Pathogenesis Of Porcine Circovirus Type 2 And Porcine Parvovirus Coinfection In Vitro

Porcine circovirus type 2 is the major causative agent of porcine circovirus associated disease, which is an important immunosuppressive diseases, has been caused serious economic losses of the pig industry all over the world. Porcine circovirus type 2 infection alone does not cause the typical clinical symptoms, when coinfect with other pathogens can promote diseases. Porcine circovirus type 2 and porcine parvovirus have the same target cells, and the coinfection pathogenesis remains unclear. Coinfection of the porcine circovirus type 2 and porcine parvovirus is a typical model for Postweaning Multisystemic Wasting Syndrome. This study by detecting the cytokine levels of different cells in porcine circovirus type 2 and porcine parvovirus coinfection models in vitro, and provide a preliminary study on its mechanism of coinfections.1. The establishment of real-time PCR detection method for PCV2, PPV, TNF-a, IFN-γ, IL-10 and p-actinIn this study, the primers of PCV2, PPV, porcine IFN-γ, TNF-a, IL-10 and β-actin were designed according to the gene sequences published in GenBank. The corresponding target gene fragment from porcine alveolar macrophages was amplificate by RT-PCR, and then connected with the pMD18-T vector, verify the correctness of the gene sequence by sequencing. The primers of PCV2, PPV, porcine IFN-y, TNF-a, IL-10 and p-actin were designed for real-time PCR. The recombinant plasmid was used as a template above, The reaction system and the primer concentration was optimizate of real-time PCR, the methed to detect PCV2, PPV, porcine IFN-y, TNF-a, IL-10 and β-actin gene was established, and the methodology was evaluated.The result show that, the parameters of the standard curve were meet the requirements by optimize real-time PCR conditions. The coefficient of determination R2 of the standard curve were greater than 0.98. The amplification reaction were very good when the template concentration between 10 copies/μL and 108copies/μL, and have good repeatability, stability and specificity.2. The impact of PCV2 and PPV coinfection on primary PAMIn this study, it was detect the viral nucleic acids copy number of PCV2 and PPV, the mRNA expression of IFN-y, TNF-a and IL-10 at different time after PCV2 and PPV coinfection, and the effects on the immune response of cells by PCV2 and PPV coinfection was analyzed. The PAM is divided into four groups:PPV infection alone, PCV2 infection alone, PCV2 and PPV coinfection and control. At 12 hpi,24 hpi,36 hpi,48 hpi,60 hpi and 72 hpi the viral DNA and total RNA of the PAM was extracted. The viral nucleic acids copy number and IFN-y, TNF-a and IL-10 mRNA expression dynamic changes were detected by real-time PCR.The results showed that, after PCV2 and PPVcoinfections, PCV2 and PPV group PCV2 viral nucleic acid copy number were higher than PCV2 group, and in PCV2 and PPV group, the PPV virus nucleic acid copy number was significantly higher than PPV group after 60 hpi; The expression levels of IFN-y mRNA were raised in PCV2 group and PPV group, the expression levels of IFN-y mRNA were downregulated in PCV2 and PPV group; The viral infection can cause the expression levels of TNF-a mRNA raised, before 36 hpi, PCV2 infection plays a major role, and PPV infection plays a major role after 48 hpi, the expression levels of TNF-a mRNA in PCV2 and PPV group were significantly higher than single infected group. At 24 hpi and 36 hpi, the PCV2 and PPV infection alone can cause the expression levels of IL-10 mRNA raised, PCV2 and PPV coinfection cause the expression levels of IL-10 mRNA significantly upregulated, the other times were not significant.3. The impact of PCV2 and PPV coinfection on 3D4/21In this study, it was detect the viral nucleic acids copy number of PCV2 and PPV, the mRNA expression of IFN-y, TNF-a and IL-10 at different time after PCV2 and PPV coinfection, and the effects on the immune response of cells by PCV2 and PPV coinfection was analyzed. The 3D4/21 is divided into four groups:PPV infection alone, PCV2 infection alone, PCV2 and PPV coinfection and control. At 12 hpi,24 hpi,36 hpi,48 hpi,60 hpi and 72 hpi the viral DNA and total RNA of the 3D4/21 was extracted.The viral nucleic acids copy number and IFN-y, TNF-a and IL-10 mRNA expression dynamic changes were detected by real-time PCR.The results showed that, the PCV2 viral nucleic acid copy numbers at 36 hpi and the PPV viral nucleic acid copy numbers at 24 hpi in PCV2 and PPV group were higher than singly infected group. The expression levels of IFN-y mRNA in PPV group were higher than the expression levels of IFN-y mRNA in PCV2 group, the expression levels of IFN-y mRNA in PCV2 and PPV group were higher than PCV2 group at 12 hpi, and then after 24 hpi, were lower than control group and singly infectioned group. The expression levels of TNF-a mRNA in PPVgroup were raised, the expression levels of TNF-a mRNA in PCV2 group were raised, and were higher than PPVgroup after 48 hpi, the expression levels of TNF-a mRNA in PCV2 and PPV group were raised after 36 hpi, and were significantly higher than singly infectioned group. The expression levels of IL-10 mRNA were slight raised at 24 hpi and after 48 hpi in PCV2 group, PPV infection has no effect on the expression levels of IL-10 mRNA, the expression levels of IL-10 mRNA in PCV2 and PPV group were significantly upregulated after 24 hpi, and peaked at 48 hpi.In summary, when PCV2 and PPV coinfect, the replication of two kinds viral were promote and the expression of IFN-y mRNA were inhibit, the expression of TNF-a and IL-10 mRNA were promote, and thus the coinfction may cause immune dysfunction, promote the disease occurrence.