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.
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.
Fenaux
M
M
Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0342, USA.
Halbur
P G
PG
Haqshenas
G
G
Royer
R
R
Thomas
P
P
Nawagitgul
P
P
Gill
M
M
Toth
T E
TE
Meng
X J
XJ
D000281:Administration, Intranasal; D000818:Animals; D000956:Antigens, Viral / Q000032:analysis; D002460:Cell Line; D018173:Circoviridae Infections / Q000473:pathology* / Q000662:veterinary / Q000821:virology*; D017925:Circovirus / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity / Q000502:physiology*; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000502:physiology; D016679:Genome, Viral; D007150:Immunohistochemistry; D008099:Liver / Q000821:virology*; D008168:Lung / Q000473:pathology / Q000821:virology; D008198:Lymph Nodes / Q000473:pathology / Q000821:virology*; D009336:Necrosis; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D013552:Swine / Q000821:virology*; D013553:Swine Diseases / Q000473:pathology / Q000821:virology; D014162:Transfection; D014766:Viremia / Q000473:pathology / Q000821:virology
D000956:Antigens, Viral; D004274:DNA, Recombinant; D004279:DNA, Viral