犬攻毒用狂犬病街毒株BD06的特性

Characteristics of rabies street virus strain BD06 for challenge test in dogs

目的对狂犬病街毒株BD06进行了毒株特异性分析,并评价其对犬的攻毒效果。方法将BD06株病毒于比格犬脑内传至第10代,参照《中国兽药典》三部(2010版)进行外源病毒(犬瘟热病毒、细小病毒、冠状病毒、Ⅰ型和Ⅱ型腺病毒)、支原体检测和无菌检验;提取传至第10代的犬脑组织总RNA,进行RT-PCR扩增反应,并进行全基因序列测序;经小鼠脑内传代后,测定小鼠的半数致死量(MICLD50);用2×104、5×104和10×104MICLD50/ml的鼠脑悬液,分别采用后肢肌注法和咬肌注射法进行犬攻毒试验。结果 BD06株病毒在犬脑中传至第10代,无外源病毒、支原体和细菌污染,基因序列未发生变化;经鼠脑传代1次后,MICLD50达105.32/ml;经咬肌注射5×104MICLD50/ml的BD06株病毒悬液可获得最佳犬攻毒效果。结论 BD06株病毒传代简单,对犬致病性强,可作为我国犬攻毒用狂犬病病毒的候选株。

Objective To analyze the specificity of rabies street virus strain BD06 and evaluate its effect in challenge test in dogs. Methods BD06 was subcultured to passage 10 in brains of Beagle dogs, and tested for adventitious agents including foreign viruses such as dog distemper virus, parvovirus, coronavirus as well as adenovirus types I and II , mycoplasma and sterility according to the requirements in Chinese Veterinary Pharmacopoeia （Volume HI, 2010 edition）. Total RNA was extracted from the brain tissue of dogs, in which the virus was subcuhured to passage 10, for amplification by RT-PCR and sequencing. The virus was subcuhured in the brains of mice and determined for MICLD50. Dogs were challenged i. m. with the mouse brain suspensions at dosages of 2 ×104, 5 ×104 和 10 ×104 MICLD50/ml in behind limbs and masseter separately. Results No foreign viruses, mycoplasma or bacteria were observed in BD06 strain after subculture in dog brain to passage 10, while the gene sequence showed no change. The MICLD50 of the strain after subculture in mouse brain for one passage reached 105. 32/ml. The challenge test in dogs with the strain at a dosage of 5 ×104 MICLD50/ml by injection in masseter showed satisfactory effect. Conclusion Rabies virus strain BD06 was simple to be subcuhured and highly pathogenic to dogs, which might be used as a candidate for challenge test in dogs.