Primary Cell Culture Of Cyprinus Carpio L. And Isolation And Identification Of Kio Herpesvirus

The Kio herpesvirus disease has caused severe financial losses to fish breeders. Isolation of KHV was the key of prevent and control this viral disease. And the susceptive cells line was the antecedent of isolating KHV. Cyprinus carpio var. specularis were chosen for experimental materials in this study. Snout and tail fin were taken to culture by tissue explant in vitro. In order to found and optimite culture conditions for the both cells, and get some cell characteristics of MSC and MFC in vitro. And we isolate the KHV using the MSC or MFC.The snout and tail fin were cultured under different mediun (MEM,DMEM,RPMI1640,M199), serum concentration (5%,10%,15%,20%), temperature(20℃,26℃,30℃,37℃).Drawed the growth curve and paired comparison the means of the distance of cells migration by LSD method, the result showed that snout and tail fin cells were most quickly multiplied under 26℃～30℃,20% serum concentration, M199.The cell migrated from snout and tail fin after cultured 3 days, and formed the outgrowth. After about a month, the cell monolayer could be subcultured. The 3rd generations could adapt the vitro conditions. The MSC were moer quickly multiplied than MFC. The MFC were like epithelial cells, deying light by H.E., but the MSC were fiber-like cell. They had the potential ability to become the tissue source of establishing continuous cell line. The both cells were cryopreserved with 10% DMSO in -80℃and liquid nitrogen. The result show that the survival rate of the cell cryopreserved in liquid nitrogen was lower than in -80℃,and MFC were higher than MSC. Both cells were sensitive with SVCV, could be observed CPE, and the virus titer increased markedly.Kio herpesvirus was isolated from the infected carp in order to research and prevent Kio herpesvirus disease. The virus was isolated by co-cultivation of MFC (or MSC) monolayers with cells taken from kidneys of infected living fish. Cytopathic effects (CPE) were observed 5-6 days post co-cultivation of fresh MFC with the kidney cell.Virus induced formation of endoplasmic vacuoles in the infected cells. The KHV particles were reviewed under electron microscope by negatively stained the sediment of virus culture with 0.5% phosphotungstate. A 849bp DNA fragment of ORF25 gene was ampliped by PCR. The PCR product was cloned and sequenced. It was the same homology of 99.9% with three strain of KHV in GenBank. The phylogenetic tree analysis indicated that there was a close genetic relationship between KHV-cj and KHV-J. The symptom and pathological of the fish that were infected by intraperitoneal injected with lml virus culture(TCID50=10-6.75) was the same with natural infection. The morbility and mortality were 100% and 75%, respectively. This study demonstrated that we isolated for the first time KHV from the naturally infected fish in Chian.