Type Ⅰ Interferon Induction By Prrsv Infection And Mechanisms Study Of Activation Of P38 MAPK Signaling Pathway

Porcine reproductive and respiratory syndrome(PRRS)is a viral infectious disease,which is caused by porcine reproductive and respiratory syndrome virus(PRRSV).This disease can result in reproductive disorder of sow and high mortality and respiratory symptoms of piglets and fattening pigs,which has become one of the serious threats to development of pig industry worldwide.PRRSV is a single-strand RNA virus and its genome is about 15 kb encoding more than 20 mature proteins.In the host,PRRSV inhibits type Ⅰinterferon(IFN)intracellular or extracellular expression,which make the host lack intracellular antiviral activity and have week natural immune response leading to persistent viral infection and inadequate vaccine affects.In 2011,Dr.YanjinZhang from department of veterinary medicine in University of Maryland found a novel natural mutation PRRSV strain inducing type Ⅰ IFN production in host,however,the mechanism how to induce IFN production was still unknown.In this study,the novel PRRSV strain could not induce type ⅠIFN production in host cells after continuous subcultures for 26th generations.To explore this mechanism,we used reverse genetic technology to construct cDNA clone successfully and rescued virus pA2MC2.Compared to the parental strain,the biological characteristics of rescued virus pA2MC2 were further performed to preliminary explore the mechanisms on the induction of type Ⅰ IFN production.In addition,we also studied the mechanism on p38 MAPK(mitogen-activated protein kinases,p38MAPKs)signal transduction infected North American strain VR-2385,which also provided theoretical basis to further clarify the pathogenesis of PRRSV.More details are as follows:The parental strain A2MC2 was continuously passaged to attenuate virulence.MARC-145,susceptible cells to PRRSV,was used as a subculture medium.When cultured to 26th generation,the A2MC2 strain did not induce type Ⅰ IFN production in cells.In plague assay,the size of plagues formed by 26th generation were larger than those of patental strain,suggested that the characteristics of virus has changed.Based on the above results,we speculated the mutation of nuclear acid and amino acids in genomes of virus resulting in the changes of biological characteristics.The viral RNA of parental strain A2MC2 was extracted and reverse transcribed to obtain cDNA.Based on the reported genome sequence of strain A2MC2,we designed several specific primers and obtained the four fragments by PCR.By ligation and transformation,the four fragments were cloned into vector pCAGEN orderly.Finally,a DNA-launched A2MC2 infectious cDNA clone plasmid was successfully constructed.The plasmid was then transfected into MARC-145 cells,and the novel viral was successfully rescued,named pA2MC2.We investigated the biological characteristics of rescued virus pA2MC2,and found that the rescued virus pA2MC2 can still induce type Ⅰ IFN production in cells.In growth curve,the amount of virus reaches the peak in the third day,which was similar to the parental strain A2MC2.In addition,the sizes of plagues in pA2MC2 were identical to that of A2MC2.Therefore,the above results exhibited that the biological characteristics of rescued virus pA2MC2 were similar with the parental strain,which indicated that we successfully rescued virus pA2MC2.In animal study,we inoculated piglets by parental strain virus A2MC2 and rescued virus pA2MC2.The results showed that these two viruses infection has minimal adverse effect on pig weight in this study.In addition,visible macroscopic lung lesions were scored and the results showed that the level of visible macroscopic lung lesions in pigs inculcated the two viruses at 14 DPI were much higher than those of PBS(phosphate buffered saline,PBS)group.However,the level of visible macroscopic lung lesions between the two viruses infected pigs was almost the identical.At 56 DPI,the lung lesions were gradually recovered.Microscopic lung pathology of two viruses infected pigs were scored via HE(hematoxylin-eosin staining,HE)staining.The levels of interstitial pneumonia between those infected pigs were similar,but still higher than those of PBS group at 14 DPI.The microscopic lung lesions were restored at 56 DPI.By detecting viral RNA in pigs serum,the trend of viral growth between these two viruses were similar and the viral load reached the peak at 7 DPI.As the time extended to 42 DPI,the viral load was minimum.The expression of some cytokines and chemokines genes,such as IL-1β(interleukin 1β,IL-1β)IL-8(interleukin 8,IL-8),IL-10(interleukin 10,IL-10),CCL2(chemokine ligand 2,CCL2)and CXCL10(C-X-C motif chemokine 10,CXCL10)were induced by these two viruses in PAM(pluggable authentication module,PAM)cells.Meanwhile,the genes expression of IL-8 and CXCL10 were also induced in lung tissues isolated from two viruses infected pigs.These results suggest that parental strain virus A2MC2 and rescued virus pA2MC2 can cause the similar hopathogenesis.The virulent North American PRRSV VR-2385 can induce STAT1(signal transducers and activators of transcription,STAT1)phosphorylation at serine 727 in MARC-145 cells.The pSTAT1-S727 elevation was in a dose dependent and time dependent manner.However,the elevation of pSTAT1-S727 induced by VR-2385 was inhibited by SB203580 which is the specific inhibitor of p38 MAPK.Otherwise,the pSTAT1-S727 elevation also induced cytokine genes expression:IL-1β,IL-8 and IL-10,which were also inhibited by SB203580.This result indicated that the elevation of pSTAT1-S727 was highly associated with p38 MAPK signaling pathway,which was confirmed by methyltransferase MTA(methyltransferases,MTA).MTA can not only inhibit the elevation of pSTAT1-S727 induced by VR-2385,but also inhibit those genes expression.All results showed that the elevation of pSTAT1-S727 was via p38 MAPK signaling pathway.These results have been verified in PAM cells.The VR-2385 can induce a series cytokines and chemokines genes expression:IL-1β,IL-8,IL-10,CCL2 and CXCL10,but the increase can be inhibited by SB203580.We co-transfected exogenous STAT1 and VR-2385 Nsps/structural protein into HEK293 cells and found that Nsp12 increased exogenous pSTAT1-S727 level.To confirm above results,the Nsp 12 protein was transfected into HEK293 cells.It showed that Nsp 12 could induce the endogenous elevation of pSTAT1-S727.The above results suggested that the activation of p38 MAPK signaling pathway might be an important way for the increased phosphorylation level after VR-2385 infection.The downstream genes expression of several cytokines and chemokines was activated.Therefore,VR-2385 is not only able to induce pSTAT1-S727,but also unregulate inflammatory cytokines and chemokines genes,which play an important role in study of PRRSV pathogenesis.