Synergistic Pathogenicity by Coinfection and Sequential Infection with NADC30-like PRRSV and PCV2 in Post-Weaned Pigs

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCVs) are two major viruses that affect pigs. Coinfections between PRRSV and PCV2 are frequently reported in most outbreaks, with clinical presentations involving dyspnea, fever, reduced feed intake, weight loss, and death in fattening pigs. The NADC30-like PRRSV and PCV2d are the main circulating virus strains found in China. This study determines the impact of NADC30-like PRRSV and PCV2d mono-infection and coinfection on the immune system, organ pathology, and viral shedding in five-week-old post-weaned pigs. Pigs were randomly divided into six groups: PBS, PRRSV, PCV2, PRRSV-PCV2 coinfection (co), and PRRSV-PCV2 or PCV2-PRRSV sequential infections. Fever, dyspnea, decreased feed intake, weight loss, and pig deaths occurred in groups infected with PRRSV, Co-PRRSV-PCV2, and PRRSV-PCV2. The viral load was higher in Co-PRRSV-PCV2, PRRSV-PCV2, and PCV2-PRRSV than those mono-infected with PRRSV or PCV2. Additionally, cytokines (IFN-γ, TNF-α, IL-4, and IL-10) produced by pigs under Co-PRRSV-PCV2 and PRRSV-PCV2 groups were more intense than the other groups. Necropsy findings showed hemorrhage, emphysema, and pulmonary adhesions in the lungs of pigs infected with PRRSV. Smaller alveoli and widened lung interstitium were found in the Co-PRRSV-PCV2 and PRRSV-PCV2 groups. In conclusion, PRRSV and PCV2 coinfection and sequential infection significantly increased viral pathogenicity and cytokine responses, resulting in severe clinical signs, lung pathology, and death.

Keywords: NADC30-like PRRSV; PCV2; coinfection; pathogenicity; sequential infection.

Figure 1

Evaluation of clinical symptoms in pigs after challenged. (A) Rectal temperatures of pigs were shown, and the temperature for fever in pigs was set at 39.5 °C; (B) clinical symptom scores were recorded for 21 days. Clinical symptoms were assessed, with higher scores indicating more severe clinical symptoms, and 0 representing no obvious clinical symptoms. (C) Mortality of pigs after challenge; (D) the average daily weight gain of the infected pigs. The following p values were indicated: * indicates p < 0.05.

Figure 2

Specific antibody of PCV2 and PRRSV were monitored. (A) Trends level of PCV2-specific antibody, S/P < 0.4 were considered negative, and S/P ≥ 0.4 were considered positive; (B) the trend of PRRSV-specific antibodies changes with the time of PRRSV infection in pigs, KQ < 40 were judged to be negative, and KQ ≥ 40 were judged to be positive.

Figure 3

Viremia detection and viral detection in tissues of PRRSV and PCV2. (A) Trends in viremia with PRRSV, with peak viral loads 3–7 days post challenge; (B) the viral load of PRRSV in lung and lymph node was measured; (C) time-course of viremia with PCV2, high viral load at 7–14 dpi; (D) the viral load of PCV2 in lung and lymph node. The following p values were indicated: * indicates p < 0.05, ** indicates p < 0.01.

Figure 4

Cytokine detection in serum of each experimentally infected group. (A) TNF-α detection in serum, the cytokine of TNF-α was significantly TNF-α cytokine levels were significantly higher in pigs infected with PRRSV; (B) IFN-γ detection in serum, cytokines were significantly elevated 7–14 days after infection with PRRSV, and high levels of cytokines persisted for 21 days after PCV2-PRRSV infection with PRRSV; (C) IL-4 detection in serum, cytokine levels were significantly higher in the Co-PRRSV-PCV2 group than in the other groups on 7–21 dpi; (D) IL-10 detection in serum, cytokines were significantly elevated 7–14 days after infection with PRRSV. The following p values were indicated: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

Figure 5

Representative histopathological sections of lung from each infected group (200×). (A) Healthy control group (PBS group); (B) PCV2 infected group, low-grade interstitial pneumonia (black arrow); (C) PCV2-PRRSV infected group, the alveoli were smaller and the lung interstitium was thickened (black arrow); (D) Co-PRRSV-PCV2 infected group, hemorrhagic spots were present in the lung, severe interstitial pneumonia (black arrow) with effacement of alveoli; (E) PRRSV-PCV2 infected group, hemorrhagic spots, severe interstitial pneumonia (black arrow) with effacement of alveoli were present in the lung; (F) PRRSV infected group, interstitial pneumonia (black arrow), partial alveolar effacement.