Abstract

Hepatitis A virus (HAV) is a causative agent of acute hepatitis and can occasionally induce acute liver failure. However, specific potent anti-HAV drug is not available on the market currently. Thus, we investigated several novel therapeutic drugs through a drug repositioning approach, targeting ribonucleic acid (RNA)-dependent RNA polymerase and RNA-dependent deoxyribonucleic acid polymerase. In the present study, we examined the anti-HAV activity of 18 drugs by measuring the HAV subgenomic replicon and HAV HA11-1299 genotype IIIA replication in human hepatoma cell lines, using a reporter assay and real-time reverse transcription polymerase chain reaction, respectively. Mutagenesis of the HAV 5’ untranslated region was also examined by next-generation sequencing. These specific parameters were explored because lethal mutagenesis has emerged as a novel potential therapeutic approach to treat RNA virus infections. Favipiravir inhibited HAV replication in both Huh7 and PLC/PRF/5 cells, although ribavirin inhibited HAV replication in only Huh7 cells. Next-generation sequencing demonstrated that favipiravir could introduce nucleotide mutations into the HAV genome more than ribavirin. In conclusion, favipiravir could introduce nucleotide mutations into the HAV genome and work as an antiviral against HAV infection. Provided that further in vivo experiments confirm its efficacy, favipiravir would be useful for the treatment of severe HAV infection.

Highlights

  • The hepatitis A virus (HAV) genome consists of a single-stranded, positive-sense ribonucleic acid (RNA), approximately 7.5 kilobases in length, encoding a large open reading frame (ORF) that is flanked by highly conserved 5 and 3 untranslated regions (UTRs), and encodes four structural proteins (VP4, VP2, VP3, and VP1) and seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D)

  • We examined whether HAV RNA levels could be inhibited by favipiravir or ribaWvireinailnsoPLeCxa/mPRinFe/d5 cwelhlse.tFhaevripHirAavVirRaNt aAcolnecveenlstractoiounldofb1e00inμhMibriteesdultbeyd ifnavaip30ir–a4v0%ir or rirbeadvuicrtiinonininPtLhCe /HPARVF/R5NceAlllse.vFealsviinpiPrLavCi/rPaRtFa/5cocnelcles,nwtrhaetiroenasoHf A1V00RμNMA lreevseulsltewderiennaot30– 40re%durecdeducbtyio1n00inμtMherHibAavVirRinNinAPlLevCe/lPs RinF/P5LCce/lPlsR

  • We found that favipiravir decreased HAV replication in a dosedependent manner and induced nucleotide mutations in the HAV genome (Figure 3, Table 1)

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Summary

Introduction

The hepatitis A virus (HAV) genome consists of a single-stranded, positive-sense RNA, approximately 7.5 kilobases in length, encoding a large open reading frame (ORF) that is flanked by highly conserved 5 and 3 untranslated regions (UTRs), and encodes four structural proteins (VP4, VP2, VP3, and VP1) and seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). Reported risk factors for severe acute hepatitis or for higher mortality induced by HAV infection include an age of more than 40 years, preexisting liver disease, diabetes mellitus, and cardiovascular disease [3,4]. Favipiravir, ribavirin, and foscarnet sodium treatment significantly downregu4latoefd 17 HAV subgenomic replicon replication in a dose-dependent manner (Figure 2A–R). Favipiravir treatment at a concentration of 1 μM resulted in a >60% reduction in HAV subgenomic replicon replication in HuhT7 cells, whereas 10 μM favipiravir was required for a 67% (Dd)elcormeaibseuvinir,H(EA)VPSsuI-b62g0e6n,o(mF)iscorfeopsbliucvoinr,r(eGp)lifcoastciaornne(Ft isgoudriuem2A, ()H. DVaulsaicnygcalotvwiro-htayidlerdocShtluodreidnet’strtetaetsmt.ent at a concentration of 10 μM resulted in a 64% reduction in HAV subgenomic replicon replication (Figure 2H). VTireinatimnheinbtitws HithAVribavreirpinlicaantidonfo[1s5ca].rnet sodium at a concentration of 1 μM resulted in a 53% and a 53% reduction, respectively, in HAV subgenomic replicon replication, whereas 10 μM ribavirin anG2d.e2n.fooFtsaycvpaieprIinrIIaeAvtisrRoaednplidiucRamtibioawnvaiirnsinHreSuqihgu7niirCfiecedlalnsftolyr aDo6w6%nreagnudlatae H58e%patditeiscrAeaVsieru, sreHspAe1c1t-i1v2e9l9y, in HAV subgenAosmdiecpriecpteldicoinn rFeigpulirceat3iAon–C(F,ifgauvriepi2rBav,Gir),.

Favipiravir and Ribavirin Significantly Downregulate Hepatitis A Virus HA11-1299
Discussion
Cell Lines and Reagents
Transfection of the HAV Subgenomic Replicon into HuhT7 Cells and Reporter
Infection of Huh7
RNA Extraction and Quantification of HAV RNA
Dimethylthiazol Carboxymethoxyphenyl Sulfophenyl Tetrazolium (MTS) Assays
Targeted Deep Sequencing
Calculation of the Half Maximal Inhibitory Concentration (IC50)
Findings
Conclusions
Full Text
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