Establishment And Preliminary Application Of KOI Herpesvirus Nested-PCR Detection Methods

Koi Herpesvirus (KHV) is the pathogen of the Koi Herpesvirus Disease (KHVD). It mainly infects koi and carp. The mortality is higher than80%in both fry and adult fish. KHV was identified in1998among koi and common carp cultured in Israel, United States and Germany, etc. The virus has been found associated with mass mortality events on most continents. During the past two years, the KHVD also occurred in the northeastern region of China. The disease can cause fish gills necrosis, nephritis, sunken eyes, and other symptoms. The high mortality and long-term latent infection brought by KHV has drawn great attention of the Office International des Epizooties (OIE). In2008, Ministry of Agriculture of China listed the Koi Herpesvirus Disease as one of the B animal diseases in1125announcement.Based on previous KHV detection researches, this study established KHV Nested-PCR detection methods. After trying different primer concentrations, annealing temperatures and cycle times, the optimal reaction conditions were determined. This method could detect KHV DNA genome in the level of0.01pg. Amplified target fragment were isolated, purified and sequenced. The sequences were compared with the three completely sequenced KHV strains and homology was higher than98%. Protein BLAST results also showed that the experimental protein belonged to the KHV virus particle-associated protein. After software analysis, the amplified ORF26gene fragments which indicated good antigenicity may serve as a target fragment of the genetically engineered vaccine.In this study, the Nested-PCR method was used in epidemiology investigation of the koi, common carp and crucian carp in Jiangsu, Zhejiang, Anhui and Shandong etc. KHV genome DNA has been detected in Nanjing and other8cities. In addition, the experiment for the first time detected the existence of KHV in China’s common food fish crucian carp. Most of the samples of fish in the test were clinically normal, no obvious lesions, but through the Nested-PCR detection method we can detect KHV’s latent existence in the ponds of China. Latent KHV existence may lead to disease outbreaks. Considering the high mortality rate and the long-term latent features of the virus, it is necessary to strengthen the monitoring of the Koi Herpesvirus Disease with Nested-PCR method, in order to minimize the possibility of the outbreak of the southern China, and to prevent significant economic losses of aquaculture industry.