This thesis reports for the first time the detection of porcine circovirus virus (PCV) in the Australian pig herd. PCV DNA was detected in the tissues of pigs from several Australian states using a multiplex polymerase chain reaction (PCR) assay, the primers for which were based on the sequence of PCV1 and PCV2 strains detected in North America and Europe. PCV type 1 or 2 was detected in 80 of 367 (21.7%) pigs tested. In the 80 positives, both PCV1 and PCV2 were detected in 14 samples. Virus was detected in pigs from all states from which samples were obtained: Western Australia, South Australia, New South Wales and Queensland. The complete genomes of 13 strains of Australian PCV were sequenced. Analysis of the data indicated there was extremely high homology between the Australian strains of PCV1 and PCV2 and previously published sequences of PCV1 and PCV2 strains from North America and Europe.There were no consistent differences between the genome of the Australian strains and strains in North America and Europe. The widespread occurrence of PCV in the tissues of pigs was confirmed by a small scale serological study of the Western Australian pig herd using an immunofluorescence assay, which did not discriminate antibody to PCV1 and PCV2. This assay detected PCV antibody in 11 of 14 pig herds in Western Australia, with a prevalence rate in positive herds varying from 25 to 47%, but it was unable to differentiate antibody to PCV1 and PCV2. A PCV2-specific recombinant viral capsid protein was produced in insect cells with a baculovirus expression system and this was used to develop a PCV2-specific ELISA and a Western immunoblotting assay. These assays were applied to samples from a national pig serum bank and detected PCV2 antibody in 33% of 3933 serum samples. The highest seroprevalence to the recombinant PCV2 capsid antigen was detected in the samples from Victoria where there was a 51.3% seroprevalence rate, and the lowest in Western Australia where there was an 11.4% seroprevalence rate. An in situ hybridisation (ISH) technique was developed for the detection of PCV in tissues of infected pigs and infected cell cultures. A monoclonal antibody specific for the capsid protein of PCV2 was also produced and has application for the development of immunocytochemical procedures for the detection of PCV2 in tissues and cell cultures. The high prevalence of PCV in the Australian pig herd and the absence of reports of PMWS suggested that the Australian strains of PMWS detected may have been of low virulence. To examine the pathogenicity of Australian strains, two animal experiments were conducted where the type species of PCV1 present in persistently-infected PK15 pig kidney cells and an Australian PCV2 strain were cultured in vitro in cell cultures and inoculated into weaner pigs. As expected, the PCV1 replicated well in pigs but did not result in the induction of clinical signs or lesions in the inoculated pigs. The inoculation into weaner pigs of cell culture replicated PCV2 with an apparent virus titre of 103 virus particles/mL resulted in infection of only some of the inoculated pigs and it was concluded that the PCV2 inoculum contained insufficient virus to infect all pigs into which it was inoculated. The PCV2 did not induce any disease syndrome and could not be visualised in tissue sections of infected pigs using immunohistochemical techniques. In conclusion, techniques were developed for the detection of PCV in the Australian pig herd. PCV of both genetic types were detected at prevalence rates similar to those reported in other countries where PMWS has occurred, and the widespread occurrence of PCV was confirmed by serological assays. The PCV strains present were genetically indistinguishable from those present in North America and Europe. The reason for the absence of PMWS in Australia is most likely not due to differences in the characteristics of the PCV strains present.

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. Since its initial detection in a Canadian commercial swine herd in 1991, PMWS has been detected in all swine producing regions of the world and is now a serious economic problem to the swine industry. The objectives of this dissertation were to biologically, genetically and experimentally characterize both PCV1 and PCV2, to identify the genetic determinant(s) for virulence and replication, and to develop an effective genetically-engineered vaccine against PCV2 infection and PMWS. The genetic heterogeneity of PCV2 and PCV1 isolates from different geographic origins were determined. We found that, although PCV1 and PCV2 genomes were very conserved, some minor genomic variation exists among PCV1 isolates and PCV2 isolates. The nonpathogenic PCV1 and pathogenic PCV2 share only about 76% nucleotide sequence identity but have similar genomic organization. The highest sequence variability among PCV isolates is found in the immunogenic ORF2 capsid gene. Based on the sequence data in this dissertation, a universal polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed that is capable of detecting all known PCV isolates and differentiating between infections by nonpathogenic PCV1 and pathogenic PCV2. In order to study the structural and functional relationship of PCV genes and to develop a genetically-engineered vaccine, we constructed infectious DNA clones of both PCV1 and PCV2. By using the PCV2 infectious clone, we showed that pigs can be infected by direct intrahepatic injection of PCV2 infectious DNA clone. The pathological lesions and clinical disease associated with PCV2 infection were more definitively characterized by using the infectious DNA clone. We found that PCV2 is the primary but not the sole causative agent of PMWS, as the full spectrum of clinical PMWS was not reproduced by the infectious PCV2 DNA clone although pathological lesions characteristic of PMWS were reproduced. A chimeric vaccine was constructed by cloning the immunogenic capsid gene of the pathogenic PCV2 into the genomic backbone of the non-pathogenic PCV1 virus. We showed that the resulting chimeric PCV1-2 vaccine virus, retained the non-pathogenic nature of PCV1 but induced a protective immune response against a wild-type PCV2 challenge. In vaccinated pigs, the chimeric PCV1-2 vaccine reduced PCV2 viremia length and serum virus loads and reduced pathological lesions such as lymphoid depletion (LD) and histiocytic replacement (HR) in lymphoid tissues, inflammation and discoloration of the lymph nodes. The amounts of PCV2 antigen and PCV2 genomic copy loads in lymph node tissues were also significantly reduced. Our results indicated that the attenuated chimeric PCV1-2 virus induces protective immunity against PCV2 infection and thus could serve as an effective vaccine against PCV2 and PMWS. To improve the safety of the vaccine, we attempted to identify the genetic determinant(s) for PCV2 virulence. An isolate of PCV2 was serially passaged for 120 times in PK-15 cells. After 120 passages, a total of two amino acid mutations were identified in the capsid protein of the passage 120 virus (VP120), P110A and R191S. Compared to other known PCV1 and PCV2 sequences, the two amino acid mutations in PCV2 VP120 are unique. The VP120 virus was biologically characterized in vitro and experimentally characterized in specific-pathogen-free (SPF) pigs. The two amino acid mutations resulted in an enhanced replication ability of PCV2 VP120 in PK-15 cells and an attenuated phenotype in infected pigs. The P110A and R191S mutations in the capsid protein either alone or collectively are likely important for PCV2 virulence and replication. In summary, we genetically characterized PCV2 isolates from different geographic regions and developed a PCR-RFLP assay. We constructed and characterized infectious DNA clones of PCV1 and PCV2, and developed a genetically engineered vaccine against PCV2 infection. We also identified the genetic determinants for PCV2 virulence and replication. The vaccine developed in this study, when it becomes available, will help the swine industry control this important pathogen. / Ph. D.