Epidemiologic Survey Of Grass Carp(Ctenopharyngodon Idella)Reovirus In South China And Establishment Of Duplex Real-time RT-PCR

Grass carp (Ctenopharyngodon idellus) is a most popular commercial fish species in south China. Grass Carp Hemorrhage is one of the most serious diseases of grass carp, causing more than90%mortality and huge economic loss to the aquaculture industry. Grass carp reovirus (GCRV) is the causative agent of the grass carp hemorrhage. The virion has a nonenveloped double capsid shell with an icosahedral morphological structure and consists of11segments of double stranded RNA.Currently, there is no effective treatment method against GCRV infection, the immunization schedule is especially important. In order to scientifically guide immunization, the epidemiology and variations of this virus are necessary. In last report, the existences of two distinct genotypes of GCRV were confirmed. GCRV-873is the classic strain and GCRV HZ08is the new genetype strain.1. Epidemiologic survey and genetic variations of VP6geneFor recognizing the molecular biology character, phylogenetic relationship and the state quo of grass carp reovirus (GCRV) prevalent isolates, eight GCRV isolates were isolated in five provinces which suffering hemorrhage disease in China from2011to2012year. Experimental results showed these isolates were very different to classic strain-GCRV873. The tissue suspension was inoculated in Ctenopharyngodon idellus kidney (CIK) cells, but no obvious cytopathic effects (CPE) were observed. However, plenty of nonenveloped virus particles with subsphaeroidal shape, which were highly similar to reported GCRV, were observed under electron microscope (EM). The SDS-PAGE electropherosis further suggested the virus possessed11segments of dsRNA, which has the typical characteristics of GCRV. VP6gene among new genotype of GCRV had high similarity, sharing97.1%-100%identity of nucleotides and97.6%-100%identity of the deduced amino acid. But compared with classic strains, it showed significant variations. Based on amino acid sequence phylogenetic tree of VP6gene, the eight GCRV isolates all belonged to subgroup1. These results indicated that this new genotype of GCRV remained an extensive pandemic and revealed new genetic diversity in China.2. Rapid detection of GCRV by duplex real-time RT-PCR assayTwo pairs of primers were chosen to establish a duplex real-time RT-PCR assay method. No cross-reaction was detected for other aquatic virus, such as MDRV, IHNV and ARV, and the limit detection of real-time RT-PCR was about10order of magnitude copies/μL for the target gene which was100times higher sensitivity than conventional PCR. The reproducibility tests in inter-assay and intra-assay indicated that the coefficients of variation were less than1.2%. The result of detecting50clinical samples showed the fluorescent quantitative assay could detect more positive samples.This study showed that duplex real-time RT-PCR assay is reliable, sensitive and fast for simultaneous detecting GCRV classic strain and new type and especially applicable to the production quantities on the sample testing of grass carp.