| Koi herpes virus disease（KHVD）has caused koi gill rot disease and high mortality onage kois and carp, including common variants of carp such as frame mirror carp, which wereinfected by herpes simplex virus type3(Cyprinid herpesvirus3, CyHV-3). It has led greatconcern around the world. KHVD is a highly contagious disease which could cause by80-100%mortality rate of the fish. Once there is an outbreak anywhere in the country, it isvery difficult to control. The disease was first discovered in1998in Israel, then in a dozencountries and regions all over the world with the popular spread, causing huge economiclosses. Carp feeding is a traditional breeding project, it is widely distributed. KHVD isfrequently happened in the country, coupled with the transport and commercial prosperity. ifthere is a disease outbreak, it could soon threaten the entire region and the country carpfeeding industry, and even other freshwater fish farming, resulting in losses to the nationaleconomy and paralysis of the entire chain of carp breeding,it could also give the China’simport and export trade a heavey infullence and brought immeasurable losses. In2007,AQSIQ issued forecast to strengthen the imported koi carp monitoring, requiring the KHVDdetection as essential items in the the import and export trade activities.The current research shows that vaccines are effective methods of prevention andtreatment of koi herpes virus disease, but China has not yet developed a really effectivevaccine, the koi herpes virus disease vaccine development in our country is still in theexploratory stage. Reported in the literature, there are a variety of diagnostic methods of thedisease. The common clinical application of the clinical symptoms and autopsy by thechange, simply make a preliminary analysis of the pathological diagnosis, final diagnosisrequires application of laboratory diagnostic methods, including virus isolation techniques,indirect enzyme-linked immunosorbent assay, in situ hybridization technology, scanningelectron microscopy, polymerase chain reaction and loop-mediated isothermal amplificationmethod,etc..The methods above have advantages and disadvantages of poor sensitivity andspecificity, some diagnosed process is too cumbersome, and the false-positive rate isrelatively high, there are still some problems such as environmental pollution difficult tomeet the needs of clinical testing. Therefore,we should imminentlly make a comprehensiveand effective control of koi herpes virus disease to strengthen the monitoring of the disease,improve the koi herpes virus detection rates and the efficient and specific detection methodsfor carp herpes virus type3.It is urgent to establish a rapid detection test of sensitive, specificity, low cost and quantity sample volume on the clinical detection of the carp herpesvirus epidemic,so that it can accurately and timely response the epidemiologicalcharacteristics of the disease, as far as possible to meet the feed export requirements.As the clinical need of improvements in the cost, detection rate and the amount of testsample, we have established the detection method used the SYBR Green I CyHV-3real-timePCR, according to the conservative gene ORF108of the pandemic strain. The recombinantplasmid pMD18-T-81was constructed by the PCR products of ORF81gene as the standardwith high stability and good repeatability tested in the real-time PCR. Specific tests haveshown that the establishment of SYBR Green I CyHV-3real-time PCR has good specificity;reproducibility tests showed carp herpes virus type3Real-time PCR on coefficient ofvariation (CV)of the positive plasmid with the concentration were4.1×107,4.1×106,4.1×105were0.95%,0.83%and0.85%, with good stability. It is showed that the detectionsensitivity of the SYBR Green I CyHV-3real-time PCR could be10TCID50of virus, whichis an order of magnitude higher than the conventional PCR.Secondly, we had an epidemiological investigation on the koi herpesvirus disease inparts of Jilin Province using real-time quantitative PCR method. Through the survey,wefound that the incidence of Koi herpesvirus disease was more and more earlie, it coulehappened with the temperature at13-15℃. The incidence of fish primarily for adult fish, fishfarms and fish ponds were acute with the same mutual infection, almost the same disease,mass death. Cage culture is also a high incidence of mortality. In a tributary of Yalu River, theincidence of mortality and morbidity of all the cage aquaculture carp are not very different.The morbidity and mortality rates of ponds feedingare are relatively low, it may be associatedwith breeding density, low stocking density might make the result of low morbidity.The139samples collected were tested using SYBR Green I real-time PCR method, inwhich there were96positive samples, the positive rate was69%. Part of the positive PCRproducts were sequenced,the sequencing results were analyzed on NCBI Blast.the homologyresults with the standard strain between98-100%.20samples were retested by fluorescencequantitative PCR after conventional PCR test which showed all negative, six of which werepositive. It is proved that SYBR Green I real-time PCR method is more sensitive andaccurate than the conventional PCR detection methods.We have detected the DNA vaccine (pIRES-ORF81) distribution in vivo metabolism andtissue of carp using fluorescence quantitative methods. Intramuscular injection ofrecombinant plasmid pIRES-ORF811ug in carp, the presence of the recombinant plasmidcan be detected in vivo various organs after24h, and the copy number of plasmids are the highest level of the organization detected, the plasmid concentrations of each organization arereduced with time. In the first six weeks, no DNA has been detected in the carphepatopancreas and intestine. DNA vaccine could still be detected9×104copies after6weeks,and is always the highest and limit in the muscle, followed by blood, kidneys, spleen,intestine, liver and pancreas. The lowest detection rate and limit is in the liver and pancreas,itcouldn’t be detected in the6thweek.In order to gain positive antiserum to detect the carp herpes virus type3, we amplifiedthe CyHV-3ORF108gene with the genome template of CyHV-3China Jilin strains(KHV-CJ),and constructed the recombinant plasmid pET32a-108,which was expressed in E.coli BL21bacteria. The ELISA test confirmed that the initial murine antibody of theexpression protein could response to the virus. This study proved that, the serum proteinprepared by the pET32a-108expression products might be used as antibody to establishimmunological detection technicals. |
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