Antiviral Activity And Mechanism Of Arctigenin Derivatives Against Spring Viraemia Of Carp Virus

Spring viraemia of carp(SVC)triggered by spring viraemia of carp virus(SVCV),is an acute and highly contagious viral disease.In the cultivation of cyprinid fishes,its outbreaks usually induce mass morbidity and mortality,bringing huge economic losses to the field of aquaculture.Up to now,the use of drugs has been an effective way for prevention and control of diseases due to the lack and limitations of vaccines.Since antiviral drugs such as moroxydine hydrochloride and ribavirin were banned,few antiviral drugs have been used in production.Hence,innovative research on aquatic antiviral drugs has received great attention and is urgently needed by the industry.Herbal medicines are easily degradable,environmentally friendly and have a variety of biological activities such as antibacterial,anthelmintic,antitumor and antiviral activities.And this makes them an optimized choice for new drug development.In this study,we firstly investigated the anti-SVCV activity of 37medicinal plants,including Arctium lappa L.(ALL),Ligusticum chuanxiong hort and so on.Among these medicinal plants,ALL was found to possess the highest anti-SVCV activity,with arctigenin(ARG)being its active pharmaceutical ingredient.The anti-SVCV activities of the derivatives synthesized with ARG as the lead compound were then evaluated.Furthermore,based on the transcriptomics data,the antiviral mechanism of the derivatives was eventually analyzed to provide a theoretical basis for the development of aquatic antiviral drugs.The main results are described as follows.1.Evaluation on antiviral activities of medicinal plants and active ingredients against SVCVPlant extracts were screened based on the maximum safety concentration.Results of real time quantitative PCR(RT-q PCR)revealed that more than 90%of SVCV was inhibited by seven plant extracts,including ALL,Ligusticum chuanxiong hort,Cynanchum otophyllum Schneid.and so on.Among these extracts,ALL showed the highest inhibition on SVCV replication.The result of high-performance liquid chromatography(HPLC)showed that arctiin(ART)and ARG were the main components of ALL and the contents of the two components were 233.68 and 12.28 mg/g.The result of antiviral activity showed that ARG significantly inhibited SVCV-induced cytopathy effect,indicating that ARG may be the main active ingredient of ALL.The anti-SVCV activity of ARG was further confirmed by RT-q PCR,titer assay,cell nuclear damage assay and ultrastructural observation.Results showed that ARG significantly reduced the titer of SVCV with half maximal inhibitory concentration(IC₅₀)of 0.81?M.It also provided morphological protection to EPC cells by weakening SVCV-induced cellular damage.Finally,the anti-SVCV activity of ARG in vivo was tested by intraperitoneally injection manner.Results showed that SVCV gene expression in spleen and kidney were significantly inhibited at 1^st day post infection(dpi).Other data of titer assay also found that SVCV were significantly inhibited,and the cumulative mortality was decreased by 20%after ARG treatment.The results above confirmed that ARG was suitable to be a lead compound against SVCV.2.Evaluation on antiviral activities of ARG derivatives against SVCVThe relative gene expression of SVCV G protein were tested after treatment with 27synthesized ARG derivatives using EPC cells as a model.By analyzing the structure-activity relationship,18 derivatives were found to have anti-SVCV activities and 12 of these derivatives were nitrogen-containing heterocyclic derivatives.Four derivatives(derivatives 8,20,25 and 27)showed higher antiviral activities than ARG by comparing IC₅₀.Among them,4-(8-(2-methylimidazole)octyloxy)-arctigenin(MON,derivative 27)possessed the highest anti-SVCV activity with IC₅₀ of 0.18?M.Therewith,SVCV-infected zebrafish were treated with MON by injection manner,and results revealed that MON was also more effective to SVCV infection than ARG in vivo.The inhibition of MON on SVCV titers continued to 6^thdpi,and its antiviral activity in spleen and kidney maintained until 7^th dpi.Moreover,MON effectively improved SVCV-induced tissue damage and increased the survival rate by 36.7%.Taken together,the anti-SVCV activity of MON was