Phages bearing affinity peptides to bovine rotavirus differentiate the virus from other viruses

Abstract

The aim of this study was to identify potential ligands and develop a novel diagnostic test to pathogenic bovine rotavirus (BRV) using phage display technology. The viruses were used as an immobilized target followed by incubation with a 12-mer phage display random peptide library. After five rounds of biopanning, phages had a specific binding activity to BRV were isolated. DNA sequencing indicated that phage displayed peptides HVHPPLRPHSDK, HATNHLPTPHNR or YPTHHAHTTPVR were potential ligands to BRV. Using the specific peptide-expressing phages, we developed a phage-based ELISA to differentiate BRV from other viruses. Compared with quantitative real-time PCR (qPCR), the phage-mediated ELISA was more suitable for the capture of BRV and the detection limitation of this approach was 0.1 µg/ml of samples. The high sensitivity, specificity and low cross-reactivity for the phage-based ELISA were confirmed in receiver operating characteristics (ROC) analysis.

Figure 1. Binding analysis of the selected phages to BRV in ELISA.

Twelve selected phages named phages 1 to 12 were incubated with the BRV in ELISA plates to test their binding activities to the viruses as described in Materials and methods. The individual phage and control are indicated in the x axis. The control is phage complex from the phage library. The OD₄₉₂ values of the tested individual phages are shown on the y axis. Bars show the standard deviation from three independent assays.

Figure 2. Phage-based ELISA differentiating BRV from other viruses.

Three phages harboring specific peptides recognizing BRV were identified from the 12 selected phages. Three phages bearing different peptide sequences were identified and designated HV, YP and HA. Bovine rotavirus (BRV), bovine herpesvirus type 1 (BHV-1), bovine viral diarrhea virus (BVDV), bovine coronavirus (BCV), porcine rotavirus (PRV), porcine transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV) were coated onto ELISA plates followed by incubation with above-mentioned phages. Subsequent incubation included addition of anti-M13 antibody and HRP-conjugated antibody. The ratio between the OD₄₉₂ value of individual phage and the OD₄₉₂ value of the control phage complex from the phage library is shown on the y axis, respectively. “*” means p<0.01, compared with other groups. Bars show the standard deviation from three independent assays.

Figure 3. Detection of serial dilutions of purified BRV using phage-mediated ELISA and qPCR.

Serially diluted BRV was used as coating antigen followed by successive incubation with phages YP, HV or HA, anti-M13 antibody and HRP-conjugated goat anti-rabbit antibody. The OD₄₉₂ of detected phage wells is shown in the left y axis. Viral RNA was also extracted from the serially diluted viruses, subsequently, the cDNA was achieved by reverse transcription and the resulting cDNA was ten-fold serially diluted and subjected to qPCR. The DNA copies are shown in the right y axis. The concentration of the viruses is indicated in the x axis. The ELISA and qPCR assays were performed in triplicate. Bars show the standard deviation from three independent assays.

Figure 4. ROC curve for detection of BRV of the phage-mediated ELISA in comparison to the qPCR.

The ROC (receiver operating characteristics) analysis for all samples to each phage detection (YP, HV or HA) was performed, respectively according to the area under the ROC curves (AUC). The results of the qPCR analysis of these samples were used as “gold standard” reference. The ROC plots the true positive rate (sensitivity) against the false positive rate (1-specificity). The diagonal indicates no discriminatory power.