Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post‐weaning multisystemic wasting syndrome in the Netherlands

205

201

Infection of animals with a molecular viral clone is critical to study the genetic determinants of viral replication and virulence in the host. Type 2 porcine circovirus (PCV2) has been incriminated as the cause of postweaning multisystemic wasting syndrome (PMWS), an emerging disease in pigs. We report here for the first time the construction and use of an infectious molecular DNA clone of PCV2 to characterize the disease and pathologic lesions associated with PCV2 infection by direct in vivo transfection of pigs with the molecular clone. The PCV2 molecular clone was generated by ligating two copies of the complete PCV2 genome in tandem into the pBluescript SK (pSK) vector and was shown to be infectious in vitro when transfected into PK-15 cells. Forty specific-pathogen-free pigs at 4 weeks of age were randomly assigned to four groups of 10 each. Group 1 pigs served as uninoculated controls. Pigs in group 2 were each inoculated intranasally with about 1.9 ؋ 10 5 50% tissue culture infective doses of a homogeneous PCV2 live virus stock derived from the molecular clone. Pigs in group 3 were each injected intrahepatically with 200 g of the cloned PCV2 plasmid DNA, and pigs in group 4 were each injected into the superficial iliac lymph nodes with 200 g of the cloned PCV2 plasmid DNA. Animals injected with the cloned PCV2 plasmid DNA developed infection resembling that induced by intranasal inoculation with PCV2 live virus stock. Seroconversion to PCV2-specific antibody was detected in the majority of pigs from the three inoculated groups at 35 days postinoculation (DPI). Viremia, beginning at 14 DPI and lasting 2 to 4 weeks, was detected in the majority of the pigs from all three inoculated groups. There were no remarkable clinical signs of PMWS in control or any of the inoculated pigs. Gross lesions in pigs of the three inoculated groups were similar and were characterized by systemically enlarged, tan lymph nodes and lungs that failed to collapse. Histopathological lesions and PCV2-specific antigen were detected in numerous tissues and organs, including brain, lung, heart, kidney, tonsil, lymph nodes, spleen, ileum, and liver of infected pigs. This study more definitively characterizes the clinical course and pathologic lesions exclusively attributable to PCV2 infection. The data from this study indicate that the cloned PCV2 genomic DNA may replace infectious virus for future PCV2 pathogenesis and immunization studies. The data also suggest that PCV2, although essential for development of PMWS, may require other factors or agents to induce the full spectrum of clinical signs and lesions associated with advanced cases of PMWS.Porcine circovirus (PCV) was originally isolated as a cell culture contaminant of a porcine kidney cell line (PK-15) (56,60). PCV is a small, nonenveloped virus that contains a singlestranded circular DNA genome of about 1.76 kb. PCV is classified in the family of Circoviridae, which consists of three other animal circoviruses (chicken anemia virus [CAV], psittacine beak and feather disease v...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Cloned Genomic DNA of Type 2 Porcine Circovirus Is Infectious When Injected Directly into the Liver and Lymph Nodes of Pigs: Characterization of Clinical Disease, Virus Distribution, and Pathologic Lesions

Fenaux

Halbur

Haqshenas

et al. 2002

205

201

“…PCV2 replicates in the lymph nodes, lungs, and livers of infected pigs, followed by the impairment of the immune system by degradation of the lymphoid structures (1,2,15,21,23,48), as well as changes in the proportions of lymphocyte subsets of peripheral blood (11). In the present study, we showed that the depletion of CD4 ϩ and CD8 ϩ T cells by the wild-type virus might contribute to higher viral load of the wild-type virus compared to the mutant virus.…”

Section: Discussionmentioning

confidence: 99%

The ORF3 Protein of Porcine Circovirus Type 2 Is Involved in Viral Pathogenesis In Vivo

