Distribution of PRRSV and PCV2 in different organs
At 7 dpi of PRRSV and PCV2, tissues of inguinal lymph nodes, spleen, liver, heart, lungs, kidneys and thymus were collected to determine the distribution of PRRSV and PCV2 by PCR to amplify a 196 bp fragment of PRRSV N gene and 148 bp fragment of PCV2 CAP. Both PRRSV N gene (Fig. 1A) and PCV2 CAP gene (Fig. 1B) were detected in all tissues collected above. As shown in Table 1, only one in five mice was positive for PRRSV in inguinal lymph node while PRRSV was detected in all other tissues collected from 5 mice. For PCV2 CAP gene, at least three mice were positive in the tissues collected from 6 mice. According to the PCR results, both PRRSV and PCV2 transmitted to most of the collected mice tissues.
Table 1
The tissue distribution of PRRSV and PCV2 in co-infected mice by PCR
| #of positive mice | #of positive tissues |
| Inguinal lymph nodes | Lung | Liver | Spleen | Kidney | Heart | Thymus |
PRRSV | 5/5 | 1/5 | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 |
PCV2 | 6/6 | 5/6 | 5/6 | 6/6 | 5/6 | 3/6 | 6/6 | 5/6 |
PRRSV infection enhanced PCV2 replication and induced more severe lung lesions
At 7 dpi, PCV2 CAP gene copy numbers were the highest in liver, followed by thymus and spleen as shown in Fig. 1C. These results were similar both in PRRSV/PCV2 co-infected and PCV2 infected groups, suggesting that PCV2 has tissue tropism in mouse model. Furthermore, the results showed that PCV2 CAP gene copies in liver (p < 0.05), thymus (p > 0.05) and spleen (p < 0.01) of PRRSV/PCV2 co-infected mice were significantly higher than PCV2 infected mice, indicating PRRSV infection enhanced PCV2 infection in mouse model. In addition, compared with normal control group, obvious lung lesions were observed in both PRRSV/PCV2 co-infected and PCV2 infected mice (Fig. 2A). Particularly, the hemorrhagic area was larger in PRRSV/PCV2 co-infected mice than PCV2 infected mice. H&E staining of lungs (Fig. 2B) revealed that both PRRSV/PCV2 co-infected and PCV2 infected mice displayed typical characteristics of interstitial pneumonia including thickened alveoli septa and decreased alveolar area. Taken all these histopathological observations, the lesions of lungs were more severe in PRRSV/PCV2 co-infected mice, suggesting that PRRSV infection enhanced PCV2 induced lungs lesions.
Matrine restrained PCV2 replication in liver and alleviated lungs lesions induced by virus infection
PCV2 CAP gene copy numbers in liver were measured by qPCR as shown in Fig. 3A. At 10 dpi, compared with virus control group, the CAP gene copies were significantly decreased in Matrine 40 mg/kg treatment group (p < 0.01). The CAP gene in Ribavirin treatment group was lower but no significant difference was observed (p > 0.05). At 13 dpi, CAP gene copies in Ribavirin (p < 0.01), Matrine 40 mg/kg (p < 0.05) and 20 mg/kg (p < 0.05) treatment groups were significantly lowered than that in virus control. Compared with virus control at 16 and 19 dpi, CAP gene copies were decreased in drug treatment groups but without statistical difference (p > 0.05). Compared with normal control, the lungs of PRRSV/PCV2 co-infected mice displayed typical characteristics of interstitial pneumonia including thickened alveoli septa and decreased alveolar area. However, both Ribavirin and Matrine treatments (especially 40 and 20 mg/kg) could significantly alleviated these pathological changes as shown in Fig. 3B.
Measurement of body weight gain, spleen and thymus indices in different treatments
Compared with normal control, the body weight gain in PRRSV/PCV2 group was significantly declined at 19 dpi (p < 0.01), while increased with varying degrees in Ribavirin and Matrine treatments, especially Matrine 10 mg/kg treatment group (p < 0.001) as shown in Fig. 4A. Moreover, the body weight gain in Matrine 10 mg/kg was higher at each indicated time point: 10 (p > 0.05), 13 (p < 0.01) and 16 dpi (p < 0.05), suggesting that Matrine has an effect of gaining body weight with the dose of 10 mg/kg. The spleen index in PRRSV/PCV2 group was much higher than that in normal control at 10 (p < 0.01), 13 (p < 0.05), 16 (p = 0.067) and 19 (p < 0.05) dpi. However, at 13 dpi, Matrine with the dose of 40 (p < 0.05) and 10 (p < 0.05) mg/kg significantly decreased the spleen index induced by virus infection (Fig. 4B). As shown in Fig. 4C, compared with normal control, the thymus index in PRRSV/PCV2 group was significantly higher both at 10 (p < 0.01) and 13 (p < 0.01) dpi and there were no remarkable changes at 16 and 19 dpi. Meanwhile, both Ribavirin and Martine declined the increasing trend with varying degrees.
Matrine Regulate Peritoneal Macrophages Phagocytosis
Phagocytic ability of peritoneal macrophages was evaluated using MFI at the percentage of FITC-positive cells gated on F4/80+/CD11b+ cells. As shown in Fig. 5A and B, at 10 dpi, the MFI and the ratio of F4/80+/CD11b+ in PRRSV/PCV2 group was significantly higher than that of normal control (p < 0.01). These results indicated that virus infection promoted an increase in macrophages and enhanced their phagocytic ability. Compared with PRRSV/PCV2 group, the percentage of F4/80+/CD11b+ in Ribavirin and Matrine treatment groups were not changed significantly, while the MFI in these groups were remarkable decreased (p < 0.01). In addition, at 13 and 16 dpi, the MFI in PRRSV/PCV2 group was much lowered than that in normal control which means that there were no remarkable changes in macrophages, indicating that persistent infection of virus has leaded to the decrease of the phagocytic ability of macrophages. However, Matrine with the dose of 40 and 20 mg/kg has promoted the phagocytic ability of macrophages compared with PRRSV/PCV2 group.
Effects Of Matrine On The Proliferation Of Splenic Lymphocytes
At 10 dpi, the proliferation index of splenic lymphocytes with and without mitogen stimulus in PRRSV/PCV2 group was remarkably higher compared with the normal control, while it decreased in Ribavirin both with and without LPS stimulus compared with PRRSV/PCV2 group (p < 0.05). Moreover, the proliferation indices in Matrine 40 and 10 mg/kg treatment groups stimulated with both ConA and LPS were much lowered than those in PRRSV/PCV2 group as shown in Fig. 6. In addition, at 16 dpi, Matrine with a dose of 40 mg/kg enhanced the proliferation index with and without mitogen stimulus compared with PRRSV/PCV2 group. The proliferation index showed no-significant changes among all the groups at 19 dpi.