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Open Access Methodology

Development of an in situ assay for simultaneous detection of the genomic and replicative form of PCV2 using padlock probes and rolling circle amplification

Sara Henriksson1, Anne-Lie Blomström2, Lisbeth Fuxler3, Caroline Fossum3, Mikael Berg2 and Mats Nilsson1*

Author Affiliations

1 Department of Genetics and Pathology, Rudbeck laboratory, Uppsala University, SE-75185 Uppsala, Sweden

2 Department of Biomedical Sciences and Veterinary Public Health, Section of Virology, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden

3 Department of Biomedical Sciences and Veterinary Public Health, Section of Immunology, SLU, Uppsala, Sweden

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Virology Journal 2011, 8:37  doi:10.1186/1743-422X-8-37

Published: 24 January 2011

Abstract

Background

In this study we utilized padlock probes and rolling circle amplification as a mean to detect and study the replication of porcine circovirus type 2 (PCV2) in cultured cells and in infected tissue. Porcine circovirus type 2 is a single-stranded circular DNA virus associated with several severe diseases, porcine circovirus diseases (PCVD) in pigs, such as postweaning multisystemic wasting syndrome. The exact reason and mechanisms behind the trigger of PCV2 replication that is associated with these diseases is not well-known. The virus replicates with rolling circle replication and thus also exists as a double-stranded replicative form.

Results

By applying padlock probes and rolling circle amplification we could not only visualise the viral genome but also discriminate between the genomic and the replicative strand in situ. The genomic strand existed in higher numbers than the replicative strand. The virus accumulated in certain nuclei but also spread into the cytoplasm of cells in the surrounding tissue. In cultured cells the average number of signals increased with time after infection.

Conclusions

We have developed a method for detection of both strands of PCV2 in situ that can be useful for studies of replication and in situ detection of PCV2 as well as of DNA viruses in general.