Inhibition of Enterovirus 71 by Adenosine Analog NITD008

Published Ahead of Print 6 August 2014. 2014, 88(20):11915. DOI: 10.1128/JVI.01207-14. J. Virol. Bo Zhangand Bao-Di Shang, Peng Gong, Sylvie Alonso, Pei-Yong Shi Cheng-Lin Deng, Huimin Yeo, Han-Qing Ye, Si-Qing Liu,Analog NITD008Inhibition of Enterovirus 71 by Adenosine /content/88/20/11915Updated information and services can be found at: These include:REFERENCES/content/88/20/11915#ref-list-1at: This article cites 44 articles, 28 of which can be accessed free CONTENT ALERTSmore»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new /site/misc/reprints.xhtml Information about commercial reprint orders: /site/subscriptions/To subscribe to to another ASM Journal go to: on October 31, 2014 by Tsinghua University/Downloaded fromInhibition of Enterovirus71by Adenosine Analog NITD008Cheng-Lin Deng,a,b Huimin Yeo,c Han-Qing Ye,a Si-Qing Liu,a,b Bao-Di Shang,a Peng Gong,a Sylvie Alonso,c Pei-Yong Shi,dBo Zhang a,bCAS Key Laboratory of Special Pathogens and Biosafety,Center for Emerging Infectious Diseases,Wuhan Institute of Virology,Chinese Academy of Sciences,Wuhan, China a;Key Laboratory of Agricultural and Environmental Microbiology,Wuhan Institute of Virology,Chinese Academy of Sciences,Wuhan,China b;Department of Microbiology,Yong Loo Lin School of Medicine,Immunology Programme,Life Sciences Institute,National University of Singapore,Singapore c;Novartis Institute for Tropical Diseases,Singapore dABSTRACTEnterovirus71(EV71)is a major viral pathogen in China and Southeast Asia.There is no clinically approved vaccine or antiviral therapy for EV71infection.NITD008,an adenosine analog,is an inhibitor offlavivirus that blocks viral RNA synthesis.Here we report that NITD008has potent antiviral activity against EV71.In cell culture,the compound inhibits EV71at a50%effective concentration of0.67␮M and a50%cytotoxic concentration of119.97␮M.When administered at5mg/kg in an EV71mouse 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 EV71with increasing concentrations of NITD008.Resistance mutations were reproducibly mapped to the viral3A and3D polymerase regions.Resistance analysis with recombinant viruses demonstrated that either a3A or a3D mutation alone could lead to resistance to NITD008.A combination of both3A and3D mutations conferred higher resistance,suggesting a collaborative interplay between the3A and3D proteins during viral replication.The resistance results underline the importance of combination therapy required for EV71treatment. IMPORTANCEHuman enterovirus71(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 EV71infections.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 EV71infections.Mechanistically,we showed that mutations in the viral3A and3D polymerases alone or in combination could confer resistance to NITD008.The resistance results suggest an intrinsic interaction between viral proteins3A and3D during replication,as well as the importance of combination therapy for the treatment of EV71 infections.E nterovirus71(EV71),a member of genus Enterovirus in thefamily Picornaviridae,is one of the major causative agents of diarrhea,rashes,and hand,foot,and mouth disease(HFMD)in children.Besides EV71,HFMD could also be caused by coxsacki-evirus A16(CV-A16)infection(1).Infection with EV71is some-times associated with severe central nervous system diseases(2). Since itsfirst isolation in1969(3),EV71outbreaks have occurred frequently in western Pacific region countries,including China, Japan,Malaysia,and Singapore.In China alone,EV71caused over 1million infections and hundreds of deaths each year from2009 to2011(/tjsj/fdcrbbg/).No effective anti-EV71drug is available,although several clinical trials of inacti-vated EV71vaccine have recently been completed(4,5).In a phase III trial,an inactivated alum adjuvant EV71vaccine showed effi-cacies of90%against EV71-associated HFMD and80.4%against EV71-associated diseases(including HFMD,herpangina,menin-gitis or encephalitis,febrile illness,viral