Development of an in situ assay for simultaneous detection of the genomic and replicative form of PCV2 using padlock probes and rolling circle amplification.
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.
Department of Genetics and Pathology, Rudbeck laboratory, Uppsala University, SE-75185 Uppsala, Sweden.
Sara
S
Henriksson
Anne-Lie
AL
Blomström
Lisbeth
L
Fuxler
Caroline
C
Fossum
Mikael
M
Berg
Mats
M
Nilsson
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000821:virology; D018173:Circoviridae Infections / Q000662:veterinary / Q000821:virology; D017925:Circovirus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D003593:Cytoplasm / Q000821:virology; D004279:DNA, Viral / Q000235:genetics*; D008198:Lymph Nodes / Q000821:virology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics*; D013552:Swine; D013553:Swine Diseases / Q000821:virology; D014773:Virology / Q000379:methods*; D014779:Virus Replication
D004279:DNA, Viral; D015345:Oligonucleotide Probes
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