Establishment Of A Rare Minnow(Gobiocypris Rarus)Infection And Vaccine Evaluation Model For Grass Carp Reovirus Genotype â…¡

Grass carp reovirus（GCRV）,firstly isolated in China,is a double-stranded RNA virus that belongs to the genus Aquareovirus of the family Reoviridae.It can provoke grass carp haemorrhagic diseases（GCHD）in grass carp fingerlings and yearling populations.,cause mass death of grass carp,a serious threat to security grass carp aquaculture industry.The outbreaks of GCHD are mainly related to genotype â…¡.Effective vaccines will probably become an important way to resolve the situation regarding GCRV-â…¡ in the successful development of the intensive grass carp farming industry.Rare minnow was reported to be sensitive to GCRV infection.Its breeding cycle is short and genetic background is clear,which are recognized as useful candidate of a disease model.In this study,we established a disease model of GCRV-â…¡ in grass carp through histoppathological and molecular pathology studies,and immunized grass carp and rare minnow with attenuated and inactivated vaccines,respectively,to observe their immunogenicity and protective effects,and establish a vaccine evaluation model for GCRV-â…¡.The results lay a foundation for further studies of the pathogenic mechanism and the development of a vaccine of GCRV-â…¡ in grass carp.The full text is mainly divided into the following two parts:1.Establishment of a rare minnow disease model for GCRV-â…¡:in the present study,the experimental group of rare minnow was injected intraperitoneally with 20 ul of Hu Nan1307 with a concentration of 10 LD₅₀,and the control group was intraperitoneally injected with the same dose of M199 medium.we investigated clinical characteristics,tissue tropism,histopathology,and relative expression of immune-related genes in rare minnow infected with GCRV-â…¡ virulent strain Hu Nan1307.Hemorrhage of rare minnow was induced by the GCRV-â…¡,which could proliferate in the fishes,and the viral load peaked on the day 7.The histopathologic changes in rare minnow were primarily vasodilation and hyperaemia in multiple organs,cell degeneration and necrosis,as well as loose intercellular in spleen,kidney,brain and heart.Electron microscopic observation in the kidney revealed virus similar in shape and size to those GCRV-â…¡ infections in grass carp.Genes encoding IL-8,Mx,TLR5,Ig M and IFNαwere relatively high up-regulated in different tissues,while multiple genes in liver and spleen were significantly up-regulated,which was consistent with GCRV-â…¡ infection in grass carp（P<0.05）.Our study suggested that rare minnow can be used as a disease model of GCRV-â…¡ as a tool to study pathogenesis and develop vaccine against GCRV-â…¡.2.Establishment of a rare minnow model for evaluation of experimental vaccines against GCRV-â…¡:In this study,grass carp was used as parallel control,grass carp and rare minnow were immunized with adjuvant inactivated and attenuated viruses,which were divided into adjuvant inactivated virus immunization group,attenuated virus immunization group,adjuvant control group and M199 control group.In the inactivated virus immunization group,5×10³copy/μL of the inactivated virus preparation was taken with a volume of 20μL by intraperitoneal injection.And the attenuated virus immunization group was administered with 1×10³copy/μL of the attenuated virus preparation in a volume of 20μL.The control group was injected with same volume of M199.The adjuvant control group was injected with adjuvant 1:1 diluted with M199.The immune conditions of grass carp were consistent with the immune conditions of rare minnows except for different immune dosages（200μL per fish）.The expression of immune-related genes,changes of specific antibodies and evaluation of intra-abdominal lesions were compared after immunization.At 28 dpi,each group was injected with lethal dose of Hu Nan1307,and the changes of viral load and the relative percent survival in each group were parallelly evaluated.The results show that the expression of immune-related genes increased statistically significant after immunization.With the rise of specific serum antibody titers,the results of rare minnow and grass carp were consistent.In addition,there was no significant residue of adjuvant observed in both fishes injected with adjuvanted inactivated virus.Challenge of immunized grass carp and rare minnow with Hu Nan1307 resulted in protection rates of 95.8%and 92.6%for attenuated virus,81.4%and 77.7%for inactivated virus,respectively,as well as viral load changed consistently.The results indicated that rare minnow can be used as a model for evaluation of experimental vaccines against GCRV-â…¡.In this study,a disease infection model of GCRV-â…¡ was established,and an evaluation model of GCRV-â…¡ vaccines were established by comparing the immunogenicity and protection efficiency of the inactivated and attenuated viruses between grass carp and rare minnow.This study provides a foundation for the future research and development of GCRV-â…¡ vaccines and provides a reference for the establishment of aquatic animal model.