Isolation And Identification Of Mink Enteritis Parvovirus （MEV） Strain From Shandong Province And The Inhibition Of Interferon βagainst Its Replication In Vitro

China is a big country in mink breeding,and shandong province accounts for more than60%of the total.Mink breeding has become a characteristic industry in Shandong province,which also drives the local economic development of Shandong province.Mink enteritis parvovirus disease（ME）is one of the three major infectious diseases that harm mink breeding industry.It mainly causes severe diarrhea,dehydration and even death of infected mink,which brings serious losses to mink breeding industry every year.Interferon（IFN）is an important innate immune defense factor in the body.Under normal circumstances,the Interferon gene is inhibited.Once stimulated by the virus,it will induce the expression of a variety of antiviral factors and play an antiviral role.IFN-βis a kind of type I interferon,as an important intermediate inducer and effector molecule in the immune process,its antiviral effect has been widely concerned.This study mainly investigated and detected mink enteritis parvovirus infection in Shandong Province,isolated and identified the main epidemic strains,and analyzed the inhibitory effect of mink IFN-βon the strains,providing scientific basis for clinical prevention and control of mink enteritis parvovirus disease.First,mink disease materials were collected from five regions in Shandong Province,and THE MEV infection was detected by PCR method.The MEV strain was isolated by CRFK cell culture,and the gene homology and biological evolution relationship between the isolated strain and other MEV strains were analyzed by biological software.Secondly,primers were designed according to the mink IFN-βgene sequence published in Gen Bank and the mink IFN-βgene was amplified.The expression product of minkβinterferon was prepared by prokaryotic expression technique of Escherichia coli.After purification,renaturation and concentration,VSV/CRFK system combined with cytopathic detection was used to detect antiviral activity.Thirdly,MEV was proliferated by CRFK cells,and the expression product ofβinterferon was added to detect the changes of cytopathic changes and virus content,and the antiviral activity of the protein against MEV was analyzed.The results showed that the positive rate of MEV was 45%（45/100）in mink samples from five cities in Shandong Province.A new MEV strain was isolated from the positive samples,named MEV-SD-Z-2020,which showed obvious cytopaplasia on CRFK cells with TCID₅₀of 10^-3.75/0.1 m L.The full length of VP2 gene was 1755 bp.Bioinformatics analysis showed that this strain had96.4%-98.1%homology with the domestic and foreign strains,and was closest to the heilongjiang（HLJ）strain reported in China.Primers were designed to amplify the 498 bp gene fragment of interferonβfrom mink liver samples,and the recombinant expression vector containing interferonβgene was successfully constructed.The 37 KDa recombinant interferonβprotein product was induced by IPTG,and its concentration was 0.8 mg/m L after purification.After renaturation,the expressed protein of mink IFN-βhad obvious antiviral activity against VSV,and the inhibition effect was the highest when the concentration was 2μg/m L,and the inhibition efficiency was about 94%.The inhibition rate of MEV replication was about 72%,and the inhibition effect lasted for 48 h,with the highest inhibition efficiency up to 83%.The results above,mink farming areas in shandong province MEV infections are more common,the separation of strain and strain homology is above 95%of reported in recent years,developed by using the mink IFN-βprotein products can significantly inhibit the replication of the strain,the mink enteritis parvovirus disease of shandong and even the whole China will provide a scientific basis for clinical prevention and treatment.