The Modes Of Entry Into Host Cells And Proliferation Of White Spot Syndrome Virus (WSSV)

White spot syndrome virus (WSSV) is a rod-shaped, circular double-strandedDNA virus, belonging to the Nimaviridae family. With a wide range of hosts incrustaceans, WSSV is the major pathogen of cultured shrimp because of its highlethality. This thesis mainly focuses on different proliferation of white spot syndromevirus (WSSV) in hemocytes and hematopoietic tissue cells. In addition, a fixationmethod of WSSV virions for transmission electron microscopy analysis wasestablished. The main results of this thesis are as follows:(1) The replication and proliferation of white spot syndrome virus (WSSV)were analyzed in hemocytes and hematopoietic tissue cells in vivo or in vitro. In thisstudy, the replication of viral genomic DNA was determined by using real-time PCRand the gene transcription was examined by using RT-PCR. The results showed thatthe viral replication and transcription were detected in the both cells after virusinfection. In hemocytes, the level of the genome replication was significantly lowerthan in hematopoietic tissue cells. Similarly, the initial time of transcription for somegenes in hemocytes was later than in hematopoietic tissue cells. Electron microscopyimages showed that at2h post-infection, multiple virus particles were contained inlarge vesicles in hemocytes, while single or several virus particle(s) was present insmall vesicles in hematopoietic tissue cells. In the nucleus of hematopoietic tissuescells, mature progeny virions were observed at12h post-infection and presentedregular arrangement in clusters with the increase of the time. However, no progenyvirus was observed in nucleus of hemocyte.(2) The modes of entry into cells of WSSV virions were characterized bytransmission electron microscopy. First, virions attached to the cell surface and thenwere wrapped by the invagination of the plasma membrane to form avirus–containing vesicle in hematopoietic tissue cells. However, in hemocytes, theplasma membrane extended pseudopodia and surrounded the virus particles to form“phagosomes”. In addition, we compared the entry processes of UV-inactivated WSSV, formalin-fixed inactivated WSSV and the viral nucleocapsid. Results showedthat all of them could be phagocytosed into hemocyte, but only UV-inactivatedWSSV could enter into hematopoietic tissue cells by endocytosis. The results of thisstudy indicated that the virus entered hemocyte by means of non-specificphagocytosis, while the entry of WSSV into hematopoietic tissue cells is dependenton endocytosis and some viral envelope proteins may play crucial roles during entryprocess.(3) Electron microscopic observation is an important method for morphologyanalysis of white spot syndrome virus (WSSV). In this study, to optimize the fixationmethod, purified WSSV virions were treated with different concentration ofparaformaldehyde or glutaraldehyde. The results showed that, paraformaldehyde andglutaraldehyde treatment could help to preserve the structure of WSSV in the processof TEM sample preparation. Fixation with1%paraformaldehyde for20minutes hadyielded the best result. Further analysis showed that virions fixed under thiscondition was suitable for immune electron microscopy analysis.