Isolation And Identification Of A Novel Grass Carp Reovirus And Detection Method Establishment Of RPA-LFD

Grass carp（Ctenopharyngodon idella）is an important freshwater aquaculture fish that is cultured widely in our countries.However,grass carp are susceptible to variable diseases,especially the grass carp haemorrhagic disease（GCHD）caused by the GCRV composed of ds RNA.GCHD occur frequently in fingerlings and yearlings,with a long onset season and high mortality,causing great economic losses in aquaculture industry.Until now,more than 50 strains of GCRV have been isolated in China.Based on differences in the genome sequence,these isolates are mainly divided into three genotypic groups,namely GCRV-Ⅰ（representative strain GCRV-873）,GCRV-Ⅱ（representative strain GCRV-HZ08）,and GCRV-Ⅲ（representative strain HGDRV）.Due to different genotypic GCRV isolates have been observed to vary in genome sequence,the nucleotide similarity was generally less than 50%.Epidemiological investigation of GCRVs showed that there is not only single infection of different genotypes,but also mixed infections.Therefore,it is necessary for the early detection and prevention and control of GCHV about the genome analysis of new strains of different genotypes and the establishment of rapid detection methods.A new grass carp reovirus,tentatively named HGCRV（healthy grass carp reovirus）,was found in the monitored grass carp samples in Hubei Province.Then we carried out cell culture,electron microscope observation,physical and chemical properties analysis,and SDS-PAGE band patterns analysis,complete genome sequencing and sequence alignment.Then dynamic proliferation in different cells and in different tissues of fish was analyzed by RT-q PCR,according to the standard curve.Moreover,visual detection method was contructed using the recombinant polymerase amplification（RPA）technique combined with colloidal gold strip method.The study provided a founding for the molecular epidemiology,genetic diversity of GCRV strains and rapid diagnosis of GCHV in China.The main results are as follows:1.Isolation,identification,and genomic analysis of HGCRVThe whole genome sequence was obtained by Illumina sequencing and RACE amplification.The result showed that it contained 11 ds RNAs with a total size of23,688 bp,and 57.2 mol%G+C content,encoding twelve proteins.All segments had conserved 5’and 3’termini（5’-GUUAUU……UCAUC-3’）.Sequence comparisons showed that HGCRV was closely related to GCRV-873（GCRV-I;69.57–96.71%protein sequence identity）,but shared only 22.65–45.85%and 23.37–43.39%identities with GCRV-HZ08 and Hubei grass carp disease reovirus（HGDRV）,respectively.Phylogenetic analysis based on Rd Rp protein showed that the known species of aquareoviruses clustered in a branch.HGCRV was clustered more closely with Aq RV-C,suggesting that HGCRV might belong to the genus Aq RV-C.Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that grass carp reovirus strains clustered into three groups.Compared with other clusters,HGCRV was more closely related to the GCRV-I（representative strain GCRV-873）,so HGCRV may be attributed to GCRV-I.2.Proliferation characteristics analysis of HGCRV in vitro and in vivoGCO,CIK,L8824,EPC and Gi CB cells were infected with 10² copies/μL of HGCRV,respectively.The results showed that passaged HGCRV caused typical CPE in CIK,L8824,EPC,and Gi CB cell lines.Different sensitivities to HGCRV were showed in different cells.According to the time of occurrence of CPE,the earliest and fastest cells were Gi CB and L8824.RT-q PCR analysis showed that the HGCRV can proliferate in GCO,CIK,L8824,EPC,and Gi CB cell lines,especially better in L8824and Gi CB cell lines,in which the virus copies reached 7.5×10⁴ and 6.4×10⁴copies/μL,respectively.Interestingly,the virus began to proliferate at 24 h in all cells.However,virus proliferation reached its highest level in L8824,CIK,and EPC cell lines at 72 h,while in Gi CB and GCO cells,proliferation peaked at 96 h.In grass carp,HGCRV can proliferate in the liver,kidney,and spleen tissues,in which tissues,the content of viral RNA increased at first and then decreased.The viral RNA expression level peaked in the spleen and kidney tissues on the 3rd day after infection,while in the liver,it peaked on the 4th day after infection.3.Establishment of RPA-LFD detection method for HGCRVHGCRV VP6 gene was used to design primers and probes and the reaction conditions were optimized.Reaction temperature optimization showed that the optimal reaction temperature is 38℃.The specificity of the method showed that no positive signal was observed in GCRV-106,GCRV-104,SVCV,Cre RV,and Cy HV₂and negative template,except HGCRV,which indicated that this method has high specificity.The sensitivity of the assay showed that the lowest limit of detection was10¹copies/μL,and the repeated assay was tested by 10²copies/μL and 10³copies/μL,which indicated that the RPA-LFD method is feasible.