The Effect Of Different Immunogens On PRRSV-induced Neutralizing Antibody And Role Of SHP1 In PRRSV Infection

Porcine reproductive and respiratory syndrome(PRRS) is an infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV).PRRSV is seriously contagious and can cause mainly respiratory disorders and reproductive failure in sows and piglets. An important biological characteristics of PRRSV is to have the tropism for porcine alveolar macrophages.PRRSV-specific neutralizing antibody has an important role in the immune protection against PRRSV,and is also an important index to assess protective immunity of PRRS.Therefore,It is important that establishment of detective way of neutralizing antibody anti-PRRSV based on PAMs,which is meaning for prevention of PRRSV.SHP1 is a tyrosine phosphatase in alveolar macrophages, and can inhibit TLR-mediated activation of NF-κB and MARK, thereby inhibiting inflammatory cytokine.But studies have been shown that SHP1 has an active role for TLR and RIG-I-mediatedⅠtype interferon. SHP1 can regulate pro-inflammatory cytokines andⅠtype interferon in the opposite way. In order to study the role of SHP1 in PRRSV-induced antiviral immune response, SHP1-mediated signaling pathways were studied in PRRSV infectionThe aim of this study is to develop PRRSV NA assay to determine NA titers in pig serum samples serum.The collected standard serums were inactivated.two-fold diluted tested serum and negative serums and equal amount of PRRSV were mixed for inoculating PAM cells.after cultured for 48 h, The m RNA level of PRRSV was detected by real-time fluorescent quantitative PCR to establish detective methods of PRRSV NA. In order to verify this method, this study collected 34 clinical samples to PRRSV NA titers. The results showed that we developed and validated porcine serum-based PRRSV NA assay,which had good specificity and reproducibility.The study can be used to assess herd immunity against PRRSV.In order to further assess the level of herd immunity protection of PRRS, we explored the effect of PRRSV-induced neutralizing antibody in the different immune ways.we established five experimental groups to monitor neutralizing antibody.they were respectively attenuated immunity(PRRSV CH-1R was vaccined on 0 d),inactivated immunity(PRRSV inactivated vaccine was vaccined on 0d),inactivated immunity after virulent immunity(after PRRSVvirulent infection 7d, we vaccined PRRSV inactivated vaccine),inactivated immunity after attenuated immunity(after PRRSV CH-1R infection 7d,we vaccined PRRSV inactivated vaccine),and the healthy control group. The results showed PRRSV NA titers of two pigs at 28 days were 1: 2,and 3 pigs at 42 days respectively were 1: 32,1: 4,1: 16 in the first group;PRRSV NA titers of three pigs at 28 days were less than 1: 2, and 3 pigs at 42 days respectively were 1: 4,1: 8,1: 8 in the second group; Three pigs PRRSV virulently infected had clinical signs, then after PRRSV nactivated vaccine, and we can not detect PRRSV NA titers in the third group.PRRSV NA titers of two pigs at 28 days were 1: 2,and 3 pigs at 42 days were 1: 2 in the fourth group;The results demonstrated PRRSV NA titers of PRRSV attenuated vaccine were higher than PRRSV attenuated vaccine after PRRSV infection;PRRSV NA titers of PRRSV inactivated vaccine were higher than PRRSV attenuated vaccine after PRRSV infection;PRRSV NA titers of PRRSV attenuated vaccine were higher than PRRSV inactivated vaccine.Therefore,the results indicated immunity of inactivated vaccine after PRRSV infection could enhance the replication of PRRSV,and inhibited the production of NA.To study the role of SHP1 in PRRSV infection,porcine alveolar macrophage(PAM) was prepared and cultured with PRRSV solution 200 TCID50. we collected cells and supernatants after treated 12, 24, 36 and 48 h respectively. The level of the virus,IRF3、IRF7、TRAF6、NF-κB、SHP1、IFN-β m RNA in the PAM cells were quantitated by Real-time q PCR includingthe cell control group,poly(I:C)stimulated cells control group and LPSstimulated cells control group.We used ELISA to detect the expression of SHP1 after PRRSV infection PAM The results showed activated SHP1 could improve m RNA level of IRF7、NF-k B、SHP1、IFN-β,and inhibited m RNA level of IRF3 and TRAF6.The m RNA levels of IRF3、IRF7、TRAF6、NF-κB、SHP1 and IFN-β of PAM cells treated with poly(I:C) and LPS respectively were both improved during 12-48h; The m RNA levels of IRF3、IRF7、TRAF6、NF-κB、SHP1 and IFN-β of poly(I:C)or LPS stimulation of PRRSV infected PAM cells were both up-regulated during 12-48 h compared with PRRSV infection control group.The result suggested SHP1 could adjust m RNA level of cytokines in PAMs infected with PRRSV by TLR-mediated signaling pathways or unknown pathways. LPS,or Poly(I: C) stimulation of PRRSV infection PAM could inhibit the replication of PRRSV, and enhance m RNA level of the certain cytokines.