Study On The Effect Of Leflunomide Against Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus(PEDV),a pathogen of porcine epidemic diarrhea(PED),belongs to the genus coronavirus of the family Coronaviridae.PEDV is a single-stranded positive-strand RNA virus.The disease is an acute and highly contagious intestinal infectious disease.Classic symptoms include vomiting,diarrhea,dehydration,and high mortality in piglets.PED was first reported in the Great Britain in 1971 and then rapidly spread to many pig-raising countries worldwide and has caused huge economic losses to the pig industry.PED is mainly controlled by vaccination.However,due to the viral antigen variation among strains,the efficacy of the vaccine is compromised.Although many antibiotics have been used to treat bacterial diseases in animals and humans,currently there is no effective antiviral drug for farm animals.It is imperative to develop novel antiviral agents to treat viral diseases caused by a lethal virus such as PEDV.Leflunomide is an immunosuppressant that is used for treating rheumatoid arthritis(RA)Its active metabolite A77 1726 is a multi-target inhibitor and inhibits the activity of p70 S6 kinase(S6K1),dihydroorotic dehydrogenase(DHO-DHase),and protein tyrosine kinases(PTK).Recent studies have shown that A77 1726 has a wide-spectrum antiviral activity.The mechanisms of A77 1726-medaited antiviral effects remain controversial.Our present study aims to determine if leflunomide could be repurposed as an antiviral drug to treat PED.IPEC-DQ,a porcine intestinal epithelial cell line,and Vero cells,a simian kidney cell line,were infected with an attenuated PEDV strain(HLJBY)and two PEDV virulent isolates(HXLV and CAJ),respectively.After incubation in the absence or presence of different concentrations of A77 1726,the levels of PEDV virus S(spike)and N(nucleoprotein)were analzyed by Western blot.We found that A77 1726 inhibited the expression of viral S and N proteins in a dose-dependent manner.Uridine,which was used to restore the levels of pyrimidine nucleotides in cells,did not reverse the decreased levels of viral S and N protein synthesis in A77 1726-treated cells.Uridine did not restore the virus titers in the conditioned media of A77 1726-treated cells.These results suggest that the antiviral activity of A77 1726 is not related to its inhibitory effect on pyrimidine nucleotide synthesis.To further investigate how A77 1726 inhibited PEDV replication,we examined the inhibitory effects of S6K1 kinase inhibitor PF-4708671,DHO-DHase inhibitor Brequinar sodium(BQR),JAK kinase inhibitor Ruxolitinib(Rux),and Src kinase inhibitor SU6656 on PEDV replication.Western blot revealed that PF-4708671 and BQR were unab to inhibit the synthesis of viral S and N proteins,whereas Rux and SU6656 inhibited the synthesis of viral S and N proteins in a dose-dependent manner and reduced the titers of PEDV in the conditioned media.These results suggest that A77 1726 inhibits PEDV replication by inhibiting JAK and Src tyrosine kinase activity.We further investigated the ability of A77 1726 to inhibit the activity of tyrosine kinases in infected and non-infected PEDV cells by Western blot.A77 1726 inhibited the phosphorylation of JAK1,JAK2,STAT3,Src and total protein tyrosine phosphorylation in uninfected Vero cells.In cells infected with PEDV,A77 1726 effectively inhibited JAK1 phosphorylation but had no effect on the phosphorylation of JAK2 and STAT3.Further experiments revealed ethat PEDV infection itself inhibited STAT3 phosphorylation.To verify that A77 1726 inhibited virus replication by inhibiting JAK and Src kinase activity,we tested whether overexpression of these genes enhanced PEDV replication.We found that overexpressing Src,JAK2 or STAT3 gene increased the viral protein levels and the phosphorylation levels of these proteins,whereas the antiviral activity of A77 1726 was significantly attenuated.In summary,our study provides evidence that A77 1726 effectively inhibited PEDV replication in two cell lines.A77 1726-mediated antiviral activity is largely mediated by inhibiting the activity of JAK and Src activity.Our study suggests that leflunomide has the potential to treat PEDV infections in piglets.