Screening And Identification Of Peptides Blocking Mink Enteritis Virus Inefction Of F81Cell

Mink enteritis virus (MEV) belongs to parvoviridae family, parvovirus genus. Itis the causative pathogen of mink viral gastroenteritis, an acute, violent and highlycontagious disease that causes violent diarrhea as the main clinical symptom. Thedisease was initially reported in Canada by Schofield, and the causative virus wasconfirmed and named as mink enteritis virus by Wills. The disease later spread to theUnited States, Denmark, Finland, Norway, Sweden, England and Japan. The firstreport of this disease in the People’s Republic of China was in1974. At present,viral enteritis is one of the most serious diseases of mink and is an obstacle to thedevelopment of the animal fur farming industry. Phage display is based on theexpression of many random peptides as fusion products with the protein present onthe surface of the bacteriophage. It has recently become a powerful molecularbiological technique with which to screen for novel and useful antiviral peptides.The aim of this study was to select specific peptides capable of binding to MEVand inhibiting its infection of the F81cell line using the method of phage displaytechnology, and lay the foundation for the manufacture of novel feed additives andantiviral agents.A virus was isolated from a dead mink which was suspected of being infectedwith the mink enteritis virus (MEV). It was eventually identified as MEV using thePCR assay, and cell culture. The VP2gene of this parvovirus isolate was cloned intopMD18-T vector, sequenced and analyzed. As a result, the VP2protein possessed onesbustitution,328A-T. The result of VP2gene phylogenetic anaysis illustrated that thisisolate and other six wild strains had the same ancestor. Then, a large number of viruswas propagated in the F81cell line. The concentration and purification was executedby the method of PEG precipitation for the screening of the phage display peptide library. The pure virion was used for the screening of the phage display peptide library.After three rounds of biopanning, the phage enrichment was117times higher thanthat after the first round. Twelve phage clones that showed threefold higherMEV-binding affinity than controls were selected by ELISA from the30phage clones.After evaluating the capacity of12selected phages to inhibit MEV propagation invitro, the phenomenon that the ability of phages to inhibit infection did not show astrict linear correlation with MEV-binding affinity was found. Single-stranded DNA(ssDNA) was extracted from the M13phages and sequenced. Peptide sequences wereanalyzed using the antimicrobial peptide predictor website. After homology analysis，Pr and Pl were chemically synthesized and examined for antiviral activity.The cytotoxicity of the two peptides Pr and Pl was evaluated after synthesis ofthem. By evaluating the TCID50of the virus, we got the result that these two peptideshad antiviral ability against MEV. To determine if the peptides inhibited a binding orpost-binding step in viral entry to the cell, two different experiments were performed.When the virus was incubated with cells in advance, the synthetic peptides did notinhibit virus infection. However, when the virus was pre-incubated with the peptides,the infectivity of the virus appeared to drop. This experiment demonstrated that theselected peptides could indeed block infection of cells by MEV. This may be becausethe peptides combine with functional epitopes which play a crucial role in cellinfection. To confirm whether the two peptides inhibit the virus infection specifically,peptides were tested for infection effects against CPV, FPV and CDV. The resultshown that these two peptides displayed remarkably antiviral ability against CPV andFPV. But neither of them posses significant inhibitory effect against CDV replication.In this study a MEV virus was isolated successfully. Tow peptides which hadantiviral ability was selected using the method of phage display technology. We alsoproofed the manner of two peptides antiviral action. All of these laid a experimentalfoundation for the prevention and therapeutics of MEV in vivo.