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Updated: Jun 18, 2026
Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'
Published on: May 26, 2013
Multiply-primed rolling-circle amplification (MPRCA) of PCV2 genomes: applications on detection, sequencing and virus
Diogenes Dezen1, Franciscus Antonius Maria Rijsewijk, Thais Fumaco Teixeira
1Laboratório de Virologia, FEPAGRO Saúde Animal, Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF), Caixa Postal 47, Eldorado do Sul, 92990-000 RS, Brazil. ddezen@gmail.com
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 10(5.55) TCID(50)/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.
Insights
Multiply-primed rolling-circle amplification (MPRCA) effectively amplifies porcine circovirus type 2 (PCV2) genomes for sequencing and virus isolation. This method enables the creation of infectious PCV2 clones from amplified DNA, though it
Area of Science:
- Veterinary Virology
- Molecular Biology
- Animal Pathology
Background:
- Porcine circovirus type 2 (PCV2) causes post-weaning multisystemic wasting syndrome (PMWS) in pigs.
- Accurate amplification and characterization of PCV2 genomes are crucial for understanding and controlling the disease.
Purpose of the Study:
- To evaluate multiply-primed rolling-circle amplification (MPRCA) for amplifying PCV2 genomes.
- To assess the utility of MPRCA-amplified PCV2 for sequencing, virus isolation, and infectious clone construction.
Main Methods:
- MPRCA was employed to amplify PCV2 genomes from infected pig tissues.
- Amplified genomes were cloned into plasmids, sequenced, and compared to existing isolates.
- Recircularized cloned genomes were transfected into PK-15 cells to produce infectious virus.
Main Results:
- MPRCA successfully amplified PCV2 genomes with high nucleotide similarity to other isolates (93-99%).
- Sequenced PCV2 genomes were nearly identical, with only one silent substitution in the ORF2 region.
- Transfection of recircularized MPRCA-amplified genomes yielded infectious PCV2 up to 10(5.55) TCID(50)/mL.
Conclusions:
- MPRCA is a valuable tool for PCV2 genome amplification, facilitating sequencing and infectious clone creation.
- The method allows straightforward construction of infectious PCV2 clones from amplified viral DNA.
- MPRCA demonstrated lower sensitivity compared to PCR for diagnostic applications.
