Since 1993, the White Spot Syndrome Virus (Wssv) disrasc occurted in China among cultured shrimps resulting in mass mortality. Eirootiological surveys trndertaken during the outbreak period of 1993-1994 indicated that all stages of peeus chitensts, P. uems and p. were infected. Conssquent bo the transport of contaminated shrlmp seedlings and seawaler, the diseasc sprcad all over the farrns. of china. The disease was more rapidly transmitted at lemeratures above 25℃, Challenge experiments showed the causative agent wss highly virulent, White sots appeared on the carqpace of both spon-taneous. and experimentally infected shrimps. Moribund shrimps contalned burbld hemolymph, hy-perticleg, in the gills, stomach, lymphold organ, and epidermal tissuc of the infected sherim. The virlons were sllghtly ovold with an envelope and avcragcd 350×150nm, nucleocapoids measurd 375×157nm. With discontinuous suceose gradient of 35,50 and 60% (W/v),the virus was separated from hemolymph of the infected shrimp. The estimated molecular weight of genomk DNA was 237Kb with EcoR I,247Kb with hind Ⅲ and 241kb with Pst1. A total of 9 hybridoma colones secrting monoclonal antibodies (MAbs) were produced from mouse myelonu and spleen cells lmnunlzed with wssv. the immunofluorescence assay of gill tissue showed that the mabs reacted with discascd but not with healthy shrimp. The Mabs belonged to IgG1, IgG2b subclass and IgM class, all with kappa light Lmmuno-electron-mderoseopy with colloidal gold markcr showcd the prcsence of 5 Mabs epitopes on the envelope and one on the capsid of the virus Baculoviral mid-gut gland necrosis showed the specltlclty of the MAbs produced, for diagnosis 5 different methods were selected. Using Kimura Primers for PcR, or Mabs for immunoblot, ELISA or FAT method im sith hybridiztion was carried out to show the gene, All these methods detected WSSV in the organ ramples of the diseased shrimp but not in helthy ons.

d development of lymphocystis cells in flounder Paralichthys olivaceus, and some his- tochemical features are studied using light and electron microscopy and histochemical methods. ibrob-lasts engulf virus particles forming phagocytosis vacuole in the cytoplasm. The infected cells proliferateand turn round and hypertrophied; basophilic inclusion bodies, Feulgen-positive, are present in the cyto-plasm, and a hyaline capsule, containing acid ploysaccharide and protein, appears outside cell mem-brane. The mature lymphocystis cells possess numerous virus particles (200～220nm in diameter with a central core 120～140nm) and high electron-dense inclusions with budding virions at the surface in the cytoplasm, and show margination of chromatin. Moderate electron density particles (70～80nm in diame-ter) fill out perinuclear cisterna and arrange in crystalloid arrays in nucleus, liberated into the cytoplasm by rupture of nuclear membrane. No virus particles are observed in nucleus. Therefore, inclusion bodies are the sites of virus assembly and maturity, while replication processes of lymphocystis virus are involved in both nuclear and cytoplasmic phases. Besides, strongly basophilic link2like structures are present be-tween adjacent cells or through connective tissue, and virus particles are observed in somewhere of the capsule, thus where may be the spread route of virus from one cell to another or to the environment. The senile lymphocystis cells release virus by the rupture of cells.

Nine bybaldoma clcmes secreting monoclonal antlbodies (Mabs) were peodineed from monse myeloma and spleen cells immunized with while spot syndrome virus (WSSV), An imdirect pathogen of ealtured penaeld shrimp Asin since 1993 Am imdirect immonoftucrescence essay was standardixed to evaluate the usefulness of the Mabs for rapid diagmostic techniques to idemlify WSSV and for further mudy of this virus. tsoryping revealed that the brabs were of immunoglobulin G (IgG) and immunaoglobulin M (IgG)class. All with kappoa light chains. Four Mabs were examlned by using immune elecron microssopy and colloidal gold as a markee, Three Mabs recogniged epitoped on the envelope of the virus and one Mab recognleed an eplnope on the copsld. The epitopes on the envelope of the virus and one Mab recognleed an eplanope on the copsld, The specilieity of the Mabs to WSSV was eltermined by a lack of recktivity with vissue comtuingg a second pemenld shrimp agens. The baculovirul mld*gat gland necrosls virus.

