Functional Analysis Of The Cells And The Gene Related With WSSV Infection In Litopenaeus Vannamei

White spot syndrome virus (WSSV) is a major viral pathogen of shrimp, which caused severedamage in shrimp culture all around the world since early1990s.WSSV is an enveloped virus. The first step of infecting the host cells is that envelopeproteins binding to the host cell membrane and then they were internalized by cells to initiate theinfection process. Until now, several structural proteins were reported to take part in infecting cells.While the molecular mechanism of viral infection is unclear and there were no effective means ofprevention and control. To analyze the molecule mechanism of WSSV infection, our paper focuson some WSSV envelope proteins and the transcription factor Runt which were related withhemopoiesis of shrimp. In the test, we used fluorescence microscope to observe whether theWSSV envelope proteins could induce the endocytosis of host cells in Litopenaeus vannamei.Through fluorescent microsphere technique, we carried out the cell endocytosis inhibition tests ofsome known receptor proteins to have a clear knowledge of the interaction of WSSV and theproteins. At the same time, we examined the expression levels of Runt in Litopenaeus vannameiafter WSSV infection and the relative protective roles of recombined Runt protein in vivo towardsWSSV infection. This paper is to lay a foundation for a clear knowledge of the infectionmechanisms of WSSV.The five parts of the research are as follows:1. Identification of the uptake of FITC labeled WSSV envelope proteins VP24，VP26， VP28， VP31and VP37via endocytosis by different host cells inLitopenaeus vannameiFirstly the cells of hemocytes, lymphoid organs, hematopoietic tissues and epithelialtissues were obtained by primary cell culture. After cells emigrated and adsorbed the plate, theywere incubated with FITC labeled recombinant WSSV envelope proteins. Observation throughfluorescence microscope showed that rVP24, rVP28, rVP31and rVP37could be seen in thecytoplasm of hemocytes. While only signals of rVP28and rVP37could be seen in the cytoplasmof lymphoid cells, and signals of rVP28, rVP31and rVP37could be detected in the cytoplasm of epithelial cells. What’s more, rVP24, rVP28and rVP37could enter the hematopoietic cells viaendocytosis. In conclusion, VP28and VP37could alone induce the endocytosis of host cells, andmay play a vital role in entering and infecting host cells.2. Appling fluorescent microspheres technique to study the interaction of shrimphemocytes in Litopenaeus vannamei with recombinant WSSV envelope proteinscoupled with fluorescent microspheres and the effect of AK，Rab7, BP53,polyclonal antibody on the uptake of VP31，VP28and VP37cells.In the study, we coupled the WSSV envelope proteins with fluorescent microspheres by EDCmethod. The results of flow cytometry analysis showed that BP53could inhibit the uptake ofVP37by hemocytes obviously, while Rab7and AK didn’t affect the internalizing of VP28andVP31. The polyclonal antibody of VP28and VP37could inhibit the uptake of VP28and VP37byhemocytes respectively. On the contrary, the antibody of VP31didn’t affect the endocytosis ofhemocytes, which may result from the internalizing of VP31was non-specific. What’s more, wecan conclude that BP53(F1ATPaseβsubunit) act as a receptor protein and participate in the processof WSSV infection. Rab7may work as a cytokines interactive with rVP28to mediate the WSSVinfection.3. Molecular cloning of runt gene cDNA in Litopenaeus vannameiWe report the cDNA cloning of the full Runt gene of Litopenaeus vannamei (lvrunt) by rapidamplification of cDNA ends (RACE) PCR. The resulting full-length gene consisted1755bp witha663bp ORF, encoding221amino acids with the calculated molecular mass of23.6kDa. Thededuced amino acid sequence contained a runt-family domain. The amino acid sequence of Lvruntshowed88.1%homology with the amino acid sequence of Runt in Pacifastacus leniusculus. Thehomology with other species was30%to52.4%, and this protein kept high sequence conservationamong different species.4. Analysis of the specific expression of lvrunt in different tissues in Litopenaeusvannamei and the influence of injecting Lvast and WSSV on the transcription oflvrunt of the hemocytes in Litopenaeus vannameiTranscription level was analyzed in seven kinds of tissues of shrimp, including gill, gut,hepatopancreas, lymphoid organs, heart, muscle and hemocytes. By comparison, the resultsrevealed that the transcription of lvrunt mostly happened in hemocytes, rarely detected in the other tissues.Expression levels of lvrunt in hemocytes significantly were increased by recombined astakineprotein or WSSV injection. The highest levels occurred in6h post injecting Astakine and12h postinjecting WSSV respectively.5. The prokaryotic expression of LvRunt and its neutralization assay in vivo onWSSV infecting.The recombined vector pBAD/gIIIA-lvrunt containing the Runt ORF was constructed, andtransformed into E.coli. After0.02%L-arabinose induction at37℃, the fusion protein wassuccessfully obtained, which were identified by SDS-PAGE and Western blot analysis. Then wepurify the LvRunt using Co-NTA resin by affinity chromatography. After injecting renatured Runt,we injected WSSV to infect shrimp. Calculate the cumulative motality rate and compare thefunction of Runt. Based on the statistical results, it can be concluded that LvRunt didn’t protectthe shrimp from death in general.