| Porcine reproductive and respiratory syndrome(PRRS)is the most economically important infectious disease for the swine industry worldwide caused by porcine reproductive and respiratory syndrome virus(PRRSV)and characterized with reproductive failure and preterm birth in sows as well as dyspnea of piglets and fattening pigs.PRRS firstly emerged in America and Europe in the 1980s while it was firstly reported in 1996 in China.Since the emergence of PRRS more than three decades ago,numerous efforts have been made to develop effective measures to manage this challenging disease.Currently,vaccination is main measure to prevent PRRS in China.However,the use of commercial vaccines with either modified live virus(MLV)or inactivated virus is inadequate to control PRRSV infection.Antiviral therapeutics could be an alternative or additional measure to combat this infection,especially when available vaccines do not match the circulating virus.Therefore,the development of antiviral agents against PRRSV remains to be urgent needed for pig industry.Ursolic acid(UA)is one of Pentacyclic triterpenoids(PT),which possesses important biological effects,including anti-inflammatory,anticancer,antidiabetic,antioxidant and antibacterial effects.It is reported that ursolic acid derivatives have the better biological effects than ursolic acid.In this thesis,the anti-PRRSV activity of 15 ursolic acid derivatives was evaluated.We further confirmed the anti-PRRSV activity and investigated the preliminary mechanism of action of two representative effective compoundsIn this study,a model of PRRSV infecting MRAC-145 cells was established.Using indirect immunofluorescence assay,two ursolic acid derivatives,compound-9 and compound-12 with significant anti-PRRSV activity were identified out of more than 40 ursolic acid,oleanolic acid and their derivatives.Based on this finding,the anti-PRRSV activities of compound-9 and compound-12 were systematically evaluated in vitro.Firstly,the effects of the two compounds on cell viability were determined using MTT assay.Next,the effects of two compounds on PRRSV virus titer and PRRSV-NSP9 mRNA expression were further evaluated by the end point dilution assay and q RT-PCR assay,respectively.Finally,we examined the effects of the two compounds on cell sensitivity and direct inactivation on PRRSV.In addition,the effects of two compounds on the adsorption,internalization,viral protein and RNA synthesis,assemblly and release of PRRSV replication cycles in MARC-145 cells were also investigated.The results showed that compound-9 and compound-12 were not toxic to MARC-145 cells at a concentration of ≤40 μM,and were not toxic to PAMs at a concentration of ≤120 μM.Compound-9 and compound-12 significantly inhibited the replication of PRRSV on MARC-145 cells and PAMs cultures in a concentration range of 5-20 μM in a does-dependent manner,and compound-9 and compound-12 sustainly inhibited the replication of PRRSV on MARC-145 cells and PAMs.Furthermore,compound-9 and compound-12 exhibited broadspectrum anti-PRRSV activities due to their inhibition on replication of other three different subtypes of PRRSV strains.The mechanism studies showed that Pre-treatment of compound-9 and compound-12 did not inhibit the replication of PRRSV on MARC-145 cells,indicating that compound-9 and compound-12 did not impair the susceptibility of MARC-145 cells to PRRSV infection.However,in Co-treatment mode and Post-treatment mode,compound-9and compound-12 inhibited the replication of PRRSV on MARC-145 cells.Moreover,compounds-9 and compound-12 could block the stages of adsorption,internalization,viral protein and RNA synthesis,assemblly and release during the PRRSV replication cycle.Importantly,compounds 9 and 12 were able to directly interact with the virus and therefore inactivate PRRSV.As far as the best of our knowledge,this study is the first time to confirm antiviral activities of ursolic acid and its derivatives against PRRSV infection in vitro,and provide experimental evidence for the development of new anti-PRRSV drugs. |
