The Studies On The Mechanism Of Rifapentine Against Yellow Fever Virus

Backgrounds and objectivesMosquito-borne yellow fever virus（YFV）,which belongs to the Flaviviridae family,is the etiological pathogen of yellow fever（YF）,a serious infectious disease that is mostly prevalent in South America and Sub-Saharan Africa.Vaccination and mosquito control can significantly reduce the incidence of YF.However,low coverage of vaccine and a large number of vectors result in periodic outbreaks of YF in risky areas like South America and Sub-Saharan Africa.The fatality rate of YF cases is about 30%as no treatment for YF is available in clinic.With the progress of economic globalization,there is an increasing number of people exchange worldwide and this may lead to imported transmitssion of YF.In recent years,due to the impact of climate change,there have been Aedes aegypti mosquitoes that can transmit YFV in the southern region of China.Our citizens are susceptible to YFV,it will be likely to spread broadly due to the YF imported case,which will seriously threat people’s lives and health,and affect economic development and social stability.Therefore,it is of great necessity to further study YFV infection and develop effective antiviral drugs for the treatment of YF.YFV infection is a highly coordinated and complex process,providing multiple potential targets for antiviral drug development.Cell entry is the initial stage of the virus life cycle,when the viral genome has not been released yet.Drugs that interfere with YFV entry may have both prophylactic and therapeutic effects.Viral replication is the key step during the entire viral life cycle,controlling the rate of infection.Therefore,drugs that inhibit viral replication can significantly reduce YFV infection,and have important clinical application value.Studies have shown that drugs targeting YFV entry or replication have good antiviral activity in vitro and in vivo.However,their exact antiviral effect and safety issues in humans still need further investigation.Thus,it is hard to apply them in the clinic in a short period of time.Here,through high-throughput screening of small-molecule compound libraries approved by FDA,we found that rifapentine has significant antiviral activity.Therefore,we had tested the antiviral activity of rifapentine using the infection model of YFV in vivo and in vitro and deciphered the mechanism and target of rifapentine against YFV.This may provide theoretical and experimental basis for the clinical application of rifapentine in the treatment of YF patients.Methods1.The YFV-infected targeted cells were used to dected the antiviral effect and cytotoxicity of rifapentine.2.The experiment of time-of-drug-addition assay was employed to pinpoint the probable working stage of rifapentine involved in YFV infection.3.Sucrose density gradient ultracentrifugation was applied to remove the rifapentine incubated with YFV and check whether rifapentine influences the infectivity of YFV particles.4.The binding assay,endocytosis assay,kinetic tests and membrane fusion assay were taken to monitor the effect of rifapentine in different entry stage of YFV.5.YFV replicon plasmid carrying Nano Luc reporter gene was constructed to determine whether rifapentine inhibits the replication of YFV,and Biacore were implemented to evaluate the affinity of rifapentine and YFV NS5 and RNA dependent RNA polymerase（Rd Rp）subumit and docking analysis was performed to predict druggable sites.6.Constructed virus plasmids were transfected into target cells,and intracellular and extracellular viral particles were collected to evaluate the impact of rifapentine on the assembly and release of YFV particles.7.Type I interferon receptor-deficient mice(A129^-/-)and wild-type C57BL/6 mice were both infected by intraperitoneal ways to design the best experiment condition according to the body weight and survival rate of mice.The infected mice were given rifapentine orally and then detected anti-YFV effect of rifapentine in vivo by survival analysis,symptoms observation,immunofluorescence and immunohistochemistry,the viral load in tissues,and hematoxylin-eosin（HE）.Results1.Rifapentine had potent antiviral effect with low cytotoxicity against YFV,its 50%cytotoxic concentration(CC₅₀)and 50%of effective concentration(EC₅₀)were 147.3μM and 0.562μM respective.And rifapentine had no effect for other flaviviruses except for WNV（West Nile virus）with weak suppressive activity.2.Rifapentine didn’t influence the infection of YFV particle.3.Rifapentine inhibited the binding assay of YFV,but did not influence endocytosis and membrane fusion.4.The replicon experiment,Biacore and molecular docking experiment showed rifapentine suppressed YFV RNA replication via docking onto the active center of YFV Rd Rp.5.Rifapentine didn’t inhibit assembly and release of YFV particles.6.Rifapentine could significantly improve survival rate,ameliorate clinical symptoms,abate tissue damage,reduce viral load and expression of viral proteins in tissues in YFV-infected A129^-/-and C57BL/6 mice.ConclusionsThis present study demonstrates that rifapentine has significant antiviral activity against YFV,which was able to influence the cellular binding in the early stage of YFV infection and inhibit YFV replication via docking onto YFV Rd Rp,and significantly enhances the survival rate,abates tissue damage,reduces viral load and expression of viral proteins in YFV-infected A129^-/-and C57BL/6 mice.Therefore,rifapentine can be considered as a potential candidate of anti-YFV compounds,and worth the further studies.