| Hemorrhage disease of grass carp,caused by Grass carp reovirus(GCRV),leads to severe economic losses in grass carp farm industry in China.GCRV is the first isolated aquareovirus in China with the strongest virulence and is the most harmful pathogen to grass carp.GCRV is a double-stranded RNA virus with eleven segments,frequent mutation,complicated etiology and lack of effective control measures are the main causes of a wide,rapid and continuous prevalence of this disease.The phenomenon of immunization failure are usually occurs,because of the complex etiology,different genotypes of coexistence and the epidemiology is unclear,so the development and application of vaccine are untargeted.Therefore,it is essential and necessary for us to research the molecular epidemiology of grass carp reovirus,and develop targeted vaccines basing above molecular epidemiology databases.In addition,GCRV's genome has a special electrophoretic pattern in PAGE,the sequence analysis result revealed a close relationship between electrophoretic pattern and genomic signature.The sequence analysis of different isolates with unlike electrophoretic pattern not only significant to deeply understand GCRV's genetic variation,but also provide a theoretical support for study on epidemiology and development of the prophylactic and therapeutic treatment of GCRV.Therefore,the content of the research embraces following three principal sections:(1)Studies on molecular epidemiology of grass carp reovirus2274 samples of non-diseased grass carp and 366 suspected samples of grass carp hemorrhagic disease were collected between 2010 to 2016 from 18 provinces and cities,including Jiangxi,Guangdong,Guangxi,Shanxi,Shandong,Hunan,Hubei,Chongqing,Tianjin et al.All the samples were detected and analyzed by triple RT-PCR,at the same time,S7 gene of the representive isolate was analyzed by sequence and phylogenetic analysis,aim to compare the molecular epidemiological characteristics,geographical,temporal distribution and the genetic evolution relationship of different GCRV isolates.Our goal is to tracking the origin of GCRV dominating strains,revealing the possible route of transmission,and establishing the molecular epidemiology databases of grass carp reovirus.The results showed that the total positive rate of non-diseased grass carp samples was 2.9 %,of which 2 samples were positive for genotype I and 2 positive samples for mixed infections of genotype I and II,61 samples were positive for genotype II,which account for 93.8% of the total positive samples,no genotype III of GCRV was detected;the total positive rate of suspected grass carp hemorrhage disease samples was 40%,of which 8 samples were positive for genotype I,67 samples were positive for genotype II,which account for 89.7% of the total positive samples;5 samples were positive for mixed infections of genotype I and II,2 samples were positive for genotype III.We can concluded that genotype II is the main epidemic genotype in China.The homology of the S7 gene sequence of the isolates of genotype I,genotype II and genotype III ranged from 90% to 99%,from 95% to 100% and above 99%,respectively.The genetic phylogenetic analysis of the S7 gene showed that GCRV could be divided into three genotypes,which was consistent with the previous results and GCRV II could be divided into different subtypes and there is no significant correlation with regional distribution of different GCRV isolates.(2)Genome sequence analysis of grass carp reoviruses with different electrophoresis patternThis research is mainly used SDS-PAGE to analyzed the genome electrophoretic pattern of 21 GCRV isolates which isolated in different regions,and 5 representive GCRV new isolates which have different electrophoretic pattern were selected and sequenced completely,with other 16 isolates GCRV,the total of 21 isolates' genomic sequence and whole-genome features are analyzed and compared among each other.Phylogenetic trees were constructed based on the amino acid sequences of each segments of different isolates.The results showed that the 21 isolates of GCRV have 3 different electrophoretic pattern in PAGE,in the same group which have the similar electrophoretic pattern showed the difference in various degree among each isolate.Furthermore,we found that the difference of electrophoretic pattern,genomic sequence,genome signature,et al.have nothing to do with the isolate where and when isolated.The complete genome sequences of typical isolate based on different electrophoresis pattern showed that homology of corresponding nucleotide sequence and acid amino sequence were less than 50%,while the homology of the isolates which the segments that were differences in the different position of genome based on the same electrophoresis pattern were more than 85%.Additionaly,these also share the greater differences of nucleotide sequence and acid amino sequence accompany with the greater differences of electrophoresis patterne.The analysis of genomic characteristics revealed that both 3' ends and 5' ends exsit a section of conserverd sequence in the whole GCRV isolates that including three electrophoresis band types.Especially,terminal conservative region and genomic features of different isolates that present the same electrophoresis pattern were similarly,while the isolates with different electrophoresis band types have some differences.Furthermore,the 3' ends and 5' ends of noncoding sequences were also correlated with electrophoresis pattern.The length of noncoding sequences have distinction in the different electrophoresis pattern and vice versa.Phylogenetic analysis of these grass carp reovirus strains based on their amino acid sequences of each segment showed small difference,but all virus strains clustered into 3 groups based on any segment,which means that there are at least three genotypes of GCRV exist in china.(3)Inactivated vaccine research on GCRV genotype IIMolecular epidemiologic investigation results showed that the genotype II is the predominant genotype currently circulating in china.HuNan1307 is a new virulent strain of genotype II,in the present study,the immunological effects and protective efficacy of an inactivated HuNan1307 vaccine in grass carp were evaluated.The GCRV HuNan1307 was produced by cultivation of the grass carp PSF cell line,and inactivated with 1% ?-propiolactone for 60 h at 4 °C.Grass carp were injected with inactivated GCRV vaccine,followed by challenge with Hu Nan1307 virus.The results showed that the minimum immune dose of the inactivated vaccine was 105.5 TCID50/0.2 mL.All grass carp immunized with the inactivated vaccine produced a high titer of serum antibodies and GCRV-specific neutralizing antibody.Moreover,the expression of 6 immune-related genes in the spleen and head kidney of treated grass carp was significantly enhanced,which indicated that a tissue immune response was induced by the HuNan1307 vaccine.In addition,grass carp immunized with the inactivated vaccine showed a survival rate above 80% after the viral challenge,equal to that of grass carp immunized with a commercial attenuated vaccine,and the protection lasted for 1 year.The data in this study suggested that the inactivated HuNan1307 vaccine may represent an efficient method to induce immunity against GCRV infection in grass carp.In conclusion,we established the molecular epidemiology database and illustrated the situation of genotype of GCRV in china,obtained the difference of the genome electrophoretic pattern and whole genome sequences among different isolates and developed an efficient inactivated vaccine of epidemic strains,which not only provide information for the diagnosis and prevention of GCRV,but also accumulate the actual products for the preven-tion and control of grass carp hemorrhage. |
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