White Spot Syndrome Virus (WSSV) was purified from hemolymph of infected shrimp. After nucleic acid extraction from the purified virus particles, EcoRI-digested fragments of the WSSV genome were cloned; three of these fragments were used as non-radioactive probes labeled with DIG-11-dUTP. The probes hybridized in situ, with sections located in the nuclei of all WSSV2infected tissues. The virus was de-tected in the gill, stomach, epidermis, and connective tissue and so on, but not detected in healthy shrimp tissues and epithelial cells of hepatopancreatic tubules of diseased shrimp.

Haemocyte types of the scallop (Chlamys farreri) were identified by Giemsa stain and flow cytometry (FCM). Additionally, the activities of peroxidase (POD), phenoloxidase (PO) and alkaline phosphatase (ALP) in haemocytes were analysed by immunocytochemical and biochemical methods. The results indicate that there were two types of haemocytes in the scallop, hyalinocytes and granulocytes, and that POD, PO and ALP were more abundant and more active in granulocytes than in hyalinocytes.

False positive results were obtained in immunodot blot assays to detect white spot syndrome virus when horseradish peroxidase-conjugated sheep anti-mouse immunoglobin (Ig) serum was used as a secondary antibody with 3-3'-diaminobenzine tetrahydrochloride dihydrate as the detection substrate. The cause was considered to be a reaction of shrimp endogenous peroxidase (POD) with the substrate. In experiments designed to inhibit POD activity, 9 different reagents were used at different concentrations and for different treatment times. EDTA, sodium azide, HEPES-Na, NaHSO3, H2O2 and phenylthiourea (PTU) were able to inhibit POD activity by 44, 60, 64, 67, 79, and 90%, respectively. Phenylmethylsulfonyl fluoride did not inhibit POD, and neither periodic acid nor H2O2 in methanol were appropriate due to the formation of flocculant precipitates when added to shrimp extracts. It was concluded that of the treatments tested, 10mM PTU for 2 h yielded optimal inhibition and that such pretreatment of samples eliminates false positive results in immunodot blot assays.

A mixture of six anti-WSSV Mab's (hybridoma culture fluid) was used (produced by Zhan et al. 1999a). Shrimp diseases occur more frequently and seriously as the shrimp culture industry in China expands. Comprehensive research in aquatic pathology began in 1982, focused on identifying various shrimp diseases and investigating their etiology, diagnosis, treatment, prevention, and pathogenic ecology. In the 1980s, the diseases were mainly caused by bacteria, fungi, and ciliate parasites; although the negative impact was noticed, diseases were somewhat controlled. But since early 1993, outbreak of epidemic disease caused by white spot syndrome virus (WSSV) has brought mass mortality and led to severe losses in shrimp culture. The disease has been reported in cultured penaeid shrimp such as fleshy shrimp Penaeus chinensis, kuruma shrimp P. japonicus, giant tiger shrimp P. monodon, and other crustaceans (Zhan et al. 1995). There is nearly no effective drug for this virus disease, so the prevention and control of its spread are imperative, as is the demand for sensitive and rapid diagnostic methods.

Enzyme-linked immunosorbent assay (ELISA) and immunodot blot assay, using monoclonal antibodies against white spot syndrome virus (WSSV) as primary antibody, and goat anti-mouse Ig serum conjugated with alkaline phosphatase (AP) as secondary antibody, were developed to detect WSSV in shrimp tissue samples. However, false-positive results were obtained and the cause was considered to be reaction of endogenous shrimp AP with ELISA and immunodot blot substrates. Four AP inhibitors were tested at different concentrations and treatment times to solve this problem. EDTA, NaHSO3, levamisole and HEPES-Na inhibited AP activity by 96.45%, 86.82%, 68.82% and 9%, respectively. In conclusion, sample pretreatment with 0.5M EDTA eliminated shrimp endogenous AP background in ELISA and immunodot blot assays, where AP was used for detection.

Eight monoclonal antibodies produced against haemocytes of shrimp (Litopenaeus vannamei) were used to research the antigenic cross-reactivity of crustacean haemocytes. 2C3 cross-reacted with the haemocytes of all the experimental animals, while 1H8 and 2C11 did not cross-react with the experimental animals. The other five monoclonal antibodies cross-reacted with some of the experimental animals.