1株狂犬病毒减毒株的毒力和分子特性研究

目的 对实验室研发的1株狂犬病减毒株CTN181-3的致病性和基因型特性进行研究。方法 用小鼠脑内、口腔接种法和金黄地鼠口腔接种法测定病毒毒力。将病毒通过乳鼠脑内、豚鼠颌下腺和细胞连续传多代,测定传代后病毒的遗传稳定性。对CTN181-3进行全基因组测序,并与其亲本株进行对比分析。结果 CTN181-3对实验动物毒力高度减弱,对3周龄小鼠无论脑内或口腔接种均无致病性,对2周龄小鼠也表现出脑内低毒力;口腔接种8周龄金黄地鼠亦无发病死亡。遗传稳定性方面,经1~3 d龄乳鼠脑内连续传5代或豚鼠颌下腺传4代,传代增殖后的病毒滴度虽高达7.10 lg PFU/mL或7.63 lg PFU/mL,对小鼠脑内接种仍保留无致病力的弱毒特性;在BSR细胞和Vero细胞上分别传10代,表型特性稳定。全基因组测序结果分析显示CTN181-3株与国内近年来分离株的同源性高于其他疫苗株与国内分离株的同源性。CTN181-3株与其原始的亲本株CTN-1比较,发生8个氨基酸位点的突变,其中G276 L→V和L1496 M→W氨基酸的突变在CTN181株未发生,为CTN181-3株所特有。因此,这2个位点的氨基酸突变应该是CTN181-3株比CTN181株毒力更弱、遗传稳定性更高的分子基础。结论 CTN181-3株的神经毒力高度减弱,毒力稳定,是一种很有应用前景的动物用狂犬病候选疫苗株。

Pathogenicity and molecular characteristics of an attenuated rabies virus strain

This study was aimed at studying the pathogenicity and molecular characteristics of the highly attenuated CTN181-3 rabies virus strain previously developed in our laboratory. The pathogenicity was tested by intracerebral inoculation (i.c.) or oral inoculation (o.i.) in mice, and o.i. in golden hamsters. The genetic stability was studied after several passages of the virus in suckling mouse brains, guinea pig submandibular glands and cell cultures. The full-length genome was sequenced and compared with that of the parental virus strains. CTN181-3 was highly attenuated in laboratory animals and showed no pathogenicity after i.c. or o.i in 3-week-old mice, and low pathogenic in 2-week-old mice. It also showed no pathogenicity after o.i. in 8-week-old-hamsters. The virus was highly genetic stable after ten successive passages in BSR or Vero cell cultures. After five successive passages in suckling mouse brains or four passages in Guinea pig submandibular glands, the virus still retained characteristics of non-pathogenicity. Full-length sequencing analysis showed greater sequence similarity between the CTN181-3 strain and domestic isolates in recent years than that between other vaccine strains and domestic isolates. Eight amino acid substitutions were identified in the CTN181-3 virus compared with the original parental virus strain CTN-1. Two amino acid substitutions, G276 L→V and L1496 M→W, were not found in the parent virus CTN181 strain and therefore may be responsible for the lower pathogenicity of the CTN181-3 strain than the CTN181 strain. Because of its high attenuation and genetic stability, the CTN181-3 strain may be a promising candidate for oral vaccination of domestic and wild animals.