Molecular Breeding of Porcine Circovirus Type 2 by Synthetic DNA Shuffling

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dc.contributor.authorSmith, Sara Marieen
dc.contributor.committeechairMeng, Xiang-Jinen
dc.contributor.committeememberBuechner-Maxwell, Virginia A.en
dc.contributor.committeememberLeRoith, Tanyaen
dc.contributor.departmentBiomedical and Veterinary Sciencesen
dc.date.accessioned2017-04-04T19:49:23Zen
dc.date.adate2011-07-19en
dc.date.available2017-04-04T19:49:23Zen
dc.date.issued2011-06-29en
dc.date.rdate2016-10-18en
dc.date.sdate2011-06-30en
dc.description.abstractPorcine circovirus type 2 (PCV2) is a small, non-enveloped, single-stranded DNA virus that causes disease in pigs and is an economically important pathogen affecting pig populations worldwide. PCV2 contains two major open reading frames (ORF): ORF1 encodes two replicase proteins and ORF2 encodes the immunogenic capsid protein. There are three genotypes of PCV2 (PCV2a, PCV2b, and PCV2c), but vaccines available for PCV2 infection are only targeted against PCV2a. The objective of this thesis was to create viable chimeric PCV2 viruses with an ORF2 displaying genetic diversity from all PCV2 genotypes by synthetic DNA shuffling. Variation was identified at 55 amino acid positions in the ORF2 gene among 853 PCV2 capsid gene sequences available in the GenBank database. Degenerate oligonucleotide primers spanning ORF2 were synthesized to contain this naturally observed sequence diversity. Sets of overlapping oligonucleotide primers were fused together using overlap extension PCR to create full-length shuffled ORF2 sequences. The shuffled library of the ORF2 genes was subsequently cloned into the genomic backbone of a wildtype PCV2a infectious DNA clone and transfected into porcine kidney cells (PK-15). After transfection and infection of PK-15 cells, viability of chimeric viruses was screened by immunofluorescence assay (IFA) using anti-PCV2 Rep antibodies. PCR was used to amplify the genomes of viable shuffled viruses from infected cells. PCV2 viruses containing an ORF2 displaying genetic diversity from PCV2a, PCV2b, and PCV2c were isolated in vitro. These shuffled PCV2 viruses may be used as potential candidates for a broadly-protective PCV2 vaccine, although additional studies are warranted to determine in vivo infectivity and pathogenicity.en
dc.description.degreeMaster of Scienceen
dc.identifier.otheretd-06302011-083037en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-06302011-083037/en
dc.identifier.urihttp://hdl.handle.net/10919/76809en
dc.language.isoen_USen
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectporcine circovirus type 2en
dc.subjectPCV2en
dc.subjectmolecular breedingen
dc.subjectDNA shufflingen
dc.subjectPCVADen
dc.subjectporcine circovirus-associated diseaseen
dc.titleMolecular Breeding of Porcine Circovirus Type 2 by Synthetic DNA Shufflingen
dc.typeThesisen
dc.type.dcmitypeTexten
thesis.degree.disciplineBiomedical and Veterinary Sciencesen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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