Canine Parvovirus is diagnosed and neutralized by chicken IgY-scFv generated against the virus capsid protein

Abstract

Canine parvovirus (CPV) can cause acute and highly contagious bloody enteritis in dog. To obtain antibodies against CPV, hens were immunized with virus-like particles (VLP) of CPV-VP2. The IgY single chain fragment variables (scFv) were generated by T7 phage display system and expressed in E. coli system. The titer of the primary scFv library reached to 1.5 × 10 6 pfu/mL, and 95% of the phages contained the target fragments. The CPV-VLP and CPV-VP2 protein showed similar reaction values to the purified scFv in the ELISA test, and the results of ELISA analysis using IgY-scFv toward CPV clinical samples were consistent with commercial immunochromatographic assay (ICA) and PCR detection, the scFv did not show cross reactivity with canine distemper virus (CDV) and canine coronavirus (CCV). IgY-scFv was successfully expressed in CRFK cells, and in the virus suppression assay, 55% of CPV infections were eliminated within 24 h. Docking results demonstrated that the number of amino acids of the binding sides between scFv and VP2 were AA37 and AA40, respectively. This study revealed the feasibility of a novel functional antibody fragment development strategy by generating diversified avian IgY-scFv libraries towards the pathogenic target of interest for both detection and therapeutic purposes in veterinary medicine.

Generation and evaluation of IgY-scFv based mimetics against canine parvovirus

“…Results are means of three replicates. The PCR and ICA results were in accordance to [ 4 ].

Figure 2 The protective rate of IgY-peptide against CPV-infected cells.

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“…This study builds on our findings previously published [ 4 ], CDR and framework region sequences of our previously obtained IgY-scFv against the VLP of CPV-VP2 (Figure 1 A) [ 4 ] were used to create an antibody mimetic peptide comprising two interacting V H− (VHCDR1) and V L− (VLCDR3) derived CDRs (31 AA, VHCDR1-VHFR2-VLCDR3). The framework region (VHFR2) from either the V H or V L domain orients the two CDRs in a manner resembling their disposition in native molecules (Figures 1 A and B).…”

“…Numerous attempts have been successfully made to generate avian sourced monoclonal IgY (mIgY) or recombinant functional IgY fragments, particularly the single chain fragment variables (scFv) [ 1 , 2 ], for immunological detection [ 3 ], diagnostic and therapeutic purposes [ 4 ] in veterinary and human medicine.…”

“…In our recent study [ 4 ], hens were immunized with virus-like particles (VLP) of canine parvovirus VP2 (CPV-VP2) and the specific IgY-scFv were generated by using the T7 phage display system. This result confirmed the feasibility of generating diversified avian IgY-scFv libraries against pathogenic targets of interest for both diagnostic and therapeutic purposes.…”

“…Among them, there were 24 positive samples and four negative samples (samples number 4, 6, 12 and 26) confirmed by a commercial colloidal gold test strip (ICA). Each sample was homogenized (10%, w/v) in PBS (1 mL, pH 7.2) and centrifuged, then the supernatants were analyzed by ELISA and PCR [ 4 ]. There was no difference in the detection performance of IgY-peptide and IgY-scFv on CPV clinical fecal samples.…”