Pathogenic Mechanism Of Grass Carp Reovirus(GCRV) Regulated By Temperature

Aquatic animal viral diseases with seasonality and temperature dependency endanger the aquaculture,and lead to considerable financial loss in the culture fishery.Generally,the outbreak of aquatic animal viral diseases is temperature dependency.When the ambient temperature is outside the epidemic temperature range of disease,The epidemic disease tends to cease or remission naturally.However,the mechanism that temperature affects the outbreak of aquatic animal viral diseases is still unclear,which greatly hinders the development of control and prevention measurement of aquatic animal viral diseases.Grass carp hemorrhagic disease(GCHD)triggered GCRV is a representative disease that has a intense temperature dependence.In this study,GCHD was used as the disease model to explore the molecular mechanism of the temperature dependency pathogenesis of GCRV,the contents are as follows:(1)The prevalence of GCHD is seasonal periodicity and temperature dependentCompared with the virus of endotherms,the viral diseases of poikilotherm aquatic animals are more susceptible to temperature fluctuations,the prevalence of diseases showing typical seasonal periodicity or temperature dependence.In this paper,we sending out questionnaires,real-time tracking of the incidence of GCRV in grass carp ponds,design experiments of virus infection under hot stress and the transformation experiments in GCRV-infected host cell lines between different temperatures in order to confirm the temperature dependent phenotype of grass carp hemorrhagic disease.The results show that GCHD generally peaked from June to September,with the most severe cases in June and July in Changsha and Yiyang.The highest number of grass carp deaths occurred at temperatures between 24℃ and 28℃.When the temperature was below 20℃,the grass carp recovered naturally.At the cellular level,the virus replication of GCRV-infected cells almost stopped at 18℃,and was significantly up-regulated when the cells were switched from 18℃ to 28℃.Similarly,when infected with GCRV at 28℃,the virus replicates normally,but when transferred to 18℃,viral replication almost stops.The above conclusions reveal that temperature strictly regulated the outbreak of GCHD.(2)The role of IL6-STAT3-HSP90 axis in temperature-dependent infection of GCRVWe hypothesized that the virus may take advantage of temperature-related immune responses in the host to improve its infection efficiency in order to elucidate the biological mechanism of temperature-dependent phenotype of grass carp infected by GCRV.In this research,CIK cells suffered hot stress transcriptome combined with GCRV infection of grass carp transcriptome screening analysis was conducted to conclude that the cytokines or the proteins associated with IL6-STAT3 pathway may mediate temperature-dependent GCRV infection.It was confirmed that IL6-STAT3 signaling pathway plays a key role in the temperature-dependent pathogenesis of GCRV infection,and influences GCRV infection in host cells by regulating HSP90 expression through a series of experiments such as GCRV infection,temperature stress,inhibitor/agonist stimulation,protein incubation,STRING database retrieval,STAT3 knockout,transcriptomic analysis,plasma membrane separation,in those experiments,we used the q PCR,WB,Co-IP,fluorescence localization and other techniques.The interaction between HSP90 and VP7 to mediate GCRV entry was further verified by using the purified viral coating protein VP7 through pulldown,mass spectrometry identification,far western blotting,immunofluorescence,immunoprecipitation and other techniques.These results indicate that temperature can regulate the expression of IL6-STAT3-HSP90 axis to affect the infection of GCRV in grass carp.When grass carp are suffered stimulation by the external environment,a large number of IL6-STAT3-HSP90 axis related protein HSP90 is expressed,and GCRV uses this protein to complete the invasion process.(3)The temperature dependence of HSP70 mediated GCRV infection in grass carpIn order to improve the molecular mechanism of temperature-dependent phenotype that grass carp was infected by GCRV,we found that in addition to HSP90,HSP70 also plays an important role in the temperature-dependent pathogenesis of GCRV-infected host by polytranscriptomic analysis.We future proved that HSP70 partially located on the cell membrane interacts with VP7 to promote virus entry into the cell in the early stage of GCRV infection through si RNA knockdown,overexpression,drug inhibition,plasma membrane separation and other experiments.In addition,VP7,as a key viral protein,can interact with a number of host proteins including HSP90,HSP70,STAT3,EEA1,regulate the expression of integrin,SRB1,Lam R and other receptor genes,and promote the virus entry.In this paper,we elucidate a previously unidentified GCRV immune escape mechanism in which GCRV hijacks temperature-regulated host heat shock responserelated proteins to promote viral entry efficiency.This provides a theoretical basis for the prevention of GCHD.At the same time,we used other ectothermic vertebrates(carp,giant salamander)and their corresponding DNA or RNA viruses for further verification this mechanism,and found that they also evolved similar mechanisms to promote viral infection.This provides a potential target for the development of drugs for animal viral diseases of ectotherms in the future and provides a theoretical basis for prevention and control strategy for aquatic animal viral diseases.