Antiviral Activity Of Rhein Against Fish Nervous Necrosis Virus

Viral neuronecrosis disease（VNN）is a viral infectious disease of fish caused by necrotic virus（NNV）infection.It is listed as an important fish disease by the International Organization for Epidemiology（OIE）.It is classified as a second-class epidemic disease in China.VNN is very harmful to larvae or juvenile fish,especially to the grouper culture industry,a large number of seedlings often die in a short time,and the mortality rate can reach more than 95%,which is the bottleneck of grouper culture in China.Because the immune system of larval or juvenile fish is not perfect,there are limitations in vaccine prevention and control.Therefore,drug prevention and control of VNN epidemic disease has become an effective means for the cultivation of marine fish fry such as grouper.As human antiviral raw materials are prohibited from being used in aquaculture,there are few antiviral drugs available in fishery production.Chinese herbal medicine is a natural drug source with many advantages,such as green,environment-friendly,a variety of activities and not easy to produce drug resistance.In this study,the antiviral NNV activity of extracts from rheum and forsythia was studied by cell line screening in vitro,and the antiviral activity and antiviral mechanism of Rhein,the main active component of rheum,were evaluated in order to provide theoretical basis for the development of anti-NNV specific drugs and leading compounds of fishery drugs.The results of this study are as follows:1.Screening of Anti-NNV Chinese Herbal Medicine activity.122 kinds of Chinese herbal medicines were crushed to 30-40 meshes.First,Petroleum ether was used as solvent to remove volatile oil by ultrasonic reflux extraction,then extracted with anhydrous methanol,vacuum concentration,low temperature drying were used to obtain crude extracts of Chinese herbal medicines.After NNV infection of Epinephelus fin cell line（GF-1）,the crude extract of Chinese herbal medicine was added to screen the anti-NNV activity.The results of real-time quantitative PCR（RT-q PCR）showed that the inhibition rate of Rheum,Clove and Suberect Spatholobus Stem on NNV was more than 80%,of which rheum activity was the best,and the inhibition rate was 99.6%.The results of the study on the anti-NNV activity of Rhein and Emodin showed that both of them could significantly inhibit the cytopathic effect induced by NNV,and their 50%inhibitory concentration(IC₅₀)on NNV was 8.78μM and 8.62μM,respectively.NNV titer test results showed that Rhein with higher anti-NNV activity significantly reduced the NNV titer,confirming the potential of Rhein as an anti-NNV virus active molecule.2.Evaluation of anti-NNV activity of Rhein in vitroAfter NNV infection,GF-1 cells were co-incubated with Rhein,stained with Actin-Tracker Green and Hoechst fluorescent dyes,and observed under fluorescence microscope:Rhein could significantly reduce GF-1 nuclear damage and microfilament rearrangement induced by virus;GF-1 cells were co-incubated with Rhein after 24 h infection of NNV,RT-q PCR results showed that Rhein inhibited the proliferation of NNV by 81.1%,indicating that Rhein has a good therapeutic effect on NNV infection.GF-1 cells were infected with NNV and Rhein for 1 h,2 h and 4 h,and the inhibition rates on NNV proliferation were 23.39%,25.54%and 35.82%,respectively,indicating that Rhein has a direct killing effect on NNV virions,and the killing effect is time-dependent.The time of adding Rhein in a single replication cycle（24 h）of GF-1 cells infected by the virus was determined,RT-q PCR results showed that Rhein mainly played a role in 6-8 h after NNV entered the cells and inhibited the proliferation of the virus.3.Regulation of Rhein on cell cycle arrest and ATP synthesis induced by NNVRhein was added to GF-1 cells after NNV infection,and the changes of cell cycle and the level of intracellular ATP were detected by flow cytometry and automatic enzyme labeling.the results showed that Rhein significantly inhibited the S-phase arrest of cell cycle induced by NNV and increased the proportion of cells in G2/M phase of GF-1 cell line.NNV significantly increased the level of ATP in GF-1 cells at 6 h and12 h after infection,while the addition of Rhein significantly decreased the level of ATP induced by NNV.In addition,mitochondrial respiratory chain complex inhibitor was used to reduce ATP synthesis in GF-1 cells.Flow cytometry and RT-q PCR detection showed that NNV-induced cell cycle S-phase arrest and NNV proliferation were inhibited,indicating that reducing host cell ATP synthesis can effectively change NNV-dominated cell cycle S-phase arrest and thus inhibit NNV proliferation.In short,Rhein inhibits the S-phase arrest of cell cycle induced by NNV and interferes with virus proliferation by reducing ATP synthesis of GF-1 cells in the early and middle stages of NNV replication.To sum up,Rhein,an active molecule found from rheum,has good anti-NNV activity in vitro.Rhein inhibits NNV-induced S-phase arrest of cell cycle by reducing host cell ATP synthesis in the early and middle stages of virus replication,thus interfering with virus proliferation.This study provides a theoretical basis for the research and development of new anti-NNV drugs and a research basis for the industrial application of rheum.