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目的了解狂犬病病毒减毒株CTN-181在豚鼠颌下腺的繁殖动态并获得毒力更低的病毒纯化克隆株。方法将CTN-181经刚离乳豚鼠颈部皮下注射,分别于1、2、3和4d取颌下腺分离狂犬病病毒,测定各天次颌下腺内的病毒滴度。用滴度高的病毒液进行下一次豚鼠颌下腺传代,连续传3代。将第3代颌下腺传代的病毒液在BHK21细胞上进行空斑试验,挑选单个病毒克隆,以3周龄小鼠脑内接种测病毒毒力。筛选对小鼠不致病的克隆株进一步测定其对乳鼠的脑内毒力,并以乳鼠脑内传代和豚鼠颌下腺传代检测其遗传稳定性。结果 CTN-181在豚鼠颌下腺能短暂轻度繁殖,通过颌下腺传代后病毒毒力明显有回升。获得19个病毒克隆,不同克隆病毒的毒力不同,为0~3.16lg LD50;3株脑内无致病力的克隆株对乳鼠的毒力亦有减弱;乳鼠脑内和颌下腺回传后毒力无回升返祖,即传代后的病毒对小鼠均无致病力。结论CTN-181母株对3周龄小鼠仍存在低水平毒力,经豚鼠颌下腺传代后毒力十分不稳定。筛选到的3株克隆病毒较其母株的毒力更低,遗传稳定性更高,更适合作为狂犬病兽用口服疫苗候选株。
Objective To understand the reproductive activity of rabies virus attenuated strain CTN-181 in the submandibular gland of guinea pig and to obtain a virus-purified clone with lower virulence. Methods Rabbit virus was subcutaneously injected into the submandibular glands of the nude mice by subcutaneously injecting CTN-181 into the neck of guinea pigs. The virus titers in the submandibular glands were determined every day. The virus liquid with high titers was used to transmit the next submandibular gland of guinea pigs for 3 generations. The third generation of submandibular gland of the virus liquid in BHK21 cells plaque test, select a single virus clone, 3 weeks old mice intracerebral virus inoculation test. The cloned non-pathogenic clones were further screened for their intracerebral toxicity to neonatal mice and the genetic stability was evaluated by passage through the brain of neonatal rats and passage of submandibular gland in guinea pigs. Results CTN-181 transiently mildly propagated in the submandibular gland of guinea pigs, and the virulence of CTN-181 significantly increased after passage through the submandibular gland. 19 virus clones were obtained. The virulence of different clonal viruses was 0 ~ 3.16lg LD50. The virulence of 3 strains of non-pathogenic clones in neonatal rats was weakened. After the virulence did not rise back to ancestral, that is, after passage of the virus in mice have no virulence. Conclusion The CTN-181 female parent still has low level of virulence to 3-week-old mice. The virulence of CTN-181 strain is very unstable after passing through the submandibular gland of guinea pig. The three clones screened out have lower virulence and higher genetic stability than their parent strains and are more suitable as oral vaccine candidate strains for rabies.