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

Abstract

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 x 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 microg of the cloned PCV2 plasmid DNA, and pigs in group 4 were each injected into the superficial iliac lymph nodes with 200 microg 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.

FIG. 1.

Construction of an infectious PCV2 molecular DNA clone. The relative positions of the primer pair used to amplify the complete PCV2 genome are indicated by the arrows (reverse primer, R-PCVSAC2; forward primer, F-PCVSAC2). The PCV2 genomic DNA amplified by PCR is digested with the SacII restriction enzyme and purified. The purified and SacII-digested genomic DNA was ligated to form concatemers. Ligated concatemers were separated by gel electrophoresis; the tandem genome dimmer of PCV2 was purified and cloned into the pSK vector, which is predigested with the SacII enzyme to produce a molecular PCV2 DNA clone.

FIG. 2.

The cloned PCV2 plasmid DNA is infectious when transfected in vitro in PK-15 cells. (A) Detection of PCV2 antigen by IFA in PK-15 cells transfected with the cloned PCV2 plasmid DNA. Intense immunolabeling of PCV2 antigen was visualized in the nucleus and to a lesser degree in the cytoplasm of the transfected cells. (B) Mock-transfected PK-15 cells.

FIG. 3.

(A) Lung from a pig inoculated by the intralymphoid route with PCV2 DNA and necropsied at 21 DPI. The lungs were rubbery, failed to collapse, and were mottled tan-red. Tracheobronchial lymph nodes were markedly enlarged and were tan (arrows). (B) Microscopic section of a normal lung from a control pig (magnification, ×21.25). (C) Microscopic section of the pig lung shown in Fig. 3A. Note the peribronchiolar lymphohistiocytic inflammation and mild necrotizing bronchiolitis (magnification, ×21.25). (D) Immunohistochemical staining of the lung shown in Fig. 3A. Note the PCV2 antigen in macrophages (arrows) and fibroblast-like cells (arrowhead) around airways (magnification, ×54.4).

FIG. 4.

(A) Normal lymph node from a control pig. Note the well-defined lymphoid follicles (arrows) (magnification, ×21.25). (B) Microscopic section of the tracheobronchial lymph node, from the pig whose lung is shown in Fig. 3A, inoculated 21 days previously by the intralymphoid route with cloned PCV2 genomic DNA. Lymphoid follicles are poorly defined; there is mild-to-moderate lymphoid depletion and mild, multifocal, granulomatous inflammation (magnification, ×21.25). (C) Same lymph node as shown in Fig. 4B. Note the poorly defined follicle with macrophages and giant cells (arrow) replacing follicular lymphocytes (magnification, ×54.4). (D) Same lymph node as shown in Fig. 4B. Immunohistochemical detection of PCV2 antigen in macrophages (arrow) and giant cells (small arrowhead) and dendrite-like cells (large arrowhead) in the follicles (magnification, ×54.4).