title: Inhibition of enterovirus 71 by adenosine analog NITD008.
abstract: Enterovirus 71 (EV71) is a major viral pathogen in China and Southeast Asia. There is no clinically approved vaccine or antiviral therapy for EV71 infection. NITD008, an adenosine analog, is an inhibitor of flavivirus that blocks viral RNA synthesis. Here we report that NITD008 has potent antiviral activity against EV71. In cell culture, the compound inhibits EV71 at a 50% effective concentration of 0.67 μM and a 50% cytotoxic concentration of 119.97 μM. When administered at 5 mg/kg in an EV71 mouse model, the compound reduced viral loads in various organs and completely prevented clinical symptoms and death. To study the antiviral mechanism and drug resistance, we selected escape mutant viruses by culturing EV71 with increasing concentrations of NITD008. Resistance mutations were reproducibly mapped to the viral 3A and 3D polymerase regions. Resistance analysis with recombinant viruses demonstrated that either a 3A or a 3D mutation alone could lead to resistance to NITD008. A combination of both 3A and 3D mutations conferred higher resistance, suggesting a collaborative interplay between the 3A and 3D proteins during viral replication. The resistance results underline the importance of combination therapy required for EV71 treatment. Importance: Human enterovirus 71 (EV71) has emerged as a major cause of viral encephalitis in children worldwide, especially in the Asia-Pacific region. Vaccines and antivirals are urgently needed to prevent and treat EV71 infections. In this study, we report the in vitro and in vivo efficacy of NITD008 (an adenosine analog) as an inhibitor of EV71. The efficacy results validated the potential of nucleoside analogs as antiviral drugs for EV71 infections. Mechanistically, we showed that mutations in the viral 3A and 3D polymerases alone or in combination could confer resistance to NITD008. The resistance results suggest an intrinsic interaction between viral proteins 3A and 3D during replication, as well as the importance of combination therapy for the treatment of EV71 infections.
authors.affiliation: CAS Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Cheng-Lin
authors.initials: CL
authors.lastname: Deng
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, Immunology Programme, Life Sciences Institute, National University of Singapore, Singapore.
authors.forename: Huimin
authors.initials: H
authors.lastname: Yeo
authors.affiliation: CAS Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Han-Qing
authors.initials: HQ
authors.lastname: Ye
authors.affiliation: CAS Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Si-Qing
authors.initials: SQ
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Bao-Di
authors.initials: BD
authors.lastname: Shang
authors.affiliation: CAS Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Gong
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, Immunology Programme, Life Sciences Institute, National University of Singapore, Singapore.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Alonso
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore zhangbo@wh.iov.cn pei_yong.shi@novartis.com.
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
authors.affiliation: CAS Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China zhangbo@wh.iov.cn pei_yong.shi@novartis.com.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology* / Q000627:therapeutic use; D002522:Chlorocebus aethiops; D024882:Drug Resistance, Viral / Q000235:genetics; D029821:Enterovirus A, Human / Q000187:drug effects* / Q000254:growth & development / Q000502:physiology; D004769:Enterovirus Infections / Q000188:drug therapy / Q000503:physiopathology / Q000821:virology; D051379:Mice; D009154:Mutation; D014709:Vero Cells; D019562:Viral Load; D010948:Viral Plaque Assay; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; C000593137:NITD008; D000241:Adenosine
references.citation: Drug Discov Today. 2010 Dec;15(23-24):1041-51
references.pmid: 20974282
references.citation: J Virol. 1997 Dec;71(12):9490-8
references.pmid: 9371611
references.citation: J Am Coll Cardiol. 1988 Nov;12(5):1334-41
references.pmid: 2844874
references.citation: J Virol. 1992 Oct;66(10):6045-57
references.pmid: 1326655
references.citation: N Engl J Med. 2014 Feb 27;370(9):818-28
references.pmid: 24571754
references.citation: Antimicrob Agents Chemother. 2008 Mar;52(3):971-9
references.pmid: 18180344
references.citation: Antimicrob Agents Chemother. 2004 May;48(5):1766-72
references.pmid: 15105133
references.citation: Proc Natl Acad Sci U S A. 2010 Dec 28;107(52):22505-10
references.pmid: 21148772
references.citation: Expert Rev Vaccines. 2014 May;13(5):609-18
references.pmid: 24621093
references.citation: J Virol. 1995 Nov;69(11):7169-79
references.pmid: 7474138
references.citation: J Virol. 2013 Feb;87(3):1759-69
references.pmid: 23175376
references.citation: J Virol. 2000 Nov;74(22):10359-70
references.pmid: 11044080
references.citation: Antimicrob Agents Chemother. 1992 Apr;36(4):727-32
references.pmid: 1503435
references.citation: Antiviral Res. 2013 Feb;97(2):198-205
references.pmid: 23267829
references.citation: Virology. 1996 Mar 15;217(2):459-69
references.pmid: 8610437
references.citation: J Virol. 2005 Feb;79(3):1943-7
references.pmid: 15650220
references.citation: Biochem Pharmacol. 2012 Jan 15;83(2):185-92
references.pmid: 21889497
references.citation: J Infect Dis. 1974 Mar;129(3):304-9
references.pmid: 4361245
references.citation: J Biol Chem. 1994 Nov 18;269(46):29173-81
references.pmid: 7961883
references.citation: Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7289-94
references.pmid: 12754380
references.citation: Antimicrob Agents Chemother. 2009 May;53(5):1850-7
references.pmid: 19237651
references.citation: J Biol Chem. 1998 May 22;273(21):12832-40
references.pmid: 9582311
references.citation: PLoS Negl Trop Dis. 2012;6(9):e1752
references.pmid: 23029567
references.citation: J Virol. 2008 Feb;82(3):1465-73
references.pmid: 18032495
references.citation: Antimicrob Agents Chemother. 2004 Sep;48(9):3523-9
references.pmid: 15328120
references.citation: J Virol. 2007 Jun;81(11):5669-84
references.pmid: 17360746
references.citation: Virol J. 2013 Jan 22;10:28
references.pmid: 23339605
references.citation: J Virol. 2011 Oct;85(19):10319-31
references.pmid: 21795339
references.citation: J Virol. 2011 Oct;85(19):10364-74
references.pmid: 21795353
references.citation: Hum Vaccin Immunother. 2014;10(2):360-7
references.pmid: 24231751
references.citation: PLoS One. 2013 May 08;8(5):e60272
references.pmid: 23667424
references.citation: Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20435-9
references.pmid: 19918064
references.citation: Microbiol Immunol. 2002;46(11):801-8
references.pmid: 12516778
references.citation: J Virol. 2011 Oct;85(19):10021-30
references.pmid: 21813612
references.citation: Antiviral Res. 2007 Mar;73(3):161-8
references.pmid: 17055073
references.citation: J Virol. 2012 Jan;86(1):542-52
references.pmid: 22031931
references.citation: Nat Med. 2009 Jul;15(7):794-7
references.pmid: 19543284
references.citation: EMBO J. 2004 Sep 1;23(17):3462-71
references.pmid: 15306852
references.citation: Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11000-7
references.pmid: 10500114
references.citation: J Virol. 2012 Feb;86(4):2121-31
references.pmid: 22130542
references.citation: Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6895-900
references.pmid: 11371613
references.citation: Hepatology. 2011 Jan;53(1):32-41
references.pmid: 21254160
references.citation: J Virol Methods. 2012 Jun;182(1-2):93-8
references.pmid: 22465253