体外培养及长期传代对草鱼呼肠孤病毒毒力的影响

Effect on Virulence of Grass Carp Reovirus with Culture and Long-Time Passage in Vitro

将不同培养时间及不同传代次数的病毒用PBS稀释后,感染健康草鱼(5～15 cm),取感染后具有典型症状的草鱼肾脏组织液,对健康草鱼进行重复感染,对其致病和致死率进行统计,并用抗草鱼呼肠孤病毒的单克隆抗体5F11进行间接免疫荧光检测.结果显示长期培养(>8 d)的病毒外衣壳脱落,其对草鱼的致病及致死率与完整衣壳感染无显著差异;传代不同次数(1～50代)的病毒对草鱼的致病及致死率无显著差异;复染的草鱼其致病及致死率与初染无显著性差异,间接免疫荧光检测复染的草鱼肾脏冷冻切片能观察到明亮的点状黄绿色荧光.即体外长期培养能导致外衣壳脱落,但这对草鱼呼肠孤病毒的毒力无影响,且体外传代(1～50代)也不影响草鱼呼肠孤病毒的毒力.

GCRV with different times of culture and passage was diluted by PBS, then used to infect healthy grass carp （5 - 15 cm）. Tissue fluid of kidney from infected grass carps with typical symptom was got to infect healthy grass carps again ,and the morbidity and mortality were counted. In addition, MAb 5F11 against GCRV was prepared to carry out indirect immuneofluorescence assay. The results showed that outer capsid was missing with long-time culture （ 〉 8 d）, however, compared with intact virion, the morbidity and mortality of infected grass carps revealed no significant difference. Besides, the morbidity and mortality of infected grass carps also revealed no significant difference among the different times of passage （1 st ~ 50th generation）. What＇ s more, the morbidity and mortality illustrated no significant difference between the grass carps infected with GCRV and the grass carps infected with tissue fluid, and indirect immuneofl^lorescence assay displayed the bright spot with green fluorescence on frozen section of kidney from grass carp infected with tissue fluid. Thus, long-time culture in vitro can cause the outer capsid off, but the virulence of GCRV has no influence. Moreover, it＇s also no influence to virulence of GCRV with passage （1st-50th generation） in vitro