superior to that of ARG both in vitro and in vivo.3.Transcriptome analysis revealed the molecular antiviral effects of MON in EPC cellsTranscriptome sequencing(RNA-seq)was used to study the effect of MON on the transcriptome level of EPC cells.The results obtained were as follows:(1)When comparing with the control,10776 different expression genes(DEGs)including 4538 up-regulated genes and 6238 down-regulated genes were selected in MON processed group;when compared with SVCV group,10881 DEGs were selected,of which 6567 genes were inhibited in MON treated group.In both comparison groups,the top 20 genes with the highest up-regulation and down-regulation folds all contained genes related to energy metabolism,suggesting that MON may affect energy metabolism in host cells.(2)GO term enrichment analysis found that a series of processes related to cell cycle were significantly enriched after MON treatment.(3)Cell cycle pathway were significantly enriched in KEGG pathways of the DEGs.Moreover,the PPAR pathway related to energy metabolism was also significantly enriched.Results above indicated that MON mainly affected pathways related to cell cycle and energy metabolism regulation.4.Study on the regulation of cell cycle and ATP synthesis by MONBased on the results of transcriptomic analysis,the effect of MON on cell cycle regulation and ATP synthesis was investigated to explore its antiviral mechanism.Results showed that the cell population of the G2/M phase was increased and SVCV-induced S phase arrest was significantly inhibited after MON treatment.MON significantly inhibited ATP synthesis in EPC cells at 6 h and 12 h post treatment.After ATP synthesis was reduced by mitochondrial respiratory chain complex inhibitors,both SVCV-induced cell cycle S phase arrest and SVCV proliferation were inhibited.This result indicated that reducing ATP synthesis effectively inhibited SVCV-induced cell cycle S phase arrest and interfered with SVCV proliferation.In addition,the eukaryotic expression plasmid of SVCV N protein was constructed,and MON significantly inhibited the expression of p EGFP-SVCV-N.The result revealed that MON could affect the expression of N protein related to cell cycle regulation,further confirming the regulation of MON on cell cycle.In summary,MON inhibited ATP synthesis in EPC cells and then repressed SVCV-induced S phase arrest of cell cycle,and thus interfered with SVCV proliferation at the early phase of SVCV replication.5.Study on the regulation of SVCV-induced apoptosis by MONTypical apoptotic features of EPC cells were induced by SVCV infection,including the formation of apoptotic bodies and depolymerization of microtubule and microfilament system.However,all morphological features associated with apoptosis were significantly inhibited after MON treatment.Besides,the result of flow cytometry showed that 43.7%of cells underwent apoptosis after infected with SVCV for 48 h,while the percentage of apoptotic cells was reduced to 8.7%after MON treatment.Results of Western Blot and enzyme activity assay indicated that MON effectively inhibited the expression of pro-apoptotic proteins,promoted the expression of bcl-2,and effectively restored the expression and enzyme activities of caspase 8,9 and 3.Furthermore,the anti-apoptotic effect of MON was found to be related to its inhibition of ATP synthesis by measuring the effect of respiratory chain complex inhibitors on apoptosis.Notably,the results of viral release assay showed that the virus titer in the supernatant was significantly reduced after MON treatment,which indicated that MON was able to inhibit the release of virus particles.In summary,MON inhibited the occurrence of SVCV-induced late apoptosis and thus reduced the release of viral particles.In conclusion,MON synthesized with ARG as the lead compound mainly inhibited SVCV-induced cell cycle S phase arrest by reducing ATP synthesis,and thereby interfered with SVCV proliferation at the early stage of SVCV replication.On the other hand,it suppressed the release of viral particles via inhibiting SVCV-induced apoptosis at the late stage of SVCV replication.Therefore,the current study is of great significance to the discovery of anti-SVCV herbal medicines,and it provides a theoretical basis for the development of anti-SVCV drugs.