Canine Parvovirus is diagnosed and neutralized by chicken IgY-scFv generated against the virus capsid protein
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RESEARCH ARTICLE
Canine Parvovirus is diagnosed and neutralized by chicken IgY‑scFv generated against the virus capsid protein Shikun Ge1,2, Long Xu1,2, Ben Li1, Fagang Zhong4, Xiang Liu1 and Xiaoying Zhang1,2,3,4*
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 × 106 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. Keywords: IgY-Technology, infectious animal diseases, virus diseases, parvovirus, phage display system Introduction Canine parvovirus (CPV) was first identified in 1978 [1]; it has a single-stranded DNA genome of negative polarity, about 5200 bp in length. The CPV capsid is a 25 nm diameter icosahedron containing three structural viral proteins (VP1, VP2 and VP3) and two non-structural proteins (NS1 and NS2), among them, VP2 accounts for 90% of the viral capsid and represents the major determinant of host range and virus-host interactions [2]. Although the conventional attenuated and inactivated CPV vaccines have been successful in reducing the disease outbreaks, the genus of parvovirus still causes severe
*Correspondence: [email protected] 2 Department of Biology, Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar, 4710‑057 Braga, Portugal Full list of author information is available at the end of the article
epizootics worldwide and leads to severe economic losses in dogs [3]. Antibody based approach is promising in CPV disease diagnosis, therapy and prevention, and the relevant hyper immunoglobulin G (IgG) and full-length monoclonal antibody (mAbs) have been routinely applied in the veterinary practices [4]. At present, most commercially available mAbs are produced in mammalian system using hybridoma technology. Functional antibody fragments (i.e.: scFv, Fab) generated by phage-display technology have been not yet routinely evaluated an
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