Screening Anti-WSSV Herbal Medicines Based On Cultured Cells And Evaluation The Herbal Treatment

White spot syndrome virus (WSSV) caused serious disease andeconomic losses in the world, becoming a limited factor in sustainabledevolpment of shrimp industry. Exploring the infectiong mechanism,pathogenesis and transmission of virus is the basis for prevention andtreatment of WSSV. Although many scholars have made unremittingefforts, WSSV has not been effectively controlled. At present, with thedevelopment of molecular biology, genetic engineering, computertechnology and other disciplines, appeared a lot of efficient and fast drugscreening model which played a positive role in screening and drugdevelopment. Combined with the cell culture of Chinese herbal medicinescreening of anti WSSV, to prevent the spread of viral disease and providestrong technical support for the health development of shrimp aquaculture.1. The primary culture of the cell from lymphoid organ of Metapenaeusensis and the infected feature with white spot syndrome virus (WSSV)Primary culture cell from lymphoid organ of greasyback shrimp(Metapenaeus ensis) has been established by using an improved1.5×L-15cell culture medium, providing a platform for drug screening cell. The cellcytopathic effects (CPE) has observed by inverted microscope, fluorescence microscopy and transmission electron microscopy, as theprimary cell monolayer inoculated with purified WSSV. The resultsshowed that the migration of lymphoid cells from the explants was initiatedat3h after seeding, and a80%confluent cell monolayer was formed within24-36h and remained viable for over20days in the1.5×L-15cell culturemedium supplemented with shrimp muscle extracts, fetal bovine serum,EGF, bFGF, sodium penicillin and streptomycin sulfate. Within24h postinoculation, apparent CPE was observed in the primary culture cells. Theinfected cells initially exhibited shrinkage or became aggregated, while thenetwork structure between the cells disappeared. Finally, the most infectedcells rounded up and then detached from the culture dishes. Underfluorescence microscope, Hoechst33342staining showed that the nuclei oflymphoid cell enlarged and deformed with a deepen color, after48hinoculation with WSSV. Under electron microscope, a large number ofclustered viral particles has been founded in the affected nuclei, while theorganelles were pushed to the cell edge, and the outline of the cellmembranes were vague.2. Priliminary screening anti-WSSV Chinese herbsUsing the screening model of cell level, to screen anti-WSSV Chineseherbs. Apparent cytopathic effect(CPE) was observed in the lymphoid cellsinoculated by the mixture of herbs and WSSV. Extracting the genomic DNA of the cells and determined quantitatively analyzed to calculate thecopy number of virions. Established a screening the antiviral medicinemodel through the observation of cell morphology and difference betweenthe number of viral copies. The result show that Chinese herbal medicinehas the effect of anti-WSSV were AWH-2、AWH-8、AWH-12、AWH-16、AWH-22、AWH-30, speculation may block the virus to adsorption andproliferation.3. Screening and clinical verification of Chinese herbs anti-WSSV incompoundDouble-dilluting Chinese herbal medicine to infected the cells,determining the maximum safe concentration of herbs by observing themorphology of cells. So that the maximum safe concentration of herbsAWH-2、AWH8、AWH-12、AWH-16、AWH-22、AWH-30was25mg/mL、6.25mg/mL、25mg/mL、12.5mg/mL、50mg/mL、25mg/mL, respectively.Under the maximum safe concentration, mixing the medicines by thesame proportion, to determine the best antiviral compound Chinese herbalmedicine. In the clinical trials, verdicting the effect of compoundherbs by observing feeding,swimming, body color change death ofshrimps. Research shows, in vitro and in vivo antiviral effect of compoundChinese herbal medicine is consistent.In conclusion, using an improved1.5×L-15culture medium to culture lymphoid cell of greasyback shrimp, established of antiviral drugscreening model in vitro. Screening Chinese herbal medicine has theeffect of anti-WSSV by the CPE of cells and number copies of virus. Inthe largest safety concentrion, concerted application of drugs. Verifyantiviral effect of compound Chinese medicine through clinicalapplication in vivo antiviral effect. The results of this study provide atheoretical basis for prevent large-scale outbreaks of viral diseases, inorder to achieve sustainable development of the shrimp industry.