Molecular Characterization Of Mink Enteritis Virus Isolated From Mink And Its Pathogenesis In Mink

During 2015 to 2016,the intestine samples from 176 mink were collected in China.In this study,eight MEVs were isolated from the mink exhibiting diarrhea disease,named MEV-SD1,MEV-SD2,MEV-SD3,MEV-SD4,MEV-SD5,MEV-SD6,MEV-SD7 and MEV-SD8,respectively.To investigate more precisely the genotype and genetic origin,the DNA sequences were compiled and edited using the Lasergene sequence analysis software package.The deduced amino acid sequences were also compared using DNASTAR software.The identity of the 8 VP2 genes was 99.4%-100%.The 8 MEV VP2 genes shared 97.9%-100% identity with all the reference sequences.The 8 VP2 proteins shared 97.9%-99.7% similarity with the reference sequences at amino acid level.There was 99.0%-99.8% similarity in the 8 VP2 proteins.Amino acid alignment showed these mutations were mainly distributed in ten sites.The 8 NS1 genes shared 98.7%-100% identity with all the reference sequences.The identity of the 8 NS1 genes were 99.8%-100%.The 8 NS1 proteins shared 98.1%-99.9% similarity with the reference sequences at amino acid level,99.3%-99.9% similarity in the 8 MEVs.To investigate more precisely the genetic origin of the gene segments of the 8 MEVs,we used biology software analysis and also consulted a lot of professional information.The amino acids at positions 300 is critical in distinguishing antigenicity and hosting range among parvoviruses but may vary among the carnivore parvoviruses.Those capsid regions are also highly anti-genic,and serve as the target of many neutralizing antibodies.But the amino acids residue at position 300 in MEV-SD1 to MEV-SD6 differed from those of MEV strains published previously,V300 A mutation,and had a common amino acid with FPV at position 300.The other two strains of MEV-SD7 and MEV-SD8 were the original V.Mutation of residue 300 of MEV VP2 decrease the released viral genome copies,which indicated that this mutation did significantly affect capsid assembly and viral genome packaging.Furthermore,phylogenetic analysis based on VP2 genes indicates that eight isolated MEVs form two branches,of which MEV-SD1 to MEV-SD6 were located in the same branch of all the reference strains of FPV.The 8 NS1 proteins shared 98.1%-99.9% similarity with the reference sequences at amino acid level,99.3%-99.9% similarity in the 8 MEVs.To the NS1 protein gene,our analysis indicated that only a very individual amino acid mutation occurred,and did not form a substantial mutation.The NS1 protein gene encoded by the parvovirus is relatively conserved in its genetic evolution,although it plays an important role in the replication and transcription of the virus,but it does not have a substantial effect on the selectivity of the virus host.Phylogenetic analysis indicated that the NS1 sequences were divided into 4 branches.The 6 NS1 genes and the three reference FPV NS1 genes formed one unique evolutionary branch.Animal inoculation experiments were performed to analyze the pathogenicity of the MEV-SD1 strain.Twenty hours p.i.,mink developed diarrhea and partial loss of appetite.By 4 dpi,these clinical symptoms were observed in all thirty-six infected mink.Following the appearance of diarrhea,the feces contained large quantities of mucus,intestinal casts,and even bloody fluid,progressing to yellowish green,watery fluid.The morbidity of experimental animals,using MEV-SD1,was 100% and the mortality was as high as 38.9%.The LD50 of MEV-SD1 in mink was 104.75 TCID50.Experimental infection studies in mink showed the novel MEV infection caused a high morbidity.At present,MEV vaccines are used to prevent further spread of the viral disease with significant decreases in morbidity and mortality.The titers of the 10 of the 84 serum samples(11.9%)were above 16 for anti-MEV antibody,and 74 of the 84 serum samples had no HI titers for anti-MEV antibody.The existing MEV inactivated vaccine can not stop the development of the virus.In China,mink farming has become a pillar industry in some areas,but the family-style feeding accounted for the majority pattern.the management of the crude mad,feed dogs and cats at the same time,irrationality of the vaccine,and so on,which resulted in the continuous mutation of the mink parvovirus and the recombination with other parvoviruses.The study demonstrated that MEV-SD1 to MEV-SD6 were FPV and it was implied that the FPV infection caused a high morbidity and mortality in mink and the inoculation dose had an effect on pathogenicity of MEV-SD1 in mink.The study indicated that the MEVs from China formed a variety of complex genotype.Therefore,we need to strengthen the monitoring of MEV in order to prevent the emergence of novel pandemic strains,and the more effective MEV vaccine and reasonable immunization program should be developed to prevent parvovirus disease.