Abstract. Porcine circoviruses (PCV) are small nonenveloped DNA viruses containing a unique single-stranded circular genome. Previously, no recognized link was found between PCV infection of pigs and disease, and PCV was considered a nonpathogenic agent. Over the last 5 years, a ''novel'' PCV, designated PCV2, has been associated with various disease syndromes in pigs, primarily postweaning multisystemic wasting syndrome (PMWS). Pigs with PMWS have a variety of clinical signs, including debility, dyspnea, palpable lymphadenopathy, diarrhea, and pallor or icterus. Lesions associated with the presence of PCV2 in a variety of cell types include lymphohistiocytic to granulomatous interstitial pneumonia, hepatitis, nephritis, myocarditis, enteritis, and pancreatitis. The lesions of PMWS have been reproduced experimentally after inoculation of piglets with PCV2 cell culture isolates, although the full expression of the disease syndrome may require the presence of other agents such as porcine parvovirus or porcine reproductive and respiratory syndrome (PRRS) virus. Recent reports have linked PCV2 to other disorders in pigs, ranging from abortion and reproductive failure to ''atypical'' PRRS. Available data indicate high seroprevalence of antibodies to PCV2 worldwide. The diagnosis of PCV2-associated disease is based on the direct demonstration of PCV2 antigens or nucleic acid in affected tissues. PCV2 is now regarded as an important emerging pathogen. Although vertical transmission has been documented, the epidemiology of PCV2 infections is poorly understood, as is the role of the immune response in controlling or augmenting disease.Porcine circovirus (PCV) was first described 25 years ago as a picornavirus-like contaminant of the permanent pig kidney cell culture PK/15 (ATCC-CCL 33). 55 Some 8 years later, the same workers reported PCV as a new, small, nonenveloped, icosahedral mammalian virus containing a circular single-stranded DNA genome. 50 Since this report, PCV has been assigned to the Circoviridae family, 33 and a small body of knowledge on PCV has accumulated in the scientific literature. Recently, the emergence of a new antigenically and genomically distinct PCV in the swine population worldwide, which has been directly associated with a number of clinical conditions, has raised the interest in PCVs. The proposal has been made that this new PCV should be designated PCV2 and the original PCV contaminant of PK/15 cell cultures should be designated PCV1. 8,40 The purpose of this article is to review the current state of knowledge on PCV and provide the swine industry, veterinarians, diagnosticians, and veterinary virologists with a better understanding of PCVs. Within this article, the nomenclature PCV1 and PCV2 will be used as defined above. Virus isolatesReplication of PCVs in vitro is best achieved by inoculation of semiconfluent monolayers of PCV-free PK/15 cells. 54 A PCV-induced cytopathic effect has not

Abstract. Samples of lung, liver, kidney, pancreas, spleen, and lymph node from pigs with postweaning multisystemic wasting syndrome from California (USA) and samples of mesenteric lymph nodes from similarly diseased pigs from Brittany (France) were examined by light microscopy, in situ hybridization (ISH), and/or virus isolation. Whole genomic probes for porcine circovirus (PCV) and chicken anemia virus (CAV) were used for ISH. Tissue homogenate supernatants were inoculated onto PK/15 cells for virus isolation, and the presence of viral antigen and viral particles was verified by indirect immunofluorescence, ISH, and electron microscopy. Histologic examination of lung from pigs from California revealed interstitial pneumonia, alveolar epithelial hyperplasia, and basophilic nuclear and cytoplasmic inclusions in mononuclear cell infiltrates and various pulmonary epithelial cells. Granulomatous lymphadenitis with syncytial cells typified the lesions seen in the pigs from France. PCV-like nucleic acid was detected by ISH in lung, pancreas, lymph node, kidney, and liver in pigs from California. Positive signal was also obtained in lymph node sections from pigs from France. Probes for CAV were consistently negative. PK/15 cell cultures inoculated with lung preparations from diseased California pigs and mesenteric lymph node preparations from pigs from France had positive fluorescence by indirect staining for PCV using pooled polyclonal pig sera and hyperimmune rabbit serum and had variable staining with a panel of 7 monoclonal antibodies specific for cell culture contaminant PCV. PCV-like nucleic acid was also detected by ISH in cell cultures. Cytopathic effect was not observed. Electron microscopic examination of inoculated cell cultures revealed 17-nm viral particles morphologically consistent with PCV. No other virus particles were observed. Although genomic analysis for the definitive identification of these viral isolates remains to be done, the evidence provided strongly suggests that these tissue isolates are closely related to, although antigenically distinct from, the original PCV cell culture contaminant.

