Matrine Inhibited Co-infection Of PRRSV/PCV2 And Its Mechanism

Co-infection with multi-pathogens is one of the epidemic characteristic of the current global swine diseases, which yield in huge economic losses worldwide. Among them, the PRRSV and PCV2 co-infection is the most commonly observed clinically. Although vaccination is commonly used in preventing swine viral diseases, PRRSV and PCV2 are both immunosuppressive viruses which often lead to failure of the vaccination. Our group has previously established the virus infected cell models to screen natural compounds possessing antiviral activity in vitro and the results demonstrated that Matrine inhibited PRRSV replication in Marc-145 cells and interfered with PCV2 infection in PK-15 cells. In this study, we first further demonstrated that Matrtine inhibited N protein to disturb PRRSV infection. And then the PRRSV/PCV2 co-infected PAM model and KM mouse model were established to evaluate the antiviral activities of Matrine against PRRSV/PCV2 co-infection and its mechanism.The results of PRRSV/PCV2 co-infected PAM cell model demonstrated that:1. The expressions of PRRSV N gene and PCV2 CAP gene were determined by qPCR assay from PRRSV infection alone group or PCV2 infection alone group and PRRSV/PCV2 co-infection group. Results showed that the variation trend of PRRSV replication in PAM cells was increased firstly and then declined. At 12 h post infection, the PRRSV N gene copies reached the highest. The N gene copies of PRRSV alone infected PAM cells were 7.82×104 copies/?l and in PRRSV/PCV2 co-infected PAM cells were 1.11×105 copies/?l. However, the PCV2 CAP gene copies were gradually increased with the longer infection time, and at 72 h post infection, it became to stabilized. The CAP gene copies in PCV2 alone infected PAM cells were 3.78×107 copies/?l and in PRRSV/PCV2 co-infected PAM cells were 6.10×107 copies/?l. Comparing with PRRSV or PCV2 alone infected PAM cells, the co-infected PAM cells inoculated with PRRSV first then with PCV2, significantly enhanced both PRRSV and PCV2 replication (p<0.05).2. The cytopathologic effect (CPE) observation and MTT method were used to determine the cytotoxicity of Matrine on PAM cells. The results showed that the maximal non-cytotoxic concentration of Matrine was 0.4 mg/ml with the concentrations employed (0.0625 mg/ml-4 mg/ml), and when the concentration was higher than 0.4 mg/ml, PAM cells displayed various degree vacuolization.3. The PRRSV N gene/protein and PCV2 CAP gene expression only were determined by qPCR and Western blot. Results showed that Matrine disturbed the expression of PRRSV N gene/protein and PCV2 CAP gene to inhibit PRRSV/PCV2 co-infection. The N gene copies in PRRSV/PCV2 co-infected PAM cells were 1.37×106 copies/?l while in 0.4 mg/ml Matrine treatment group it was decreased to 2.44×105 copies/?l. Moreover, The CAP gene copies in PRRSV/PCV2 co-infected PAM cells were 2.12×105 copies/?l while in 0.4 mg/ml Matrine treatment group it was decreased to 1.12×105 copies/?l. In addition, PRRSV/PCV2 co-infection activated NF-?B pathway, and increased TLR3, TLR4 and TNF-a expression at 12 h in PAM cells (p<0.05). The antiviral mechanism of Matrine may be mediated by inhibiting virus induced TLRs/NF-KB/TNF-a pathway activation.The study using PRRSV/PCV2 co-infected KM mouse model demonstrated that:4. The KM mice were inoculated with PRRSV first and then with PCV2 to establish PRRSV/PCV2 co-infection model. The PCR analysis demonstrated that PRRSV N gene and PCV2 CAP gene were detected in multiple tissues including heart, liver, spleen, lung, kidney, thymus and inguinal lymph nodes. Moreover, the viral load of PCV2 was the highest in liver (5.66×103 copies/?l), followed by thymus (2.21×103 copies/?l) and spleen (1.17×103 copies/?l). Although PRRSV were detected in most of tissues, it was not proliferated so significantly with qPCR analysis. Therefore, in the subsequent experiments we only monitored the viral load of PCV2. Still, comparing with PCV2 infection alone, PRRSV infection significantly enhanced PCV2 replication (p<0.05), and exacerbated PCV2 induced interstitial pneumonia.5. The safe dose of Matrine in mice was determined by intraperitoneal injection as 40 mg/kg. After 7 days of post-infection (dpi), mice were intraperitoneally injected with Matrine for 5 consecutive days. The qPCR analysis demonstrated that Matrine of 40 mg/ml could significantly attenuate PCV2 replication in liver (p<0.05) and alleviate virus induced interstitial pneumonia, suggesting that Matrine could directly inhibit virus replication to suppress the virus infection.6. The mouse body weight gain was observed by administration of Matrine at 10 mg/kg at 10,13 and 16 dpi (p<0.05). In addition, Matrine treatment enhanced the peritoneal macrophage phagocytosis at 13 and 16 dpi (p<0.05). The proliferation activity of lymphocytes was increased when using Matrine at 40 mg/kg (p<0.05). Hence, the antiviral activity of Martine may function through enhancing immune response to against viruses infection.In conclusion, our study demonstrated that Matrine is of antiviral activities against PRRSV/PCV2 co-infection and explored the potential antiviral mechanisms of Matrine against PRRSV/PCV2 co-infection. These data provided new insight on controlling PRRSV and PCV2 infection and supported to develop the Matrine as a new possible veterinary medicine.