Pathogenicity Of Shrimp White Spot Syndrome Virus ZheJiang Strain And The Protective Effects Of Recombinant Envelope Protein RVp28 Against Virus Infection In The Laboratory Animal, Procambarus Clarkii

Shrimp culture is one of the important industries of aquaculture in China. Butdiseases have become the major impediment to the sustaining development of shrimpfarming in recent years. Among them, white spot syndrome virus (WSSV) isextremely virulent and causes high mortalities, with a wide geographic distributionand host range. Since its first discovery in Southeast Asia around 1993, WSSV hascurrently been a major threat not only to shrimp culture but also to marine ecology.The mechanism of the virus entry into the shrimp and pathogenesis is not known, andviral infection in shrimp has no effective therapeutic or prohylactic counter measures.Now it will be a new way to prevent shrimp virus diseases by interrupting thecombination between virus and host cell using gene-engineering technology. In thepresent work, WSSV-ZJ strain and the recombinant envelope protein rVp28expressed in silkworm pupae infected with recombinant virus HyNPV-Vp28 wereused in crayfish, Procambarus clarkii. The trials were conducted to study thepathogenicity of virus, the effects of rVp28 on immune factors and on diseaseresistance in crayfish.The WSSV isolate used in this study originated from WSSV-infected Penaeus chinensis collected from Ningbo in 2001 and was proliferated in crayfish. It had the similar morphology and structure with but the different size to the other isolates observed by transmission electron microscope (TEM). The completed virion with double envelope was ellipse, and the size of it was 319 nm×69 nm and 318 nm×90 nm in virus solution and slice, respectively, but the purified nucleocapsids was rod-shaped with the size of (211~344) nm × (50~63) nm.The results of infection trial shown that the cumulative mortality of crayfish was 100% in 8 days via oral ingestion or intramuscular injection routes when the water temperature was 22-24℃. When virus was infecting and proliferating in crayfish, the epidermal cells of stomach, midgut, hepatopancreas, gill and epithelial tissue were susceptive to the virus, which was histologically characterized by hypertrophied nuclei and highly stained cells. The results of slice obviously shown that the infectedcells were characterized by the hypertrophied nuclei and chromatin disappeared, the denaturen and necrosis mitochondrias and the abnormal rough endoplasmic reticulum. Through in situ hybridization by DIG-labeled probe, the results shown that the epithelial cells of alimentary tract were the leading targets infected by virus. It implicated that the infection and proliferation of virus caused the metabolism of cells decayed and crayfish dead.In immune and challenge trial, crayfishes with body weight 23.0 + 0.2 g andsilkworm pupae infected with the recombinant baculovirus HyNPV-Vp28 were used.75 days experiments involving 60 WSSV-free crayfishes in each treatment wereconducted to investigate the protective effects of rVp28 on virus infection. The testpupae treatment was supplemented with 2% pupae expressed rVp28 in control feed.The control pupae treatment was supplemented with 2% normal pupae in control feed.The control treatment wasn't supplemented with any pupae. Each treatment wasdivided into two even subgroups on day 35. First subgroups were orally challenged,and the remaining subgroups were intramuscularly (IM) challenged. The resultsshown that the test pupae treatment had the significantly higher cumulative survivalcompared with control treatment and control pupae treatment in 20 days (i'<0.05),with relative percent survival (RPS) values 59.26% and 54.16% via oral challenge and49.99% and 46.12% via IM challenge, respectively. There weren't significantdifference in cumulative survival among three treatments when re-challenged on day55, but the test pupae treatment still had higher RPS values (P > 0.05). It implicatedthat the protection against WSSV could be induced in crayfish by recombinant proteinrVp28 expressed in silkworm pupae.The examination of epithelium cells of alimentary track was conducted using three methods. There was normal structure in the survivors of the test pupa treatment, but there were typical pathological changes in moribund crayfishes of the other treatments observed by histopathology. The tissues of the moribund crayfish in control treatment and control pupae treatment shown obvious positive hybridization signals but the tissues in the survivors of test pupa treatment shown negative hybridization signals by in situ hybridization and dot blotting. It implicated that the resistance presumablyresulted from the direct interaction of the recombinant envelope proteins with the binding site on the target cells and from blocking the spread of intruding virus.The results of immune analysis shown that the phagocytic percent and phagocyticindex of hemocytes significantly increased in the test pupae treatment compared withthe other treatments (PO.05). It implicated that rVp28 induced celluar immunity. TherVp28 significantly increased the antibiotic activity, bacteriolytic activity andactivities of phenoloxidase, superoxide peroxidation, acid phosphatase and catalase inserum compared with the other treatments (P<0.05). The activity of alkalinephosphatase was significantly higher compared with control treatment (P<0.05) butthere was no difference between the test pupae treatment and control treatment (P >0.05). It implicated that rVp28 improved the level of humoral immune factors. In themuscle tissue, the alkaline phosphatase activity significantly increased (PO.05). Inmuscle and hepatopancrease tissues, the activity of myelopetoxidase significantlyincreased compared with control treatment (P<0.05), but there was no differencecompared with control pupae treatment (P > 0.05). In conclusion, it implicated thatrVp28 had the role of improving the immune function of crayfish.The results above suggested that the recombinant envelope protein rVp28 expressed in silkworm pupae had higher efficacy on disease resistance, and improved the celluar and humoral immune in crayfish. Oral administration is the only practical way for delivery of the medicament protein to shrimp. Production of heterologous proteins in silkworm has several advantages such as safe, convenient for administration and inexpensive. The method is expected to provide a new approach against shrimp virus diseases. To elicit the penetration mode of rVp28 through the alimentary track, the underlying mechanisms of rVp28 recognition with target cells and the induced mechanism of immunity, more studies are needed.