Study On The Mechanisms Of Flavaspidic Acid AB And M?CD Against PRRSV

Porcine reproductive and respiratory syndrome virus(PRRSV)represents a significant challenge to the swine industry worldwide.PRRSV can cause reproductive failures in pregnant sows and respiratory diseases in newborn pigs.Recently,there have been devastating outbreaks of atypical PRRS caused by a highly pathogenic PRRSV(HP-PRRSV)strain in China,which is characterized by high fever,high morbidity,and high mortality in all age pigs.A substantial effort has been made to control and eradicate PRRSV infection since it was identified.However,not much progress has been made and PRRSV still remains the biggest challenge to swine industry.Unfortunately,the available types of PRRSV vaccines have certain drawbacks concerning safety and efficacy.Thus,there is an urgent demand for new antiviral therapies such as effective antiviral drugs.Flavaspidic acid AB is a compound derived from Dryopteris crassirhizoma,a traditional antiviral Chinese medicine.Here,we first identified its anti-PRRSV activity through targeting multiple stages in PRRSV infection in vitro.Our studies demonstrated that flavaspidic acid AB could inhibit the internalization and cell-to-cell spreading of PRRSV,but not block PRRSV binding to cells.By monitoring the kinetics of PRRSV replication,we showed that flavaspidic acid AB significantly suppressed PRRSV replication when treatment was initiated 24 h after virus infection.Furthermore,we confirmed that flavaspidic acid AB was able to significantly induce IFN-?,IFN-?,and IL1-?expression in porcine alveolar macrophages,suggesting that induction of antiviral cytokines by flavaspidic acid AB could contribute to FA-AB induced inhibition of PRRSV replication.Methyl-?-cyclodextrin(M?CD)is a drug widely used to sequester cholesterol and it was able to disrupt the lipid rafts.In this study,we investigated the importance of lipid rafts in PRRSV infection First,we showed that disruption of cellular lipid rafts significantly impaired PRRSV infection.Subsequently,we demonstrated that disruption of cellular lipid rafts inhibited PRRSV entry,and PRRSV glycoproteins,Gp3 and Gp4,were associated with lipid rafts during viral entry.Isolation of detergent-insoluble raft fractionations from cells showed that CD163 was located in raft microdomains,which might contribute to the glycoproteins-raft association.Moreover,lipid raft disruption caused a significant reduction of the intracellular viral RNA production.In addition,Nsp9(RNA-dependent RNA polymerase)was shown to be mainly distributed in lipid raft fractions,suggesting that lipid rafts probably serve as a platform for PRRSV replication.Finally,we confirmed that disassembly of lipid rafts on the virus envelope impaired PRRSV infectivity,which might affect the integrity of PRRSV particles and cause the leakage of viral proteins.These findings might provide insights on our understanding of the mechanism of PRRSV infection and provide a foundation for the possibility to develop new therapeutic agents to control PRRSV infection.