoculated with CTNCEC25 and parental CTN-1 strains to explore the cytopathic effect （CPE） and growth kinetics of CTNCEC25 on cultured cells. To determine the pathoge- nicity of CTNCEC25, suckling mice, adult mice, guinea pigs and rabbits were inoculated with CTNCEC25 via the intracerebral route and their survival monitored every day. Furthermore, the CTNCEC25 strain was passed serially in CECs for 20 passages and then 3 passages in the brains of suckling mice to determine phenotypic stability. CTNCEC25 achieved similar growth kinetics in Vero cells and N2a cells compared with parental CTN-1, but CTNCEC25 replicated more efficiently in CECs than the CTN-1 strain with a titer 72 h after infection reaching 10^7.5-7.6 FFU/mL, which was significantly higher than the 10^5.8FFU/mL achieved by its parental strain, CTN-1. Moreover, CTNCEC25 induced apparent CPE in Vero cells, CECs and N2a cells. Analyses of intracerebral inoculation demonstrated that CTNCEC25 was attenuated pro- foundly in adult mice and was completely apathogenic to guinea pigs and rabbits, though it caused death in suckling mice. The CTNCEC25 strain proliferated steadily after serial passage in CECs and the brains of suckling mice, and remained avirulent in adult mice. These results suggest that CTNCEC25 is a highly at- tenuated and genetically stable strain of the rabies virus. CTNCEC25 replicated stably and efficiently in cultured cells and achieved high titers, so it could be a promising and safe vaccine strain for rabies preven- tion in China.