Liu

Chen

et al. 2006

140

108

Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome. We previously showed that a novel identified protein, ORF3, was not essential for PCV2 replication in cultured PK15 cells and played a major role in virus-induced apoptosis. To evaluate the role of the ORF3 protein in viral pathogenesis in vivo, we inoculated 8-week-old BALB/c mice that have been developed for PCV2 replication with ORF3-deficient mutant PCV2 (mPCV2). By 42 days postinoculation, all of the mice inoculated with the mPCV2, as well as with wild-type PCV2 (wPCV2), had seroconverted to PCV2 capsid antibody, and the mutant induced levels of PCV2 antibodies that were higher than those of the wPCV2. The PCV2 genomic copy numbers in serum were significantly higher (P < 0.05) in the wPCV2-inoculated mice than in mice inoculated with the mPCV2. Also, the wPCV2 caused microscopic lesions characterized by lymphocyte depletion with histiocytic infiltration of lymphoid organs, but the mutant virus failed to induce any obvious pathological lesions. In situ hybridization and immunohistochemical analyses also showed that larger amounts of viral DNA and antigens were detected in the lymph nodes of the wPCV2-inoculated than mPCV2-inoculated mice. Furthermore, animals of the wPCV2-inoculated group showed significant downshifts of CD8 ؉ T-cell subsets of peripheral blood lymphocytes compared to the control mice (P < 0.05) at various time points postinoculation. Also, the proportions of the CD4 ؉ and CD4 ؉ CD8 ؉ cells were significantly reduced in wPCV2-inoculated mice at some time points postinoculation. In contrast, there are some reductions in the proportions of these subsets in the mutant virus-inoculated mice, but the proportions do not decrease significantly. Taken together, these results demonstrate that the ORF3 protein is also dispensable for viral replication in vivo and that it plays an important role in viral pathogenesis.

“…It is known that PCV2 replicates in the lymph nodes, lungs, and liver and that one of the major pathogenic effects is the impairment of the immune system by degradation of the lymphoid structures (10,11,20,27,33,68). Gross lesions affecting the lymph nodes in both the nonpathogenic PCV1-and the chimeric PCV1-2-inoculated pigs were mild and limited to only a few pigs, whereas the pathogenic PCV2-inoculated pigs all had moderate to severe enlargement and discoloration of lymph nodes (Table 4).…”

Section: Discussionmentioning

confidence: 99%

Immunogenicity and Pathogenicity of Chimeric Infectious DNA Clones of Pathogenic Porcine Circovirus Type 2 (PCV2) and Nonpathogenic PCV1 in Weanling Pigs

Fenaux

Opriessnig

Halbur

et al. 2003

138

120

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. We report here the construction and characterization of two chimeric infectious DNA clones of PCV1 and PCV2. The chimeric PCV1-2 clone contains the PCV2 capsid gene cloned in the backbone of the nonpathogenic PCV1 genome. A reciprocal chimeric PCV2-1 DNA clone was also constructed by replacing the PCV2 capsid gene with that of PCV1 in the backbone of the PCV2 genome. The PCV1, PCV2, and chimeric PCV1-2 and PCV2-1 DNA clones were all shown to be infectious in PK-15 cells, and their growth characteristics in vitro were determined and compared. To evaluate the immunogenicity and pathogenicity of the chimeric infectious DNA clones, 40 specific-pathogen-free (SPF) pigs were randomly assigned into five groups of eight pigs each. Group 1 pigs received phosphate-buffered saline as the negative control. Group 2 pigs were each injected in the superficial inguinal lymph nodes with 200 g of the PCV1 infectious DNA clone. Group 3 pigs were each similarly injected with 200 g of the PCV2 infectious DNA clone, group 4 pigs were each injected with 200 g of the chimeric PCV1-2 infectious DNA clone, and group 5 pigs were each injected with 200 g of the reciprocal chimeric PCV2-1 infectious DNA clone. As expected, seroconversion to antibodies to the PCV2 capsid antigen was detected in group 3 and group 4 pigs. Group 2 and 5 pigs all seroconverted to PCV1 antibody. Gross and microscopic lesions in various tissues of animals inoculated with the PCV2 infectious DNA clone were significantly more severe than those found in pigs inoculated with PCV1, chimeric PCV1-2, and reciprocal chimeric PCV2-1 infectious DNA clones. These data indicated that the chimeric PCV1-2 virus with the immunogenic ORF2 capsid gene of pathogenic PCV2 cloned into the nonpathogenic PCV1 genomic backbone induces a specific antibody response to the pathogenic PCV2 capsid antigen but is attenuated in pigs. Future studies are warranted to evaluate the usefulness of the chimeric PCV1-2 infectious DNA clone as a genetically engineered live-attenuated vaccine against PCV2 infection and PMWS.