Abstract
BackgroundPorcine epidemic diarrhea virus (PEDV) is emerging as a pathogenic coronavirus that causes a huge economic burden to the swine industry. Interaction of the viral spike (S) surface glycoprotein with the host cell receptor is recognized as the first step of infection and is the main determinant of virus tropism. The mechanisms by which neutralizing antibodies inhibit PEDV have not been defined. Isolating PEDV neutralizing antibodies are crucial to identifying the receptor-binding domains of the viral spike and elucidating the mechanism of protection against PEDV infection.MethodsB cell hybridoma technique was used to generate hybridoma cells that secrete specific antibodies. E.coli prokaryotic expression system and Bac-to-Bac expression system were used to identify the target protein of each monoclonal antibody. qPCR was performed to analyze PEDV binding to Vero E6 cells with neutralizing antibody.ResultsWe identified 10 monoclonal antibodies using hybridoma technology. Remarkably, 4 mAbs (designed 2G8, 2B11, 3D9, 1E3) neutralized virus infection potently, of which 2B11 and 1E3 targeted the conformational epitope of the PEDV S protein. qPCR results showed that both 2B11 and 2G8 blocked virus entry into Vero cells.ConclusionThe data suggested that PEDV neutralizing antibody inhibited virus infection by binding to infectious virions, which could work as a tool to find the receptor-binding domains.
Highlights
Porcine epidemic diarrhea virus (PEDV) is emerging as a pathogenic coronavirus that causes a huge economic burden to the swine industry
BALB/c mice were immunized with purified PEDV and ten Monoclonal antibodies (mAbs) were prepared via lymphocyte hybridoma technique, which could react well with the PEDV-infected Vero cells by immunofluorescence assay (IFA)
Identification of the target protein recognized by mAbs The PEDV S protein is the only identified target of PEDV Neutralizing antibodies (nAbs)
Summary
Porcine epidemic diarrhea virus (PEDV) is emerging as a pathogenic coronavirus that causes a huge economic burden to the swine industry. Interaction of the viral spike (S) surface glycoprotein with the host cell receptor is recognized as the first step of infection and is the main determinant of virus tropism. The mechanisms by which neutralizing antibodies inhibit PEDV have not been defined. Isolating PEDV neutralizing antibodies are crucial to identifying the receptor-binding domains of the viral spike and elucidating the mechanism of protection against PEDV infection. QPCR was performed to analyze PEDV binding to Vero E6 cells with neutralizing antibody. Porcine epidemic diarrhea virus (PEDV), which belongs to the Alphacoronavirus genus of the Coronaviridae family, is an etiological agent of porcine epidemic diarrhea (PED) and causes an enteric disease that affects all ages of swine [1, 2]. In spite of widespread immunization with the currently marketed vaccine, PED still persists in swine raising countries and resulted in devastating damage to the pork producers [9]
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