Study On Antiviral Activity And Mechanism Of Coumarin Compounds Against Spring Viraemia Of Carp Virus

As the traditional and common breeding industry that there is a large farming production in China,cyprinids are kinds of delicious food.However,with the expansion of aquaculture,different pathogenic microorganisms have resulted in frequent outbreaks of diseases,which bring substantial economic losses to the aquaculture industry.Spring viraemia of carp virus?SVCV?is responsible for the highly contagious spring viraemia of carp?SVC?disease associated with one of the most serious diseases in cyprinids.Since viral infections have been reported in most cyprinid fish,SVC is designated a notifiable disease by the Office International des Epizooties?OIE?and also listed A in Chinese animal epidemic diseases directory.Up to now,there is no licensed treatments available for the control of SVCV infection,making them serious public health,and thereby SVC should be closely monitored to destruct all aquatic life when the infection is established.Eventually the trend of the SVC outbreak will be limited.In order to obtain anti-SVCV drugs in this study,the newly synthesized coumarin compounds were evaluated antiviral activities by using the cell molecular screening model in vitro.The further research on the antiviral activity of active coumarins was studied through the observation on morphological and biochemical changes and tests on apoptosis after EPC cells infected by SVCV or exposed by drugs.Additionally,the effect of active coumarins on viral infection and the relationship between drugs and host antiviral response were also explored,which was contributed to provide the theoretical basis for the development of antiviral drugs in aquaculture.The main results are described as follows.1.Evaluation on antiviral activity of coumarin compounds against spring viraemia of carp virus in vitro.For the preliminary screen,the relative expression of SVCV G protein of 49 kinds of coumarin compounds were tested by quantitative real time PCR?qRT-PCR?.Among the screened coumarins,the inhibitory percentage of B4?7-[3-?benzimidazole?propane]coumarin?,C2?7-[4-?4-methyl imidazole?butoxy]coumarin?,C4?7-[4-?benzimidazole?butoxy]coumarin?and D5?7-[6-?2-methylimidazole?dioxyl]coumarin?was examined more than 90%,as the most effective agents in this test.By analyzing the structure-activity relationship,different types of imidazole derivatives?including methyl-imdazole or benzimidazole?were considered to play a key role in the anti-SVCV activity of the screened coumarins in the same length of the linker group.Furthermore,the active coumarins significantly reduced SVCV-induced cytopathic effect?CPE?,kept host cell viability,and protected EPC cells against SVCV.In comparison,the protection of C4 and D5 in 72 h was better than B4 and C2,and thus they were chosen for the further study.Importantly,C4 and D5 inhibited the expression of SVCV N,P and M more than90%,and showed a good metabolic features with the higher drug contents in cells in a certain period of time.2.Effect of C4 and D5 on SVCV-induced apoptosis.Based on the observation of fluorescence staining,scanning and translation electron microscopy that typical apoptotic features including cellular morphology disappeared,nuclear fragmentation,cytoplasmic degradation,and the collapse of cytoskeletal fiber system,the results were determined that SVCV could induce apoptosis in EPC cells,and C4/D5significantly inhibited SVCV-induced apoptosis.Flow cytometry analysis showed that more than 52%of apoptotic cells occurred in 48 h after SVCV infection.With the increase of infection time,the apoptotic rate would further enhance,reaching 63.2%.For 10 mg/L C4 and D5 treatments,apoptotic rates were reduced significantly,which the percent of normal cells was more than 80%that closed to the control group.Besides,the apoptotic rates of coumarin groups were gradually decreased with drug dose enhanced,which indicated that a negative correlation was existed between drug doses and viral-induced apoptosis to some extent.While the result of caspase activity indicated that C4 and D5 controled signal occurance to block viral-induced apoptosis.3.Antiviral effect of coumarin C4 on SVCV clearance via Nrf2/ARE signaling pathway activation regulated by PKC.In order to explore the antiviral mechanism of coumarin C4 against SVCV,the state of Nrf2-ARE signaling pathway was examined.The result showed that coumarin C4 obviously increased SOD and CAT activities and GSH content in EPC cells,which could prevent cell from oxidative damage.In addition,C4 resulted in a higher phosphorylation levels of PKC?Thr638 and PKC?Ser660 to some extent that is involved in the activation of Nrf2phosphorylation to favor Nrf2 translocation to nucleus 4-fold versus control group.The result of RNA interfere reflected that one of downstream target genes in Nrf2-ARE signaling pathway,HO-1,could affect IFNs expression,and thus inhibit SVCV replication.Further data of Western blot and qRT-PCR indicated that C4 also up-regulated HO-1 expression in addition to cellular IFN response.Therefore,C4 had an intense response on SVCV replication through the activation of PKC-Nrf2-ARE signaling pathway,which enhanced HO-1 and IFNs expression to suppress viral infection.4.D5 played an antiviral role on spring viremia of carp virus by acting on the G protein.By studying on the effect of coumarin D5 on viral replication during the early stage of SVCV infection,D5 blocked viral adhesion or viral RNA delivery from endosomes to the cytosol,and thus played an antiviral role in EPC cells.After incubating SVCV with D5 in vitro4 h and recovering the virion particles by ultracentrifugation before inoculation of cultures,the result clearly replied that D5 inhibited the relative expression of SVCV G protein?reaching90%?and significantly decreased viral titer from 10^6.5±0.1 to 10^3.4±0.2,which indicated that D5broke virus particle to reduce viral infectivity and proliferation.A similar result was obtained from the experiment using EPC cells transfected with pEGFP-SVCV G,in which D5 inhibited the expression of pEGFP-SVCV G in EPC cells.Therefore,the results presented in this section suggested that D5 target SVCV G protein and can be a virucidal agent to block SVCV replication in host cells.Furthermore,D5 also activated Akt-mTor signaling pathway by reducing phosphorylation levels of Erk1/2,which decreased the number of autophagosome and inhibited SVCV-induced autophagy.Since autophagy was necessary for SVCV proliferation in EPC cells,there was an inhibition of D5 during viral replication and virion particle release periods.5.Evaluation on antiviral activity of coumarin C4 and D5 against spring viraemia of carp virus in vivo.SVCV-infected zebrafish treated with coumarin C4 and D5 were tested for fish mortality and virus concentration in fish body by intraperitoneally injection manner.The results showed that the cumulative mortality in C4 or D5 treatments was decreased 17.5%and 20%compared with SVCV group,respectively.Other data also indicated that C4 and D5 had an antiviral effect on SVCV,because of the reduction of SVCV G protein expression in spleen and kidney at 1^stt and 4^th.Meanwhile,two coumarins promoted anti-oxidase activities and thus kept the balance of antioxidant system.Besides,C4 and D5 could activate IFNs expression against SVCV proliferation in zebrafish during the early stage of viral infection.Therefore,these results suggested that treatment with C4 or D5 was potential agents for reducing SVCV infection.In summary,coumarin C4 and D5 could effectively control SVCV proliferation by host antioxidant-antiviral response and direct antiviral effect,respectively.As the consequence,both coumarins reduced virus concentration obviously,kept host cell viability effectively,and decreased the mortality of zebrafish.Therefore,there was a broad application prospects of C4and D5 in prevention and control of SVC.