Inhibitory Mechanism Of α-glycerol Monolaurate On Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome（PRRS）,is an acute infectious disease caused by porcine reproductive and respiratory syndrome virus（PRRSV）.At present,the prevention of PRRS is mainly through the vaccine,but the protective effect of vaccine is unsatisfactory,so it is necessary to find new prevention and treatment methods of PRRS.The purpose of this study was to investigate the inhibitory effect ofα-GML on PRRSV and its mechanism.This study is mainly divided into two experiments.In experiment 1,the inhibition effect of differentα-GML treatments on PRRSV was investigated.（1）The virus was identified as PRRSV by electron microscopy,RT-PCR and Western blot,and the virus titer（TCID50）was determined at 10^-5.25/0.1m L by Reed-Muench method.（2）The maximum non-cytotoxic concentration ofα-GML was 25μg/m L by MTT assay.（3）The blank control group,α-GML control group,PRRSV group andα-GML+PRRSV group were set up,and two methods about post-treatment and co-treatment ofα-GML were adopted in this experiment.The expression of nucleocapsid protein N in PRRSV was measured by RT-q PCR,Western blot and indirect immunofluorescence.And the results showed that the post-treatment ofα-GML did not significantly inhibit the gene and protein expression of PRRSV nucleocapsid protein N（P>0.05）,while co-treatment ofα-GML and PRRSV could significantly reduce the gene and protein expression of nucleocapsid protein N（P<0.001）.These showed thatα-GML co-treatment with PRRSV could inhibit PRRSV.The experiment 2 was based on the results of the first experiment to explore the mechanism ofα-GML to inhibit the PRRSV.（1）The effect ofα-GML co-treatment on virus activity.And the blank control group,PRRSV group andα-GML+PRRSV group were set up in the experiment.The samples of the three groups were placed directly in a refrigerator at 4℃for 2h,and then the virus titer of the three groups was measured by Reed-Muench,and the virus amount of the three groups was measured by ELISA.The results showed that co-treatment ofα-GML and PRRSV could significantly reduce the virus titer（P<0.001）,and significantly reduced the amount of virus（P<0.001）,indicating thatα-GML significantly reduced the virus activity and even killed the virus directly.（2）The effect ofα-GML on the structure of viral capsule and cell receptor.The blank control group,α-GML control group,PRRSV group andα-GML+PRRSV group were set in the experiment.Results showed that co-treatment ofα-GML and PRRSV significantly reduced the gene expression of PRRSV capsular protein M and GP5（P<0.01）,and significantly down-regulated the relative gene expression levels of cell surface receptors CD163（P<0.05）,indicating thatα-GML could affect the expression of PRRSV capsule and receptor.（3）Regulation ofα-GML on cellular immune-related signaling pathways.And the gene and protein expressions of key factors in the TLR7/8-My D88-NF-κB/IFN-αsignaling pathway were determined in the blank control group,α-GML control group,PRRSV group andα-GML+PRRSV group.The results showed that,compared with PRRSV group,the co-treatment ofα-GML and PRRSV significantly down-regulated the gene expressions of TLR7,TLR8,TRAF6,TRAF3 and NF-κB1（p50）were significantly own-regulated（P<0.05）.And the gene expression of NF-κB inhibitor subunit IκKβwas significantly up-regulated（P<0.05）.The the protein expression of My D88 and NF-κB1（p50）were down-regulated,and the protein expression of NF-κB pathway inhibiting enzyme IκB was significantly up-regulated（P<0.05）,and decreased the secretion of pro-inflammatory factor TNF-α,suggesting thatα-GML inhibited the activation of NF-κB pathway induced by PRRSV infection and alleviated the associated inflammatory response.In conclusion,α-GML co-processing with PRRSV can inhibit PRRSV.The main mechanism ofα-GML inhibition of PRRSV is thatα-GML can significantly reduce the viral activity of PRRSV,reduce the expression of PRRSV capsule protein M and GP5,as well as the cellular receptors CD163,and thus realize the inhibition effect of PRRSV.Meanwhile,PRRSV inhibited the activation of NF-κB pathway and decreased the secretion of TNF-α.