The Antiviral Effect Of Allicin On Porcine Reproductive And Respiratory Syndrome Virus Infection

Porcine reproductive and respiratory syndrome(PRRS)is an acute viral infectious disease caused by the porcine reproductive and respiratory syndrome virus(PRRSV).PRRSV can infect pigs of all ages,especially pregnant sows and piglets.Pregnant sows infected with PRRSV three months after pregnancy can lead to stillbirth.Some sows also have fetal autolysis and mummification,while piglets infected with PRRSV show fever,dyspnea,anorexia,lethargy,eyelid edema,ear wing or hindquarters cyanosis,etc.Due to the genetic diversity of the PRRSV strains,the current killed vaccines and modified live vaccines can’t provide full protection against all field strains,and the live vaccine has the risk of virulence reversion.Therefore,it is urgent to develop alternative PRRS control measures,such as antiviral drugs.Allicin is one of the main OSCs in garlic that exerts antibacterial,antiparasitic,antiviral,anti-inflammatory,anticancer,and antioxidative functions.It has been widely used in livestock and poultry health care.Previous studies have shown that allicin has significant antiviral effects on a variety of enveloped viruses.However,the effect of allicin on PRRSV infection has not been reported yet.In this study,we evaluated the antiviral activity of allicin against PRRSV and explored the potential mechanisms.The antiviral effect of allicin against PRRSV was evaluated by antiviral assays using two PRRSV strains of the prevalent PRRSV lineages in China,HP-PRRSV TA-12 strain and NADC30-like PRRSV NL1207 strain.Allicin inhibits the infection of these two PRRSV strains in MARC-145 cells and porcine alveolar macrophages in a dose-dependent manner.A time-of-addition experiment showed that allicin treatment affected multiple stages of the PRRSV life cycle,but could not directly inactivate PRRSV.We further evaluated the effects of allicin on each step of the PRRSV life cycle,including viral attachment,internalization,replication,assembly,and release.Through various antiviral assays,we found that allicin significantly interfered with the internalization,replication,and assembly of PRRSV,but did not significantly affect viral attachment and release.In addition,allicin exhibited no inhibitory effects on the expression of the main receptor CD 163 involved in PRRSV internalization and the enzymatic activity of PRRSV main protease nsp4.Excessive inflammatory responses in the lungs of PRRSV-infected animals,also known as cytokine storms,lead to interstitial pneumonia which is often associated with high mortality.Based on this,inhibiting the excessive inflammatory responses can also effectively alleviate the clinical disease and mortality caused by PRRSV infection.It has been reported that allicin has anti-inflammation properties by suppressing the pro-inflammatory pathways,such as the MAPK and NF-κB signaling pathways.In the second chapter,we found that allicin can reduce the inflammatory responses induced by PRRSV infection.In PRRSV-infected MARC-145 cells,the mRNA levels of pro-inflammation cytokines were significantly up-regulated;in cells with PRRSV infection and allicin treatment,the expressions of IFN-β,IL-6,and TNF-α were significantly down-regulated.Therefore,allicin can alleviate the expression of proinflammatory cytokines induced by PRRSV infection.To analyze the mechanism by which allicin inhibits PRRSV-induced inflammatory responses,we performed transcriptomic analysis on MARC-145 cells with three different treatments,including a negative control,PRRSV infection,and PRRSV infection plus allicin treatment.KEGG pathway enrichment analysis of DEGs showed that PRRSV infection induced pro-inflammatory signaling pathways,including the TNF signaling pathway and MAPK signaling pathway,which were restored by allicin treatment.The 94 DEGs up-regulated by PRRSV infection were significantly down-regulated by allicin treatment,and 13 and 8 of them were enriched in the TNF signaling pathway and MAPK signaling pathway,including IL-6 and TNF signaling pathway-related receptor proteins TRAF1 and TNFAIP3,serine/threonine protein kinases MAP3K1 and MAP3K8 involved in the activation of MAPK signaling pathway and NF-κB signaling pathway.The above results indicated that allicin may inhibit inflammatory responses by suppressing the TNF signaling pathway and MAPK signaling pathway.In conclusion,allicin has antiviral activity against PRRSV and exerts its antiviral function by interfering with the internalization,replication,and assembly of the PRRSV life cycle.In addition,the anti-inflammation effect of allicin may be the potent antiviral mechanism against PRRSV.Data generated in this study suggest that allicin is a promising drug candidate for PRRS control.