Identification and Functional Analysis of the Novel ORF4 Protein Encoded by Porcine Circovirus Type

Porcine 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 twomouse 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 andmice. Mice infected with PCV2 had a higher serum viral load (genomic cop-ies) andmore severe lymphoid tissue damage in the spleen than those infected with wPCV2. Meanwhile, flow-cytometric analy-sis 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. Circoviruses are a family of small nonenveloped icosahedralviruses infecting pigs, geese, canaries, pigeons, and parrots (1–3). Porcine circovirus type 1 (PCV1) was initially isolated as a persistent contaminant from a porcine kidney cell line and is non