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Abstract. One-day-old gnotobiotic piglets were inoculated intranasally with in vitro passaged porcine circovirus 1 (PCV-1), PCV-2, and porcine parvovirus (PPV) alone or in combination (PCV-1/PCV-2, PCV-1/PPV, and PCV-2/PPV). Piglets were evaluated for 1) the development of porcine postweaning multisystemic wasting syndrome (PMWS), 2) distribution of viral antigens by immunochemistry, and 3) viremia and the presence of viral DNA in nasal and ocular secretions and feces. All single agent-infected piglets and piglets infected with PCV-1/PCV-2 or PCV-1/PPV were clinically asymptomatic. They were transiently viremic and seroconverted to homologous virus(es). At termination of the study on postinfection day (PID) 35, microscopic lesions were restricted to focal inflammatory cell infiltrates in livers and myocardia. One piglet given PCV-1/PPV was PPV viremic for 2 weeks after infection and had lymphangiectasia of the spiral and descending colon associated with granulomatous inflammation. All four PCV-2/PPV-inoculated piglets developed PMWS, characterized by sudden onset of depression and anorexia, icterus, and submucosal edema. One piglet became moribund on PID 27, and the remaining three piglets were euthanatized between PID 27 and PID 30 because of severe disease. Lymph nodes were small and the livers were mottled. Disseminated angiocentric granulomatous inflammation was present in all tissues examined except the brain. Multiple lightly basophilic intracytoplasmic inclusion bodies were identified in macrophages and histiocytes. PCV-2 antigen was widely distributed within macrophages; PPV antigen was sparse. Hepatocellular necrosis and bile retention were prominent. PCV-2 DNA was identified in ocular, fecal, and nasal secretions. Terminal sera contained antibodies to PPV (4/4) and PCV-2 (3/ 4). Production of PMWS in gnotobiotic swine appears to require PCV-2 and additional infectious agents such as PPV for full disease expression in gnotobiotic piglets.

Porcine circovirus (PCV)-2, a newly described single-stranded circular DNA virus pathogen of swine is the cause of postweaning multisystemic wasting syndrome (PMWS). In gnotobiotic piglets, PCV-2 infection alone produces asymptomatic infection without evidence of overt PMWS. Gnotobiotic piglets infected with PCV-2 were injected with keyhole limpet hemocyanin in incomplete Freund's adjuvant (KLH/ICFA), and the effects on virus production and development of PMWS were determined. In the first experiment, piglets were injected subcutaneously on the left hip and shoulder, and viral burden was assessed in regional lymph nodes draining the injection sites and in contralateral lymph nodes 13-14 days after infection. Immune activation increased the number of virus antigen-positive cells in draining lymph nodes and increased the amount of infectious virus recovered by 1-4 log10. In a second experiment, the effects of injections of KLH/ICFA with or without concurrent stimulation of peritoneal macrophages by intraperitoneal injections of thioglycollate broth on induction of PMWS was assessed. All immunized piglets developed moderate to severe PMWS, whereas none of the piglets infected with PCV-2 alone developed PMWS. In PMWS-affected piglets, extensive replication of PCV-2 was documented by both immunocytochemistry and quantitative viral titrations. Thus, immune activation is a key component of the pathogenesis of PCV-2-associated PMWS in swine.

Abstract. Neonatal gnotobiotic piglets were inoculated with tissue homogenates and low-and highpassage cell culture material to determine if the lesions of the newly described porcine postweaning multisystemic wasting syndrome (PMWS) could be reproduced. For this, 17 3-day-old gnotobiotic piglets were inoculated intranasally with pelleted chloroform-treated, filtered extracts from cell cultures, filter-sterilized homogenates of lymphoid tissue from PMWS-affected piglets, or control materials. Piglets were maintained in germ-free isolators for up to 5 weeks after infection prior to euthanasia and collection of samples for analysis. All piglets inoculated with the viral inocula developed lesions typical of PMWS, including generalized lymphadenopathy, hepatitis, nephritis, interstitial pneumonia, myocarditis, and gastritis. Porcine circovirus (PCV), as well as porcine parvovirus (PPV), was detected in tissues by virus reisolation, polymerase chain reaction analysis, or immunohistochemistry. All infected piglets developed moderate to high titers of antibody to PCV and moderate titers to PPV. No lesions, virus, or virus-specific antibodies were detected in sham-inoculated or uninoculated control piglets. These studies demonstrate that the lesions of PMWS can be experimentally reproduced in gnotobiotic piglets using filterable viral agents derived from pigs with PMWS and provide an experimental basis for further investigation into the pathogenesis and control of this emerging infectious disease in swine.

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