Selection And Immunogenicity Of Attenuated Strain Of Koi Herpesvirus

Cyprinid herpesvirus 3(CyHV-3), also known as koi herpesvirus(KHV) or carp nephritis and gill necrosis virus(CNGV), which is the etiological agent of a highly contagious and extremely virulent disease to common carp and koi varieties. Under KHVD affect the mortality rate of ponds will reach 70–95%, A major outbreak of the disease was first reported in Israel and in the early 1990 s the United States and European countries have been troubled the disease, in China the last decade of has also seriously threatened on the carp aquaculture, the highly pathogenic disease has spread rapidly to aquaculture around the world, and caused serious economic losses. However, no effective vaccine is available for the prevention and control of the disease. This study aimed to attenuate virulence of virulent strain of Koi herpesvirus and using to assist in the Eucalyptus leaves reduced virulence. Finally, using attenuated strain carry out immune test and challenged experiment in carp that it has a good study the effect of immune protection. The contents and results of the study are as below:1. Eucalyptus Liquid research on cell and viral replication in the cell. The experimental group were added different dose of Eucalyptus Liquid(10mg/ml、2mg/ml、1mg/ml、0.5mg/ml、0.2mg/ml、0.1mg/ml),but no any Eucalyptus Liquid in negative control group, to examine the influence of Eucalyptus Liquid on cell with MTT methods, the optical density at OD 490 nm was measured by ELISA microplate reader. At a concentration range of cells safety, by CPE observation and MTT to examine were inhibited to replication of koi herpesvirus. The results were as follows: The safety examine of drug to cell showed that Eucalyptus Liquid with the concentration of 0.2mg/ml and 0.1mg/ml. The remarkable results in inhibition of KHV replication showed that Eucalyptus Liquid with the concentration of 0.2mg/ml.2. Selection and Immunogenicity of attenuated strain of koi herpesvirus. KHV was passaged to 52 th generation on KF-1 cells, and KHV titter was determined by using the method of Reed-Muench. The 3th,21 th and 52 th generation KHV(TCID50=10-6.59;10-5.87;10-4.61),inactivated vaccine group and PBS control group were inoculated peritoneal immunity into craps. As to immune experiment, each group with 100TCID50 immunizing dose; booster immunization after three weeks; blood for the carp immunitied every 7 days, determined of antibodies by ELISA method while carry out neutralization test determined antibody levels. Set up challenged group in the end and verify that the protective immunity of attenuated strain of koi herpesvirus by biopsy. Results showed that we injected carps with viruses of 3th generation and a mortality of 90%.P3 is a virulent strain. Immunization P21 group have more than 25% mortality, indicating that strain is not completely attenuated. Inactivated vaccine group of mortality is low, it is only 10%. The immunization P52 group have all became survivors. These results indicate that P52 virus confer immunity against the KHV induced disease. ELISA results showed that P52 group for the KHV-specific antibodies more higher than inactivated vaccine group, with control group has not change. Neutralization test results showed that control group has not produce neutralizing antibodies with P52 group produced higher levels of neutralizing antibodies than inactivated vaccine group. Consistent with the ELISA results. P52 group protection rates were higher in challenged experimental. The pathology results showed that the P52 group organization has not change significantly, inactivated vaccine group organization has little change, while the organization of the control group have change significantly. Obtained an attenuated strain of KHV and protection rate was significantly Increased when attenuated strain was used for infection of craps.