Effect Of Classical Swine Fever Virus C Strain And Its Non-structural Protein On Innate Immune Response Mediated By Toll-like Receptor In Porcine Alveolar Macrophages

Classical swine fever（CSF）,caused by Classical swine fever virus（CSFV）,is a seriously infectious disease of pigs.With a high rate of mortality,infected animals exhibit leukopenia,fever,bleeding and abortion.CSF seriously hinders the normal development of pig industry,so our country and some countries adopt the Hog Cholera Lapinized vaccine（C strain vaccine）developed in China in the 1950s to prevent the burst of epidemy,and achieved a preferably effect.CSFV Shimen strain and C strain can induce different immune responses after infection.Shimen strain can inhibit host innate immunity and establish persistent infection,while C strain can stimulate the host to produce specific antibodies which play a protective role,and the mechanism is still unclear.Toll-like receptor（TLRs）,as a member of the pattern recognition receptor（PRRs）family,recognizes pathogen-associated molecular patterns（PAMPs）.Then,it induces the production of type I interferon and pro-inflammatory cytokines.Our team previous study found that CSFV Shimen strain and C strain can cause different expression of TLRs gene;Shimen strain inhibits TLR3-mediated signal pathway to affect host innate immunity.Various proteins of CSFV Shimen strain were overexpressed by lentivirus packaging system,in order to study their effects on TLRs signal pathway.Results showed that TLRs signal pathway was altered by these proteins in different degrees.Based on the fact that CSFV Shimen strain and C strain cause different host immune responses,the purpose of this study was to analyze the effects of CSFV C strain and its non-structural proteins on TLRs mediated innate immunity of porcine alveolar macrophages（3D4/21）,and the following results were obtained:（1）At different times after CSFV C strain infected 3D4/21 cells,the effect of C strain on TLRs mediated innate immune response gradually showed a dynamic regulation process.At24 h post infection,C strain significantly restrained the expression of TLR2,TLR7 and its downstream key molecules My D88 and IRF7（P<0.05 or P<0.01）,while significantly increased the expression of TLR3,TLR4 and TRIF（P<0.05）.It mainly inhibited the signal pathway mediated by TLR2 and TLR7 to avoid excessive activation of TLRs;At 48 h and 72h post infection,C strain significantly up-regulated the expression of TLR2,TLR3,TLR4,TLR7 and the key proteins downstream of TLRs signal pathway（My D88,TRIF,IRF3 and IRF7）as well as the downstream immune effector factors type I interferon and IL-6（P<0.05or P<0.01）,thus contributing to virus clearance.（2）The non-structural proteins of CSFV C strain were overexpressed in 3D4/21 cells by lentivirus packaging system,as a way to study their effects on host innate immunity through TLRs signal pathway.The results showed that p7,NS4B,NS5A and NS5B can induce phosphorylation of ERK1/2;p7 promotes the expression of TLR2,TLR3,TLR7 and TRIF,while inhibits TLR4 expression,mainly through TLR3-mediated signal pathway to affect natural immunity;N^pro up-regulated the expression of TLR2 and TLR7;NS3 promoted the expression of TLR2 and TLR7,while inhibited TLR3 expression;NS4A promoted the expression of TLR2 and TLR3;NS2 up-regulated the expression of TLR2,TLR7,My D88IRF7 and IFN-α,and mainly promoted TLR7 mediated signal pathway;NS4B up-regulated TLR3,TRIF,IRF3,and IFN-βexpressions and promotes TLR3-TRIF-IRF3-dependent signaling pathways to affect innate immunity;NS5A up-regulates the expression of My D88,IRF7 and IFN-α,promotes My D88-IRF7 mediated TLRs signaling pathway;NS5B promotes the expression of TLR2 and TLR4,and promotes the expression of key proteins downstream of TLRs signaling pathway as well as the expression of type I interferon,pro-inflammatory cytokines IL-1βand IL-6.In summary,this study shows that in the early stage of CSFV C strain infecting 3D4/21cells（24 h）,C strain prevents the overactivation of TLRs by inhibiting the signal pathway mediated by TLR2 and TLR7,thus preventing the vaccine virus from being eliminated too early;In the middle and late stage of C strain infection（48 h,72 h）,the expression of type I interferon and IL-6 was induced by promoting the signal pathways mediated by TLR2,TLR3,TLR4 and TLR7,thus helping to eliminate the virus;It is suggested that the non-structural proteins of CSFV C strain regulate the expression of downstream cytokines by promoting or inhibiting the signal pathways mediated by TLR2,TLR3,TLR4 and TLR7,thereby affecting the natural immunity of the host.These research results provide new scientific data for elucidating the effects of CSFV C strain and its non-structural proteins on innate immunity through TLRs mediated signaling pathways.