Study On The Resistance Of EGCG And Its Metabolite ECG To GCRV And WSSV

China is a big aquaculture country and often has serious epidemic.In freshwater culture,haemorrhagic disease caused by grass carp reovirus?GCRV?is highly lethal and highly infectious.It is one of the most important diseases known to cause the largest species of freshwater culture in China and the most explosive death of grass carp,which has a huge impact on the yield of freshwater culture.In marine aquaculture,white spot syndrome caused by white spot syndrome virus?WSSV?is the most harmful disease in shrimp,crab and even aquatic crustacean breeding.Epigallocatechin gallate?EGCG?,an ester catechin monomer isolated from green tea,has been shown to inhibit GCRV and WSSV.Through pharmacokinetic studies,we found that EGCG can be converted into ester-based catechin monomer ECG with the same antiviral potential in animals.Therefore,this study evaluated the safety of two ester catechin monomers?EGCG,ECG?and explored their application value in the prevention and control of aquatic animal diseases.Through transcriptome sequencing analysis,the molecular mechanism of ester catechin monomer against aquatic animal viruses was systematically analyzed with EGCG as the research object.The conclusion is as follows:1.EGCG metabolism research in vivoAccording to pharmacokinetic tests on different subjects,C_max of EGCG in grass carp plasma,liver and kidney were 103.667?g/m L,151.34?g/m L,2.12?g/m L,and AUC_?0-t?were 836.777?g/?g·h?,1204.391?g/?g·h?,38.511?g/?g·h?;The C_max of EGCG in the liver and muscle of Procambarus clarkii was 73.78?g/?g·h?and 24.87?g/?g·h?.The content of EGCG was the highest in the liver,and we speculated that the main target organ of EGCG was the liver.By comparing the value of AUC_?0-t?corresponding to the ECG of EGCG and its metabolites,The Plasma ECG conversion rate(AUC_ECG/Plasma/AUC_EGCG/Plasma)was 6.98%;The AUC_ECG conversion rate(AUC_ECG/Liver/AUC_EGCG/Liver)in the Liver of grass carps was 7.90%;The ECG conversion rate(AUC_ECG/Kidney/AUC_EGCG/Kidney)was 73.24%.The ECG conversion rate(AUC_ECG/Plasma/AUC_EGCG/Plasma)in the liver of Procambarus clarkii was 15.04%;The ECG conversion rate(AUC_ECG/Liver/AUC_EGCG/Liver)was 12.78%.Thus,the metabolic pattern of EGCG conversion to ECG in animals is universal.2.Study on anti-GCRV activity of EGCG metabolite ECGCIK cells were used as the evaluation model of antiviral capacity of the drugs.The virus titer was detected by q RT-PCR technology.It was found that ECG had a good protective effect on CIK cells infected by GCRV.The inhibition rate of the ECG on GCRV was higher than 50%at 20?g/m L,and basically completely inhibited the replication of GCRV at 40?g/m L,similar to the antiviral effect of EGCG.3.Study on anti-WSSV activity of EGCGIt was found by real-time fluorescent quantitative PCR?q RT-PCR?that EGCG had a good protective effect on WSSV-infected Procambarus clarkii,and the drug almost completely inhibited the replication of WSSV at 10?g/?g·bw?.Moreover,the drug has a good safety.It was found through pathological tissue observation that EGCG did not cause significant stress to the tissue cells when large dose of EGCG?40?g/g·bw?was added,which further confirmed the potential of EGCG in anti-WSSV application.4.Study on EGCG anti-GCRV mechanismThrough transcriptomic sequencing analysis,it was found that GCRV infection caused a general up-regulation of host cell immune-related gene expression,while the EGCG treatment group significantly reduced the GCRV-induced immune response;Through KEGG and GO enrichment analysis,laminin receptor?Lam R?related cell functions and signaling pathways were significantly enriched.Therefore,We speculated that EGCG could inhibit GCRV from invading host cells by specifically binding with Lam R receptor of host cell membrane,thus indirectly regulating host immune function.