Molecular Mechanism Of The Effect Of Porcine Reproductive And Respiratory Syndrome Virus On Innate Immunity Of Porcine Pulmonary Alveolar Macrophages

Porcine reproductive and respiratory syndrome, which is characterized by respiratory, reproductive disorders of sow and respiratory disease of piglets, is a viral disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the effect of PRRSV infection on immunity of porcine pulmonary alveolar macrophages (PAM) was systematically investigated from the view of innate immunity. The research involved in analysis of the mRNA transcription of pattern recognition receptor (PRR) , molecules associated with innate immunity, IFN-stimulated genes (ISG) and cytokines in PAM infected with PRRSV. The aim of our study is to explore the molecular mechanism of the effect of PRRSV infection on PAM immunity.Partial gene fragments of swine PRR including CD16, CD18, TLR2, TLR4, SR, molecules associated with innate immunity including Nrampl, SP-A, ISG including OAS, PKR, Mx1, and cytokines involved IFN-α, IFN-γ, IL-1β, IL-6, TNF-α, IL-8, MCP-1, IL-10, IL-12p40, IL-18 and GM-CSF were amplified by RT-PCR from PAM and cloned according to their sequences available in GenBank respectively. After sequencing, competitive deleted clones for the cDNA molecules mentioned above were generated by reverse PCR. Then, the linear regression equations were obtained after construction of the standard competitive curves respectively by quantitative competitive PCR (qcPCR) assay.The PAM from PRRSV-infected PAM and control PAM in vitro and in vivo were investigated for the mRNA transcriptions of PRR and molecules associated with innate immunity by qcPCR according to their respective standard competitive curve constructed. In vitro, all PRR and molecules associated with innate immunity, including CD16, CD18, TLR2, TLR4, SR, Nrampl, SP-A decreased sharply to the lowest level at 4h post inoculation (PI), then increased gradually to normal at 48h PI. In vivo, all molecules decreased at different level during the early stage of PRRSV infection (at day 3~14 PI). In conclusion, the mRNA transcriptions of these molecules were prohibited during the early infection, resulting in the impairment of macrophage innate immunity. During the late stage of PRRSV infection (at day 28~42 PI), the mRNA transcriptions of these molecules recovered to normal level or upregulated, suggesting the function of PAM appears to rebound and increase somewhat.The mRNA transcriptions of ISG of PRRSV-infected PAM and control PAM in vitro and in vivo were investigated by qcPCR according to their respective standard competitive curve constructed. In vitro, all ISG mRNA transcriptions including OAS, PKR and Mxl in PRRSV-infected PAM showed lower level than that in the control PAM, exhibiting that the mRNA transcriptions of these genes were suppressed by PRRSV infection. This hints that PRRSV infection might damage the antiviral function of PAM. In vivo, ISG mRNA transcriptions in PAM of PRRSV-infected piglets had no significant change compared to that of control piglets at day 3 and 7 PI; however, they showed a sharp decrease at day 14 PI, and then ascended to normal at day 28 PI, and even increased a little at day 42 PI. The data invivo indicated that ISG mRNA transcriptions in PAM are prohibited by PRRSV infection at day 14 PI, resulting in severe impairment of the antiviral function of PAM at this stage.The mRNA transcriptions of cytokines from PRRSV-infected PAM and control PAM in vitro and in vivo were observed by qcPCR according to their respective standard competitive curve constructed. Meanwhile, protein expression of cytokines IFN-a, IL-6, IL-10 and IL-12 in culture supernatant of PAM were examined by quantitative ELISA. In vitro, the mRNA transcriptions of proinflammtory cytokines upregulated and peaked at different phase postinoculation of PRRSV, which seemed that PRRSV infection could induce inflammation. In vivo, during the early stage of PRRSV infection (at day 3~7 PI) , the mRNA transcriptions of proinflammtory cytokines in PAM including IFN-a, TNF-a, IL-8, IL-1B and IL-6 reduced at different level; at metaphase (at day 14 PI) , IL-10 increased...