刀额新对虾原代淋巴细胞培养及其感染白斑综合征病毒（WSSV）的病理特征

为深入了解对虾白斑综合征病毒(WSSV)的致病机理和体外培养的对虾细胞对WSSV的敏感性,实验以1.5×L-15培养基培养刀额新对虾原代淋巴细胞,待细胞形成单层后接种WSSV,通过倒置显微镜、荧光显微镜、透射电子显微镜观察接种病毒后细胞的病理变化。结果显示,使用1.5×L-15培养基培养对虾淋巴组织,3 h后可观察到有细胞迁出,并能迅速形成单层,36 h后细胞的迁出汇合率可达80%,且能存活20 d以上。接种WSSV 24 h后,出现病变的细胞变圆、漂浮,细胞之间的网状结构消失,最后细胞破碎、溶解;接种WSSV 48 h后Hoechst 33342染色结果显示,感染的细胞核深染,且变形、膨大;电镜下,细胞核内含大量成簇分布的杆状病毒,细胞器被挤向细胞边缘,细胞膜轮廓模糊。研究表明,纯化的病毒粒子接种体外培养的淋巴细胞,能够使其产生明显病理变化,证明了WSSV对体外培养的淋巴细胞具有感染力,并且可在淋巴细胞中增殖。

The primary culture of the cell from lymphoid organ of Metapenaeus ensis and the infected features with white spot syndrome virus(WSSV)

White spot syndrome virus (WSSV)caused serious disease and economic losses in the world, becoming a limited factor in sustainable devolpment of shrimp industry. Shrimp cell culture is a simple and rapid tool to study the pathogenic mechanism of the virus and to determine the viral susceptibility to the cells. In this research, primary culture cell from lymphoid organ of greasyback shrimp Metapenaeus ensis has been established by using an improved 1.5×L-15 cell culture medium. The cell cytopathic effects (CPE) has observed, as the primary cell monolayer inoculated with WSSV. The results showed that the migration of lymphoid cells from the explants was initiated at 3h after seeding, and a 80% confluent cell monolayer was formed within 24~36h and remained viable for over 20 days. Within 24h post inoculation, apparent CPE was observed in the primary culture cells. The infected cells initially exhibited shrinkage or became aggregated, while the network structure between the cells disappeared. Finally, the most infected cells rounded up and then detached from the culture dishes. Under fluorescence microscope, Hoechst33342 staining showed that the nuclei of lymphoid cell enlarged and deformed with a deepen color, after 48h inoculation with WSSV. Under electron microscope, a large number of clustered viral particles has been founded in the affected nuclei, while the organelles were pushed to the cell edge, and the outline of the cell membranes were vague. The study illustrated that the cultured lymphoid cells had apparent CPE as inoculated with WSSV. This phenomenon proved that WSSV could infect and multiply within the primary culture cells.