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 ؋ 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...

Reproduction of PMWS in immunostimulated SPF piglets transfected with infectious cloned genomic DNA of type 2 porcine circovirus

“…As a result, the authors confirmed that although PCV2 was the agent associated with PMWS, cofactors are crucial for the development of severe clinical PMWS. The infectiousness of another PCV2 DNA clone was checked by intramuscular and intraperitoneal injections [32] with similar results [14]. Since no clinical PMWS was reproduced with this pure PCV2 inoculum, the purpose of our work was to evaluate the impact of immunostimulation on the PMWS severity level showed by six-week-old specific-pathogen-free piglets after injection of pure tandem-cloned PCV2 DNA by the intramuscular route.…”

“…AF201311) was cloned in tandem in pBluescript KS+ (Stratagene, La Jolla, CA, USA) at the SacI site as described by Fenaux et al [14]. DNA was produced at a concentration of 0.8 mg/mL without lipopolysaccharides (0.046 endotoxin unit/µg) by PlasmidFactory (Bielefeld, Germany).…”

“…With this aim, Fenaux et al [14,15] investigated the infection ability of a PCV2 molecular DNA clone when injected directly into the liver and inguinal lymph nodes. Only mild pathological lesions were observed in four-weekold SPF piglets despite widespread distribution of the virus in their organs.…”

Features of Porcine Circovirus-2 Disease: Correlations between Lesions, Amount and Distribution of Virus, and Clinical Outcome

“…Importantly, both groups of swine expressed the full spectrum of host-virus interactions (asymptomatic infection to overt PMWS), gross lesions, and characteristic histologic lesions now attributable to PCV-2 infection and identified in commercial swine production operations. 1,7,13,31,32,34,35,37 All experimental pigs were euthanized when moribund or at 4-5 weeks of age regardless of disease status. Disease status, for the swine still alive at the end of the experiment was determined ultimately by a combination of clinical presentation, gross lesions, and tissue virus burden as determined by IHC and quantitative virus recovery.…”

The ORF3 protein of porcine circovirus type 2 promotes secretion of IL-6 and IL-8 in porcine epithelial cells by facilitating proteasomal degradation of regulator of G protein signalling 16 through physical interaction

“…FR823451) was isolated from a pig spleen and propagated in PK15 cells. Tandem dimers of the PCV2 genome and PCV2 mutant were constructed and used to infect PK15 cells (Fenaux et al, 2002;Liu et al, 2005). Primer sequences used for the PCR are described in Table S1 (available in the online Supplementary Material).…”