Research On Screening Of Inhibitory Drugs For Grass Carp Reovirus

Grass carp(Ctenopharyngodon idellus)is an important economic fish in China,which accounts for about 20% of the freshwater aquaculture production,but the annual production reduction caused by disease.Among them,Grass Carp Reovirus(GCRV)is the most harmful and is classified as a Class II animal disease by the Ministry of Agriculture and Rural Affairs of China.The development of environmentally friendly drugs and vaccines with clinical value is of great significance for the prevention grass carp haemorrhage.This article mainly conducts research related to screening of grass carp reovirus inhibitory drugs.We tried to explore whether the use of macromolecular drugs related to grass carp reovirus adsorption hosts could be used to control different genotypes of grass carp reovirus infection.1.Validation the interaction between GcFibuli-4 and intact grass carp reovirus particles;Explore the inhibitory effect of GcFibuli-4 peptide fragments on grass carp reovirus infectionFibulins family is a series of extracellular matrix proteins which composed by epidermal growth factor-like domains(calcium-binding epidermal growth factor-like)and C-terminal Fibulin-type domains.GcFibuli-4 is a member of the grass carp Fibulin protein families which is mainly found in the extracellular matrix.Studies have found that the grass carp GcFibuli-4 protein may interact with grass carp reovirus outer capsid protein.If there could interact with each other,GcFibuli-4 protein may be used as an intermediate for grass carp reovirus to attach the host.The results show that it can indeed interact with intact grass carp reovirus virions.Therefore,we used the peptide fragment which can interact with the grass carp reovirus capsid protein to inhibit proliferation of grass carp reovirus.As a result,it was found that it had an inhibitory effect on the proliferation of grass carp reovirus when the dosage was above 50 μg / m L in vitro.2.Inhibitory of heparin sodium and chondroitin sulfate on grass carp reovirus in vitro proliferationIn recent years,a large number of studies have shown that heparin molecules can inhibit the infection of many viruses.To reveal whether heparin molecules and their analogues can also prevent grass carp reovirus infection,we used grass carp reovirus I and III to infect grass carp kidney cell line(CIK),the results of Real-time RT-PCR and Western blot reveals the effects of progeny virus amplification in cell supernatants and intracellular viral protein synthesis after grass carp reovirus infection,respectively.The inhibitory effect of heparin sodium and its analogue chondroitin sulfate on grass carp reovirus infection was evaluated.The results showed that heparin sodium had an inhibitory effect on grass carp reovirus infection at concentrations above 100 μg / m L.When the dosage was above 10 mg / m L,the viral protein synthesis efficiency and the supernatant progeny virus amplification efficiency were significantly reduced.Its analogue chondroitin sulfate has a lower inhibitory effect than heparin sodium,but when the dosage is more than 20 mg / m L,the viral protein synthesis efficiency and the supernatant progeny virus amplification efficiency are also significantly reduced.The results show that heparin sodium and its analogues have a broad-spectrum inhibitory effect on the infection of different genotypes of grass carp reovirus.his study paved the way for development of heparin analogue-based environmental-friendly medicine against grass carp hemorrhagic disease.3.Using the Gobiocypris rarus infection system to explore the antiviral effect of multiple drugs at the fish levelGobiocypris rarus is a small fish species which is unique in our country.According to related reports,it is very sensitive to grass carp reovirus.Therefore,we used the grass carp reovirus II(GCRV-JX02W)which is isolated by our laboratory.It was found to have obvious bleeding symptoms after fifth days.Therefore,we used the Gobiocypris rarus infection system to investigate the inhibitory effect of multiple drugs on grass carp reovirus at the fish level.Ammonium chloride(50 μg/m L),GcFibuli-4 peptide fragment(100 μg/m L),sodium heparin(20 mg/m L),chondroitin sulfate(30 mg/m L)were separately mixed with virus,then Gobiocypris rarus were infected by intraperitoneal injection,and the mortality was observed and counted.The results show that ammonium chloride can significantly reduce the lethality of grass carp reovirus to Gobiocypris rarus.Other drugs such as GcFibuli-4 peptide,heparin sodium and chondroitin sulfate also have a certain degree of grass carp reovirus infection Inhibitory.