The University of Georgia College of Veterinary Medicine

eorgia Veterinary Scholars Program

GVSP Summer 2007 Scholars

Georgia Veterinary Scholar	Faculty Mentor

Yandace Brown University of Georgia Class of 2010	Dr. Ralph Tripp

Development of Porcine Circovirus-2 (PCV-2) Vaccine Candidates

Yandace K. Brown *1, R. Jeff Hogan1, 2, S. Mark Tompkins1, Les Jones1, Frank Michel2 and Ralph A. Tripp1 Center for Disease Intervention, Department of Infectious Diseases1 and Department of Anatomy and Radiology2, University of Georgia, Athens, Georgia, USA 30602

Abstract

Porcine circovirus (PCV) belongs to the Circoviridae family whose members have virons of icosahedral symmetry, are non-enveloped, and have covalently-closed, circular, single-stranded DNA genomes from 1.8 to 2.3 kb in length. Two genotypes of PCV are recognized: PCV-1 and PCV-2. PCV-1 is a non-cytopathogenic adventitious virus of porcine cell lines. PCV-2 is a pathogenic swine virus associated with post-weaning multi-systemic wasting syndrome (PMWS), characterized by severe weight loss and generalized lymph node enlargement. Non-pathogenic PCV-1 and pathogenic PCV-2 share less than 80% nucleotide sequence identity; therefore PCV-1 cannot be used in whole vaccine strategies for PCV-2. The ORF2 gene of PCV-2 encodes for the 30 kDa capsid protein that shares > 90% amino acid sequence identity among PCV-2 isolates, therefore capsid-based subunit vaccines are plausible. The objectives of this study are to develop efficacious PCV-2 vaccines using two approaches: inactivated whole PCV-2 vaccine (iPCV2) and PCV-2 capsid-based subunit vaccine (Nvac). For iPCV2 vaccine development, a permissible swine cell line free of adventitious PCV must be used. PK15 cells are permissible for PCV-2 propagation but contain endogenous PCV. These cells were treated with PCV sequence-complimentary phosphorodiamidate morpholino oligos (PMOs); however treatment did not clear the endogenous virus. Since PCV is not integrated into the genome, and daughter cells are infected from the tissue culture of infected cells, we used limiting dilution and dimethyl sulfoxide (DMSO) to synchronize the cell cycle for limiting dilution to facilitate cloning of uninfected cells. Virus-free PK15 cells will be used to propagate PCV-2 and formalin-inactivated virus used for vaccine preparation. For Nvac development, PCV-2 ORF2 was cloned onto a pcDNA3 plasmid expression vector, bacteria transfected with the plasmid, and the Nvac protein isolated. Preliminary vaccine studies are ongoing in mice vaccinated and boosted +/- novel adjuvants, e.g. mannan to evaluate generated antibody titers. For all vaccine candidates, pilot studies in swine will be performed and disease pathogenesis and weight loss evaluated as endpoints for vaccine efficacy in challenged animals.