Studies On Chicken Antibodies Against Envelop Proteins Of White Spot Syndrome Virus And Its Antiapoptotic Gene ORF390

White spot disease (WSD) is at present the most serious viral disease for cultivated shrimp, which caused very serious and widespread losses in the shrimp industry around the world. The causative agent is commonly known as white spot syndrome virus (WSSV) owing to the main clinic sign 'white spots' in the exo-mesoderm under the carapace.The effect of water temperature on the development of white spot disease was studied. Rayfish were exposed to different temperatures (4℃, 16℃ and 25℃) after WSSV injection and the mortalities were recorded over a period of 30 days. The results clearly show that low temperature affects the WSSV pathogenicity in cryfish. Moreover, total haemocyte and granular cell counts of cryfish held at different temperatures were not significant different. The phenoloxidase activities in haemocyte lysate supernant (HLS) of crayfish at all temperature group remain similar. It is concluded that low temperature could prevent WSSV proliferation and crayfish might act as a carrier of WSSV at low temperature.The four envelope protein genes were constructed into pMAL-c2 prokaryotic expression vector and the maltose-fusion expression proteins were purified. The fusion proteins were used as immugens with chickens to generate antibodies. Results of freshwater crayfish neutralization experiment suggested that VP28 and VP19 chicken antibodies were able to neutralize WSSV infection of freshwater crayfish in aconcentration dependent manner upon intramuscular infection. The freshwater crayfishes were firstly fed WSSV infected meal and then fed with basal diet or diet supplement with VP28 and VP19 chicken antibodies. All the freshwater crayfish died fed with basal diet at 30 days. Conversely, the survival rates of freshwater crayfish fed with the diet containing VP28 and VP19 chicken antibodies can be reduced to 60% and 50%, respectively. Oral passive immunization experiment showed that fed crayfish with diet supplement with chicken antibodies could prevent the WSSV infection in a certain extent. All results showed that VP28 and VP19 are likely to play key roles in the systemic WSSV infection in freshwater crayfish and the oral passive immunization of chicken antibodies has potential for immunotherapeutic application to prevent WSSV infection.In this study, we confirmed that the apoptosis triggered by actinomycin D was inhibited by WSSV infection. As mutants of Autographa californica nucleopolyhedrovirus (AcMNPV), AcMNPV. 35k/pol+ lacks a functional p35 gene undergoing apoptosis and its infection could induce Sf9 cells apoptosis. To identify the putative apoptotic suppressor gene of WSSV, overlapping cosmid clones representing the entire WSSV genome were individually cotransfected along with genome DNA of AcMNPV. p35k/pol+. Using this marker rescue assay, a WSSV DNA fragment that was able to rescue AcMNPV. p35k/pol+ infection in Sf9 cells was isolated. By further sequence analysis and rescue assay, the ORF390 was identified as a novel anliapoptotic gene. The ORF displays two putative caspase9cleavage sites LLVETDGPS , VKLEHDGSK , a caspase3 cleavage site EEDEVDGVP . The ORF was cloned into the pIE1 vector and then the recombinant vector was transfected into Sf9 cells. The Sf9 cells didn't show obvious characteristics of apoptosis when infected with AcMNPV. p35k/pol+. And the transient expression of ORF390 allowed AcMNPV. p35k/pol+ replication in Sf9 cells and result in formation of polyhedra successfully. The results indicate that function of ORF 390 in WSSV is a kind of apoplotic suppressor like P35 in AcMNPV.