Porcine circovirus type 3 (PCV3) is a newly discovered porcine circovirus identified in 2015. It has been considered associated with clinical signs resembling porcine dermatitis and nephropathy syndrome (PDNS), reproductive disorders, cardiac pathology, and multisystemic inflammation. However, the infection biology and pathogenic mechanisms of PCV3 remain unclear. In this study, a viral strain was rescued using an infectious PCV3 DNA clone and administered intranasally to 4-week-old and 8-week-old SPF piglets to systematically assess the pathogenicity of PCV3. The results provide the first evidence that PCV3 infection alone can successfully replicate PDNS-like disease: 4-week-old piglets may die, while 8-week-old piglets develop similar lesions but do not die. This provides direct experimental evidence that PCV3 is a causative agent of PDNS-like disease.
I. Research Design and Methods
The PCV3 genome is 2.0 kb of single-stranded circular DNA, with ORF1 (rep), ORF2 (cap), and ORF3 as its main features; the cap protein shares only approximately 36%–37% amino acid homology with PCV2. The study synthesized the full-length genome based on the PCV3/CN/Hebei-LY/2015 sequence, cloned it into the pSK vector, transfected PK15 cells to rescue the virus, and passaged it 15 times, reaching a titer of 10^6.53 TCID50/ml. The animal experiments were divided into two parts:
Approximately 2 ml of PCV3 was administered intranasally (10^6.53 TCID50/ml); the KLH group received 2.0 mg of KLH emulsified in incomplete Freund’s adjuvant at four sites on day 4 after inoculation. Body temperature and clinical symptoms were recorded daily during the study; animals were weighed and blood was collected weekly to assess serum viral load, PCV3 capsid antibodies, PBMC proliferation, and cytokines. Necropsy was performed at 28 dpi, followed by histopathology and immunohistochemistry.
II. Main results
1. Clinical symptoms and mortality
After vaccination of 4-week-old piglets with PCV3, symptoms began to appear at 8 dpi and were present in all by 16 dpi. Symptoms included fever, anorexia, coughing, sneezing, and diarrhea, followed by increased respiratory rate, lethargy, redness of the skin and ears, multifocal papules, trembling and/or convulsions. There were 2/5 deaths in each of the PCV3-only and PCV3+KLH groups, with a mortality rate of 40% in both groups; the times of death were 16 and 18 dpi, and 19 and 25 dpi, respectively. 8-week-old piglets also showed PDNS-like symptoms and lesions, but there were no deaths during the 28-day observation period. The control group showed no abnormalities.
2. Viral load and antibodies
At 4 weeks of age, the serum viral load in the PCV3 group was 2.23×10^5～7.72×10^8 copies/ml, and that in the PCV3+KLH group was 3.52×10^5～8.31×10^8 copies/ml. Both groups peaked at 21 dpi, with no significant difference between groups. At 8 weeks of age, the PCV3 group reached its highest level at 21 dpi, averaging approximately 7.92×10^7 copies/ml. Inoculated pigs developed PCV3 capsid antibodies in the late stage of infection; the appearance of antibodies was associated with a decrease in viral load. The control group remained negative throughout. The proliferative response of PBMCs to PHA was significantly lower than that of the control group, suggesting that PCV3 can reduce the proliferative capacity of peripheral blood lymphocytes.
3. Pathological changes and antigen distribution
At necropsy, the lungs were rubbery and failed to collapse, with diffuse mottled reddish-brown discoloration and severe multifocal dark purplish-red consolidation; the lymph nodes were enlarged by 2–3 times and had brownish-red hemorrhage; the kidneys were edematous with pinpoint hemorrhages; the liver showed congestion, grayish-white nodules, and necrosis; the spleen was enlarged with marginal necrosis. Histologically, findings were characterized by lymphocyte necrosis and depletion, epithelioid cell proliferation, eosinophil infiltration, bronchopneumonia, renal tubular dilation, and interstitial lymphocyte/macrophage infiltration. Immunohistochemistry showed PCV3 antigen-positive cells in the lungs, heart, kidneys, lymph nodes, spleen, liver, and small intestine; the distribution was similar in pigs inoculated at 4 weeks and 8 weeks of age.
4. Cytokines and chemokines
After inoculation, pro-inflammatory cytokines and chemokines in pig serum were significantly upregulated: IFN-γ and IL-12α peaked on day 7, IL-1β peaked on day 14, and TNF-α, IL-6 and CCL5 peaked on day 21; IL-23α and IL-10 remained elevated. The high levels of inflammatory factors largely coincided with the peak viral load and the onset of clinical symptoms, suggesting that the inflammatory response contributes to PCV3 pathogenesis.
Three. Discussion and Significance
PCV2 infection alone usually cannot reproduce PDNS; immune stimulation or co-infection with PPV, PRRSV, or other viruses is needed. However, in this study, PCV3 infection alone induced PDNS-like disease, and immune stimulation with KLH did not significantly aggravate the outcome. This suggests that PCV3 may be more pathogenic to piglets than PCV2, as infection alone was sufficient to cause typical clinical and pathological changes. Piglets aged 4 weeks were more likely to die than those aged 8 weeks, suggesting that age and immune maturity affect disease severity. PCV3 was widely distributed in the lungs, heart, kidneys, lymph nodes, spleen, liver, and small intestine, and caused lymphocyte necrosis and depletion, which may lead to immunosuppression and increase the risk of secondary infections. Eosinophil infiltration and persistently elevated pro-inflammatory cytokines may be important mechanisms underlying skin rashes, nephritis, and multisystem inflammation.
IV. Conclusion
This study was the first to successfully reproduce PCV3-induced PDNS-like disease through experimental infection, demonstrating that PCV3 infection alone can cause dermatitis, nephropathy, multisystem inflammation, and death in piglets. It also revealed characteristics of viral load, antibody dynamics, tissue antigen distribution, and cytokine changes.