Molecular Basis Of Differential Cytokine Expression Induced By Nonstructural Proteins Of Different Pathogenic PRRSV Strains In Target Cells

Porcine reproductive and respiratory syndrome (PRRS), a highly contagious disease, caused by porcine reproductive and respiratory syndrome virus (PRRSV), has become one of the most economically significant diseases for the swine industry worldwide. It is characterized by severe reproductive failure such as a high rate of late abortion and early farrowing in sows and respiratory disease in piglets. In2006, the emergence and prevalence of a highly pathogenic PRRSV (HP-PRRSV) casusing high fever, high morbidity and high mortality to pigs of all ages, led great economical loss to the swine industry in China.The pathogen HP-PRRSV with a unique molecular hallmark namely a discontinuous30amino acid deletion in its Nonstructural protein2(Nsp2) coding region was geneticly different from the classical Chinese field strains. So far, the molecular mechanisms for high virulence of HP-PRRSV are not clear. Porcine pulmonary alveolar macrophages (PAMs) are major target cells of PRRSV. The destruction and function alteration of infected PAMs might be one of the factors for PRRSV pathogenesis. In order to compare the effects that different pathogenic PRRSV strains exert on PAMs, our study focused on analysing the cytokine expression in PAMs and identifying the viral proteins involved in TNF-α differential expression induced by different pathogenic PRRSV strains, which can provide the basis for understanding of the molecular mechanisms for increased virulence of HP-PRRSV.First of all, our study explored the effects that different pathogenic PRRSV strains exert on cytokine expression in PAMs. PAMs were infected with HP-PRRSV strain (JXwnO6) and low-virulence PRRSV strain (HB-1/3.9) respectively and the cytokine expression in PAMs were investigated at both mRNA and protein levels. Relative quantitation real-time PCR was used to measure the interleukin (IL)-ip, IL-8, IL-10, interferon (IFN)-a, IFN-β and tumor necrosis factor (TNF)-a mRNA in PAMs. Commercial ELISA kits were used to measure soluble proteins of cytokines aforementioned in the culture supernatant. The results showed that no statistical differences were observed at both mRNA and protein levels of IL-β and IL-8. Both of JXwnO6and HB-1/3.9down-regulated the level of type I interferons in the supernatant, but there was no significant difference. Meanwhile the HP-PRRSV strain tends to enhance the induction of IL-10and decrease the elicitation of TNF-a compared to the low-virulence strain. Enhanced induction of IL-10and decreased elicitation of TNF-a might play an important role in immune suppression and antiviral effect antagonism for HP-PPRSV.We analysed the viral proteins involved in differential TNF-a expression in PAMs induced by different pathogenic PRRSV strains. We further verified the role of these viral proteins using reverse genetic technology to construct chimeric viruses by exchanging the determinant viral protein-coding regions. By using the TNF-a promoter reporter system, Nsp7, Nsp11and Nsp12were found to be the proteins involved in differential TNF-a expression at mRNA level. By investigating the effect that each Nsp exerted on the ERK signaling pathway, Nsplf3and Nspll of HP-PRRSV were found to be more effecitive to inhibit ERK signaling pathway, which might result in greater ability in suppression of TNF-a production. Using reverse genetic technology to construct chimeric viruses by exchanging both Nsp1β-and Nspll-coding regions, the chimeric viruses could almost reverse the phenomenon of differential TNF-a production. These results demonstrated that Nsp1β and Nsp11played an important role in differential TNF-a production induced by different pathogenic PRRSV strains. Our study suggested that HP-PRRSV was able to effectively inhibit TNF-a production at protein level though its viral proteins, namely Nsp1β and Nspl1, which is beneficial for its replication in vivo. This property of HP-PRRSV may be one of the mechanisms for its increased virulence for piglets.In summary, our findings revealed the differential effects that different pathogenic PRRSV strains exert on cytokine expression in PAMs and identified that Nsp1β and Nspll were the viral proteins involved in differential TNF-a production induced by HP-PRRSV. These results provide scientific basis for understanding of the molecular mechanisms for increased pathogenicity of HP-PRRSV.