The Mechanism Of Host Responses Regulated By Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) poses significant economic losses to the pork industry and is currently a major concern for the swine industry worldwide. Especially, an outbreak of highly pathogenic PRRSV (HP-PRRSV) occurred in China and caused disastrous loses to the farmers. At present, PRRSV is sustaining evolution and is particularly difficult to control, due to the pathogenesis and immunological mechanism is in a complex manner, and development of a safe and effective vaccine for PRRSV has difficulties. Most recently, the studies of PRRSV are focus on the mechanisms that how the virus to evade the recognition and elimination of host innate immune reponses.Phosphatidylinositol-3-kinase (PI3K)/Akt is an important cellular pathway, and has been shown to participate in various replication steps of multiple viruses. To select the mutant virus which was conferred the drug resistance, GSK-3 inhibitor X was used to suppress the HuN4-F112 virus replication in MARC-145 for 14 passages. Then complete genome of the selected mutant virus which tolerated to the GSK-3 inhibitor X was sequenced by high-throughput sequencing. Complete genome sequence analysis revealed that six amino acids mutations in Nsp2, Nsp12, GP3 and GP4 were essential for the virus replication by reverse genetic technique.The sterile alpha motif and HD domain 1 (SAMHD1) protein has been identified as a novel innate immunity restriction factor, function as a triphosphohydrolase that depletes cells of deoxynucleotide triphosphates (dNTPs), inhibits lentiviral complementary DNA (cDNA) synthesis and blocks HIV-1 infection in myeloid-lineage cells. The underlying mechanisms of SAMHD1 transcriptional regulation showed that inducing SAMHD1 upregulation is part of an early intrinsic immune response via TLR3 and RIG-I/MDA5 agonists that ultimately induce the nuclear translocation of the interferon regulation factor 3 (IRF3) protein. Further studies show that IRF3 plays a major role in upregulating endogenous SAMHD1 expression in a mechanism that is independent of the classical IFN-induced JAK-STAT pathway. Both overexpression and activation of IRF3 enhanced the SAMHD1 promoter luciferase activity, and activated IRF3 was necessary for upregulating SAMHD1 expression in a type I IFN cascade. We also show that the SAMHD1 promoter is a direct target of IRF3 and an IRF3 binding site is sufficient to render this promoter responsive to stimulation.Solutions presented in this thesis can be summarized as follows:-The activity of GSK-3 was essential for the virus infection. Virus replication was significantly reduced by mutations in Nsp2 (N401D and D795N) and in Nsp2 (R121W); Virus replication and proliferation was enhanced in vitro by mutations in GP3 (P204S) and GP4 (F5L and A22V), together with the enhancement of Akt phosphorylation.-As an innate immunity restriction factor, SAMHD1 participates in the antiviral responses during PRRSV infection. The phosphorylation and nuclear translocation of IRF3 plays vitial roles in upregulation of endogenous SAMHD1 expression.