Diversity Analysis On Biological Characteristics Of Three Strains Of Grass Carp Reovirus From Different Genotype

Grass carp (Ctenopharyngodon idellus) is one of the main species of freshwater fishfarming in our country, and the National Fisheries Statistics Yearbook indicated that the totalproduction of freshwater fishwas26,445,400tons in2012, of which grass carp aquacultureproduction reached4,781,700tons. Frequent outbreak of Grass carp hemorrhagic disease notonly cause huge economic loss to grass carp aquaculture, but also result in serious damage tothe healthy development of freshwater aquaculture in China. The pathogen of this disease isGrass carp reovirus(GCRV), which was the first aquatic virus isolated and characterized inthe mainland of China. The genome of GCRV is composed of11segments of double-stranded RNA(dsRNA)result in high variance More than40strains of GCRV have beenisolated and identified. According to the banding pattern of different isolates strains andpublished gene sequences, there are at least three genotypes of GCRV which represented bytype I GCRV873, the typeII GCRV HZ08and the type III GCRV104. There are somepublications refer to virus difference in cell sensitivity, cytopathic effect, virulence, genomeband pattern and gene sequences, however, the property of virus stability, proliferation insame cell and fish is scarce. In order to establish TaqMan Real-Time PCR method fordetection of three genotypes GCRV, the primers were designed according to conservativeregion of segment6(S6). Quantitative comparison of selected three virus was carried out onthe aspects including proliferation in the cell and the fish, stability under the conditions ofdifferent pH, treatment with ethyl ether and trypsin, and several times of freezing and thawing.The present study will provide basic data for epidemiological investigation, disease controland vaccine development for grass carp hemorrhagic disease. This research mainly includesthe following contents:1. The phylogenetic tree was constructed based on S6sequences from availablesequences of aquareovirus registered in GenBank. Analysis was conducted with genome bandpattern of present GCRV isolates in our lab using SDS-PAGE. Three isolates were selected asfollows GCRV JX0901(genotype I), GCRV HZ08(genotype II) and GCRV104(genotypeIII) according to the combined results of phylogenetic tree and genome band pattern. Themethod for GCRV genotyping will be better conducted by phylogenetic and genome bandpattern together. 2. To analyze biological characteristics of different genotype GCRV, a method tomonitoring the proliferation of three genotypesusing TaqMan fluorescent quantitativepolymerase chain reaction (FQ-PCR) was established. The results showed that the methodpossessed high sensitivity and specificity with lowest limit detection was4copies,10copiesand8copies, respectively. The clinical samples from epidemiological investigation weretested using the established method, the positive detection rate was higher than conventionalRT-PCR.3. Virus stability analysis indicated that the virus titers of all three viruses varied a littlewithin the pH scope of3.0to10.0. The adaptative range of GCRV HZ08is a bit wider thanthat of GCRV JX-0901and GCRV104, possibly as one of results that the genotype II iscurrent popular type of GCRV in China. Repeated freezing and thawing affected the virustiters markedly. Three strains was not sensitive to the treatment of ether and trypsin.4. The proliferation property in same cell and fish infected by three genotypes of GCRVwas characterized. The result of GSB cell inoculation showed different obvious cytopathiceffect with JX-0901and104infection compared to HZ08which has no observed CPE. Thevirus copies after cell proliferation in GSB-F cells were higher than GSB-E. Furthermore, thevirus copies detected in JX-0901and104were significantly higher than HZ08. Proliferationcapacity in GSB cell between GCRV JX-0901strains and GCRV104strains was nosignificant difference. Gobiocypris rarus were challenged with three GCRV isolates in samedose and observed for14days. During the test period, no fish died or diseased with clinicalsigns.Virus detection results discovered that all the three virus could proliferation ingobiocypris rarus with low copies. The gobiocypris rarus were injected with virus with10times gradient dilution from100to10-4respectively, and14days later, all the treatmentgroups were challenge with one virulent wild strain GCRV from genotype II.The resultsindicated that different genotype strains had low cross protection to others, but possessed highprotection to same genotypes.In present study, the method of GCRV genotyping was optimized combined sequenceanalysis with genome band patternIn order to study the biological characteristics of GCRVfrom different genotypes, TaqMan Real-Time PCR method against three genotypes wereestablished which was not only suitable for the qualitative detection of the virus, but alsoapplied to quantitative analysis.The characteristics of proliferation in cell line and fish wereinvestigated, furthermore, the property of physicochemical and sensitivity to chemical reagentwere also studied. The present study provides theoretical data for grass carp hemorrhagicdisease prevention and control, and vaccine development.