Study On The Activity And Mechanism Of Favipiravir And Ribavirin Against Peste Des Petits Ruminants Virus

Peste des petits ruminants(PPR)is a highly contagious animal disease of small ruminants caused by the peste des petits ruminants virus(PPRV).Goats and sheep are highly susceptible to PPRV.The morbidity and mortality of sick goats and sheep can be as high as100%,which seriously threatens the healthy development of the global sheep industry.Although the outbreak of PPR can be controlled by application of the PPRV attenuated vaccine,the sporadic or subclinical cases still occur occasionally,and the situation of PPR prevention and treatment is not optimistic.Therefore,it is very important to develop effective anti-PPRV agents to ensure the healthy development of sheep industry in China.The maximum non-toxic concentration of 22 available antiviral compounds ribavirin,acyclovir,Poly I:C,moroxydine hydrochloride,amantadine,dideoxyinosine,acetylsalicylic acid,emetine,oseltamivir phosphate,zidovudine,favipiravir,RSV604,boceprevir,caffeic acid,2-Deoxy-D-glucose,mycophenolic acid,abidol,tiotropium bromide,6-azauridine,6-methylmercaptopurine riboside,buthionine sulfoximine and resiquimod were determined by MTT assay to evaluate the anti-PPRV activities.The effective anti-PPRV chemicals were selected according to the inhibition effciency of cytopathic effect(CPE)of PPRV infected Vero cells,and the anti-PPRV effects of farpiravir,ribavirin and acyclovir were subsequently detected by IFA test and Western-blot assay.The PPRV-infected Vero cells were treated with favipiravir and ribavirin respectively,and the effects of favipiravir and ribavirin on virus adsorption,invasion,replication and release were detected by Western-blot and Plaque formation test.In order to clarify the anti-PPRV mechanism of farpiravir and ribavirin,q RTPCR and Western-blot were used to detect the JAK/STAT and PI3K/Akt signaling pathways and their downstream signaling molecules respectively.The results indicated that:1.Screening of activiral chemicals against PPRVAmong the 22 candidates,favipiravir,ribavirin and acyclovir showed higher anti-PPRV activity in vitro at the final concentration of 50,12.5 and 25 μg/m L respectively,and the cytopathic effect of PPRV-infected Vero cells were inhibited obviously.Moreover,the expression of viral structural proteins H,F and N in PPRV infected Vero cells were significantly decreased owing to fapiravir and ribavirin treatment,and the phenomenon of virus proliferation in the Vero cells was accordingly reduced.While,in the acyclovir treated PPRV infected cells,the expression of H and F proteins were also significantly decreased.The anti-PPRV effciency were favipiravir,ribavirin and acyclovir in order.2.Effects of favipiravir and ribavirin on PPRV proliferation cycleThe adsorption,invasion and release of PPRV were significantly inhibited by fapiravir and ribavirin treatment,which implied that both facpiravir and ribavirin acted on the whole process of the PPRV proliferation cycle.3.Favipiravir and ribavirin mediate JAK/STAT signaling pathway to inhibit PPRV proliferationThe expression level of STAT in PPRV infected Vero cells was hugely down-regulated(P<0.05).Both favipiravir and ribavirin could hugely up-regulate the expression level of STAT in PPRV infected Vero cells(P<0.05),the expression of downstream signal molecules of JAK/STAT such as antiviral protein genes ISG54,PKR,IRF9 and Mx A were also significantly up-regulated resulting from favipiravir treatment(P<0.05).Similarly,the expression of ISG20 and Mx A were also significantly elevated in ribavirin treated PPRV infected Vero cells(P<0.05).These data suggested that the antiviral activity of favipiravir and ribavirin against PPRV were mediated by JAK/STAT signal pathway probably.4.Favipiravir and ribavirin mediate PI3K/AKT signaling pathway to inhibit PPRV proliferationThe expression of PI3K/AKT in PPRV infected Vero cells was significantly up-regulated(P<0.05)and the downstream signaling molecules p-GSK3,p-Bad,p-CREB and p-NFκB were activated(P<0.05).PI3K/AKT signaling pathway was significantly down-regulated in favipiravir and ribavirin treated Vero cells infected by PPRV(P<0.05),the expression of downstream signaling molecules p-CREB,p-GSK,p-Bad and p-NFκB(P<0.05)were significantly down-regulated,and reduced expression of Bcl-2 was also observed,suggesting that anti-PPRV activity of favipiravir and ribavirin probably mediated by PI3K/AKT signal pathway.Favipiravir and ribavirin played an antiviral role by inhibiting the survival of PPRV infected cells and promoting cell apoptosis.In summary,favipiravir and ribavirin can significantly inhibit the adsorption,invasion,replication and release of PPRV by up-regulating the expression levels of JAK/STAT in PPRV-infected Vero cells.Fapilavir up-regulated the expressions of antiviral protein groups ISG54,PKR,IRF9 and MXA.While,ribavirin promoted the expressions of ISG20 and MXA.Favipiravir and ribavirin also exhibited antiviral effiacy by inhibiting PI3K/Akt signaling pathways associated with anti-apoptosis,which inhibited the expression of its downstream signaling molecules p-GSK-3,p-CREB,p-Bad and p-NFκB,and reduced the expression of the anti-apoptotic protein Bcl-2.