Multiply-primed rolling-circle amplification (MPRCA) of PCV2 genomes: Applications on detection, sequencing and virus isolation |
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Authors: | Diogenes Dezen,Franciscus Antonius Maria Rijsewijk,Thais Fumaco Teixeira,Carine Lidiane Holz,Samuel Paulo Cibulski,Ana Clá udia Franco,Odir Antonio Dellagostin,Paulo M. Roehe |
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Affiliation: | aLaboratório de Virologia, FEPAGRO Saúde Animal, Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF), Caixa Postal 47, Eldorado do Sul, 92990-000 RS, Brazil;bDepartamento de Microbiologia, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Av. Sarmento Leite 500, Porto Alegre, 90050-170 RS, Brazil;cCIRAD, Départament Systèmes Biologiques, UR-15, Campus International de Baillarguet, 34398 Montpellier, France;dCentro de Biotecnologia, Universidade Federal de Pelotas, Pelotas, RS, Brazil |
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Abstract: | Multiply-primed rolling-circle amplification (MPRCA) was used to amplify porcine circovirus type 2 (PCV2) genomes isolated from tissues of pigs with signs of post-weaning multisystemic wasting syndrome (PMWS). Two of the amplified PCV2 genomes were cloned in prokaryotic plasmids and sequenced. Both were nearly identical (1767 nt) except for one silent substitution in the region coding for the capsid protein (ORF2). In addition, they showed high nucleotide sequence similarity with PCV2 isolates from others countries (93–99%). To investigate whether the MPRCA amplified PCV2 genomes could be used to produce infectious virus, the cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 105.55 TCID50/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes aiming at sequencing and virus isolation strategies, where particularly useful is the fact that it allows straightforward construction of PCV2 infectious clones from amplified genomes. However, it was less sensitive than PCR for diagnostic purposes. |
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Keywords: | Swine Porcine circovirus type 2 PMWS Rolling-circle amplification Infectious copy |
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