Infectious clone is a useful tool in exploring viral replication and pathogenesis. In order to prevent linear PCV2 cyclization, PCR mutagenesis was used to construct the first molecular clone (pSK-2PCV2) by ligating two copies of the complete PCV2 genome with the pBluescript SK (pSK) vector. In addition, pSK-PCV2 and ds-PCV2 were constructed. PK-15 cells were transfected with above three infectious clones. Indirect immunofluorescence assay (IFA) revealed that the virus antigen mainly localized in infected cell nucleolus and cytoplasm. PCV2 specific nucleotide fragment in cell culture was amplified by RT-PCR. Typical porcine circovirus particles with diameter about 17 nm were also observed by transmission electron microscope (TEM) in the infected cells. The rescued virus sequences from the cultures had 100% homology with the inserting PCV2 genome. The rescued virus shared similar properties with that of the parental virus. The study establishes a platform for further research on the virus molecular biology and pathogenicity.
Animals ; Cell Line ; Circoviridae Infections ; virology ; Circovirus ; genetics ; growth & development ; pathogenicity ; physiology ; Cloning, Molecular ; DNA, Viral ; genetics ; physiology ; Recombination, Genetic ; genetics ; Swine ; Transfection ; Virulence ; Virus Replication

Epidemiological characteristics of swine pulmonary Pneumocystis (P.) carinii and concurrent infections were surveyed on Jeju Island, Korea, within a designated period in 172 pigs submitted from 54 farms to the Department of Veterinary Medicine, Jeju National University. The submitted cases were evaluated by histopathology, immunohistochemistry, PCR/RT-PCR, and bacteriology. P. carinii infection was confirmed in 39 (22.7%) of the 172 pigs. Histopathologically, the lungs had moderate to severe lymphohistioctyic interstitial pneumonia with variable numbers of fungal organisms within lesions. Furthermore, porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV-2) co-infection was a common phenomenon (12.8%, 20.5%, and 48.7% were positive for PRRS, PCV-2, or both, respectively, as determined by PCR/RT-PCR). Infection was much more concentrated during winter (December to March) and 53.8% of the infected pigs were 7- to 8-weeks old. In addition, three pigs showed co-infection with bacteria such as Pasteurella multocida and Streptococcus suis. The results of the present study suggest that the secondary P. carinii infection is common following primary viral infection in swine in Korea. They further suggest that co-infection of P. carinii might be enhanced by the virulence of primary pathogens or might have synergistic effects in the pigs with chronic wasting diseases.
Aging ; Animals ; *Circovirus/pathogenicity ; Incidence ; Pasteurella Infections/complications/epidemiology/veterinary ; Pasteurella multocida/i ; *Pneumocystis carinii/immunology/pathogenicity ; Pneumonia, Pneumocystis/complications/epidemiology/physiopathology/*veterinary ; Porcine Postweaning Multisystemic Wasting Syndrome/complications/*epidemiology ; Porcine Reproductive and Respiratory Syndrome/*epidemiology ; *Porcine respiratory and reproductive syndrome virus/pathogenicity ; Prevalence ; Republic of Korea/epidemiology ; Reverse Transcriptase Polymerase Chain Reaction ; Sea ; Streptococcal Infections/complications/epidemiology/veterinary ; Streptococcus suis/i ; Sus scrofa ; Swine Diseases/epidemiology/virology