No abstract

bPorcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. To date, viral proteins Cap, Rep, Rep=, and ORF3, encoded by the PCV2 genome, have been described. Here, transcription and translation of a novel viral gene within the PCV2 genome (designated ORF4) was determined and functionally analyzed in vitro and in vivo. Northern blot analysis indicated that the RNA transcribed from the ORF4 gene is about 180 bp in length and overlaps ORF3 in the same direction. Site-directed mutagenesis confirmed that the viral ORF4 protein is not essential for virus replication in PK-15 cells and in mice infected with an ORF4-deficient PCV2 (PCV2⌬). PCV2⌬ triggered higher activity levels of caspase-3 and -8 than wild-type PCV2 (wPCV2) in PK-15 cells. The antigenic epitopes of two mouse monoclonal antibodies (MAbs) raised against the viral ORF4 protein were mapped to the same 19KSSASPR25 peptide. Expression of ORF4 was confirmed using the specific MAbs in wPCV2-infected PK-15 cells and mice. Mice infected with PCV2⌬ had a higher serum viral load (genomic copies) and more severe lymphoid tissue damage in the spleen than those infected with wPCV2. Meanwhile, flow-cytometric analysis indicated that the PCV2⌬ infection caused a significant decrease of CD4 ؉ and CD8 ؉ T lymphocytes. Our results demonstrate that ORF4 is a newly discovered viral protein that is not essential for PCV2 replication but plays a role in suppressing caspase activity and regulating CD4؉ and CD8 ؉ T lymphocytes during PCV2 infection.

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome, which is an emerging swine immunosuppressive disease. To uncover cellular protein responses in PCV2-infected PK-15 cells, the comprehensive proteome profiles were analyzed utilizing two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF identification. Multiple comparisons of 2-DE revealed that the majority of changes in protein expression occurred at 48-96 h after PCV2 infection. A total of 34 host-encoded proteins, including 15 up-regulated and 19 down-regulated proteins, were identified by MALDI-TOF/TOF analysis. According to cellular function, the differential expression proteins could be sorted into several groups: cytoskeleton proteins, stress response, macromolecular biosynthesis, energy metabolism, ubiquitin-proteasome pathway, signal transduction, gene regulation. Western blot analysis demonstrated the changes of alpha tubulin, beta actin, and cytokeratin 8 during infection. Colocalization and coimmunoprecipitation analyses confirmed that the cellular alpha tubulin interacts with the Cap protein of PCV2 in the infected PK-15 cells. These identified cellular constituents have important implications for understanding the host interactions with PCV2 and brings us a step closer to defining the cellular requirements for the underlying mechanism of PCV2 replication and pathogenesis.