Preliminary Study Of Quercetin Inhibition Of Genotype Ⅱ Grass Carp Reovirus And Its Pharmacokinetics In Grass Carp

Grass carp,Ctenopharyngodon idella is a freshwater fish mainly farmed in Asian countries,with an annual production of over 5 million tonnes in China.Grass carp reovirus（GCRV）,which is caused by grass carp hemorrhagic disease,has brought great economic pressure to the grass carp farming industry.Domestic scholars have classified dozens of strains of grass carp reovirus isolated based on the homology of S6 fragment into three genotypes,namely GCRV-Ⅰ,Ⅱ and Ⅲ,and the representative strains of the three genotypes are GCRV-873,GCRV-HZ08 and GCRV-104respectively.Genotype II GCRV is considered to be the main genotype that causes grass carp hemorrhagic disease epidemic and economic losses in China in recent years,and mixed infection of the three genotypes often occurs.There is an urgent need for green,broad-spectrum anti-GCRV drugs in aquaculture due to safety concerns for aquatic organisms and water as well as consumers.Quercetin is a plant-derived fungicide.Studies have shown that quercetin exhibits antiviral effects against a variety of viruses,but its use against GCRV has not been reported.Our previous studies have shown that Hsp70,the major heat shock protein,is induced during the heat shock response and can promote replication of grass carp reovirus（GCRV）,and that quercetin may reduce the replication efficiency of genotype I and genotype III GCRV in vitro by inhibiting the induction of Hsp70.To further elucidate the clinical potential of quercetin in antagonizing grass carp reovirus,this study investigated the in vivo antiviral effects of quercetin on genotype II GCRV using a rare carp model of genotype II GCRV infection.A rational dosing regimen was developed for the clinical application of quercetin,and pharmacokinetic parameters of quercetin in grass carp were formulated to provide a reference for later clinical use.The main findings are as follows:1.The rare minnow was injected intraperitoneally with the genotype II GCRV virus named GCRV-JX02,which is preserved in our laboratory,and the results of surface haemorrhagic symptoms and PCR assays indicated that a successful infection model was established.The results of quercetin protection in the rare minnow intraperitoneal injected with GCRV-JX02 showed that quercetin was safe for the rare minnow in the dose range of 30-480μg/g.Rare minnow infected with GCRV-JX02showed typical signs of haemorrhage and tissue damage on pathological examination.The semi-lethal dose(LD₅₀)of GCRV-JX02 was determined to be 6.0 x 10² copies by q RT-PCR.Quercetin protects rare minnow from GCRV-JX02 infection in a dose-dependent manner by reducing the production of offspring virus and increasing fish survival.As a result,quercetin exhibits broad-spectrum antiviral activity in vivo and can be used as an environmentally friendly therapeutic agent in aquaculture.2.To further evaluate the efficacy of quercetin,PCR,viral load changes and histopathological analysis were performed on various tissues of diseased rare minnow protected by quercetin,and the results showed that quercetin could effectively reduce virus replication in various tissues of diseased minnow,and the virus inhibition effect was more obvious in heart and kidney tissues.The results of histopathological sections showed that quercetin has the ability to reduce inflammation in the body,suggesting that quercetin may be used as a targeted anti-inflammatory agent to treat the inflammatory response caused by GCRV and thus exert an antiviral effect.3.The half effective inhibitory concentration of quercetin on grass carp reovirus genotype I was determined in grass carp CIK cells.The results showed that the half effective inhibitory concentration(EC₅₀)of quercetin on grass carp reovirus was4.796μg/m L.After a single oral administration of 20,40 and 60 mg/kg of quercetin crude extract,the enrichment levels in the three tissues of grass carp were distributed as hepatopancreas>kidney>blood,with maximum peak concentrations(C_max)of0.129μg/m L,0.583μg/m L and 0.666μg/m L in blood at 48 h;C_max in hepatopancreas were 3.822μg/g,5.386μg/g and 6.252μg/g,respectively;C_max in kidney were 2.437μg/g,3.140μg/g and 3.447μg/g,respectively.The drug concentration of quercetin in the hepatopancreas at 20 mg/kg dosing concentration is 1.57 times higher than that in the kidney and exceeds the blood drug concentration by about 29 times.Although the maximum drug concentration of quercetin in the blood was much lower than the half effective inhibitory concentration of quercetin,the maximum drug concentration in the hepatopancreas of the 40 or 60 mg/kg quercetin-treated group was higher than the half effective inhibitory concentration of quercetin,so that the antiviral activity of the drug in the fish tissues was guaranteed in the 40 or 60 mg/kg quercetin-treated group.Thus quercetin not only directly inhibits virus replication,but also may exert antiviral effects and reduce mortality by decreasing the inflammatory response.In conclusion,this study provides theoretical support for the further application of quercetin as an environmentally friendly anti-GCRV agent in grass carp culture.