| Koi herpesvirus disease(KHVD)with high mortalities caused by Cyprinid herpesvirus 3(Cy HV-3),which infects common carp and koi at various ages as well as common variants of carp.According to the results of the tenth classified report presented by The International Committee on Taxonomy of Viruses,ICTV,Cy HV-3 is classifed as a member of the genus Cyprinivirus within the family Alloherpesviridae in the order Herpesvirales.The mortality rate caused by the Cy HV-3 in fish reachup to 80-100 % and Cy HV-3was firstly isolated and named in Israel in 1998,which captured world attention.The first recognized case of KHVD in carp occurred in 1998,which was distributed worldwide now and results in considerable hazard in common carp and koi(Cyprinus carpio L.)farming industries.China is a large Carp farming country,and there is a long history in cooking carp and carp culture.KHVD was listed as an infectious disease that must be declared by World Organization for Animal Health(OIE).In China,the disease is classified as a secondary epidemic disease.In 2007,AQSIQ demand to strengthen the imported koi carp monitoring,and the KHVD detection is essential items during the import and export trade activities.KHV disease(KHVD)outbreaks regularly occur in spring and autumn when water temperatures are between 18° and 28 °C,and the virus has the strongest infectivity water temperatures are between 22° and 24°C.When the temperature is raised or lowered to a suitable temperature,the virus can proliferate and cause cytopathic effect,This regular phenomenon has caused wide concern over the recent years,but the mechanism has not been completely clarified.The tumor necrosis factor receptors superfamily(TNFR)encoded two tumor necrosis factor receptor-like secretory proteins,ORF4 and-ORF12,which maybe play an important role in immune evasion during virus infection.In our study,the antigenic potential was investigated.The genes encoding ORF4 and-ORF12 was respectively amplified by PCR.The PCR products were inserted into expressed plasmid PET 32 a to construct the recombinant plasmid p ET32a-ORF4 and p ET32a-ORF12.After transferring into E.coli strain BL21,the recombinant antigens of ORF4 and ORF12 were purified.Neutralization test indicated that serum from mice immunized with tow antigens can reacted with the Cy HV-3 virus.Neutralizing antibody titer of the antibody against ORF12 is 1:13The most effective way to prevent KHVD is immunization,but there is no effective vaccine against Cy HV-3 infection.The protective immunity responses of both antigens against Cy HV-3 infection was invested by constructing eukaryotic recombinant plasmid.The results showed that eukaryotic recombinant plasmids were successfully constructed and expressed in KF-1cell.When the carp were immunized with recombinant plasmids p IRES-ORF4 and p IRES-ORF12 respectively,the antibodies against both antigen were induced.Immunization with three different doses(2 mu g/tail,10 mu g/tail,40 mu g/tail)induced a certain level of antibody titer,and antibody titer in the e dose of 40 mu g/tail group is highest.The antibody titer of the immune group was significantly different from that in the control group(p < 0.05).However,there is not significant difference between three immunization group(p < 0.05).Furthermore,challenge assay showed that immunized with p IRES-ORF 4 and p IRES-ORF12 induced a significant protective immune and the protective rate is 73.3% and 83.3% respectively.The pathological section showed that there is not obvious pathological changes in the immunization group.All these results indicated that immunization with the p IRES-ORF and p IRES-ORF12 obtained ideal protective effect.Clinically,the disease was diagnosed based on clinical symptoms,autopsy change and epidemiological analysis,and the laboratory diagnosis is mainly through PCR method.The national standard for detection of Cy HV-3 infection in carp is to isolate virus,then the virus was confirmed by PCR.However,this method takes a long time,and it is easy to make false negative to miss detection.In some local standards,the PCR method is used to diagnose the disease.However,the morbidity of the herpesvirus 3 in carp farming area in our country is higher,and the cases with virus infection and without symptom often occur in improper temperature.Sometime,there are a mix infection.In these cases,the detection of the disease was greatly increased by sensitive PCR method,but the false positives is also increased.Based on our previous studies that the ORF12 genes expressed as soluble antigen and the expression quantity is very high,the Enzyme-linked immunosorbent assay(ELISA)based on the r ORF12 antigen was established.Cross reaction test showed that ELISA based on ORF12 protein can not recognized the positive serum against cypravirus,Aeromonas hydrophila,and vckers aeromonas respectively,indicating that ELISA based on ORF12 protein is specificity for Cy HV-3.The coefficients of variation(CVs)was 1.69 % and 4.54%,and the CV < 10%,indcating repeatability and stability test are better.Finally,50 samples were test by standard test method and ELISA establish in our study respectively.The positive coincidence rate of the two methods was 75%.The results indicated that the ELISA based on ORF12 protein have higher coincidence and were suitable candidates for diagnosis of Cy HV-3infection in carp. |