exanthema,and respira-tory infection)(4).Despite the promising progress in vaccine de-velopment,antiviral therapy should still be pursued as a complementary means of intervention against EV71infection.EV71is a nonenveloped,single-positive-strand RNA virus. The genomic RNA is approximately7,000nucleotides in length and contains a single,long open reading frame(ORF)that is flanked by the5=untranslated region(UTR)and the3=UTR with a poly(A)tail.VPg(3B)is covalently linked to the5=end of the viral genome and involved in the initiation of viral RNA replica-tion(6).The5=UTR consists of two functional domains,a clover-leaf and an internal ribosome entry site,which contribute to viral replication and translation,respectively.The ORF encodes a single polyprotein that is initially processed by viral proteases into three precursor proteins,P1,P2,and P3(7).Precursor P1is further cleaved to yield structural proteins VP0,VP3,and VP1;and VP0is finally cleaved into VP2and VP4during virion formation.P2and P3are proteolytically processed into various nonstructural pro-teins associated with viral replication,such as2A pro,2BC,2B,2C, 3AB,3A,3B(VPg),3CD,3C,and3D(8,9).Following viral pro-tein synthesis,the genomic RNA serves as a template for negative-strand RNA synthesis,generating the intermediate double-stranded replicative-form(RF)RNA.The double-stranded RF RNA is then used as a template to synthesize more positive-strand Received29April2014Accepted30July2014Published ahead of print6August2014Editor:S.PerlmanAddress correspondence to Bo Zhang,zhangbo@,or Pei-Yong Shi,pei_yong.shi@.S.A.,P.-Y.S.,and B.Z.are co-senior authors of this article.Copyright©2014,American Society for Microbiology.All Rights Reserved.doi:10.1128/JVI.01207-14October2014Volume88Number20Journal of Virology p.11915–11915 on October 31, 2014 by Tsinghua University / Downloaded fromRNA.The nascent positive-strand RNA is encapsidated.Viral par-ticles are released from infected cells(10).Inhibitors have been reported for distinct targets of EV71and its closely related rhinovirus,as summarized in a recent review (11).Pleconaril,picodavir,and BPROZ-194were shown to target viral capsid protein VP1(12–14).Rupintrivir was found to inhibit the3C proteases of both human rhinovirus and EV71(15,16). Nucleoside analogs(such as2=-C-methylcystidine,N-6-modified purine,and ribavirin)have been reported to inhibit the viral poly-merase of EV71(17–20).None of these inhibitors has been suc-cessfully developed into a clinically approved drug.Therefore, new chemical entities are urgently needed for EV71antiviral de-velopment.NITD008is an adenosine nucleoside analog that has been re-ported to selectively inhibit viruses in the family Flaviviridae(21). Although NITD008showed efficacy in a dengue virus mouse model,it was not advanced to clinics because of the adversefind-ings of a preclinical toxicity study(21).Here we report that NITD008potently inhibits EV71in cell culture and in a mouse model,demonstrating that this compound could potentially be developed for EV71therapy.Mechanistically,we characterized the profile of EV71resistance to the compound.The resistance results showed that mutations in the viral3A and3D polymerase regions could confer resistance to NITD008,suggesting intimate cross talk between3A and3D during viral replication. MATERIALS AND METHODSViruses,cells,and compound.Vero(African green monkey kidney)cells were cultured in Dulbecco modified Eagle medium(DMEM;Invitrogen) with10%fetal bovine serum(FBS),100U/ml of penicillin,and100␮g/ml of streptomycin.The wild-type(WT)recombinant viruses were prepared from the infectious cDNA clone of EV71(22),stored as aliquots atϪ80°C, and used for antiviral activity assays and resistant virus selection.EV71 strain5865/SIN/000009(S41)(23)was used for an animal study. NITD008was synthesized as reported previously(21).Antiviral activity assay.Vero cells were seeded into12-well plates (1ϫ105/well),incubated for24h,and then incubated with viruses at a multiplicity of infection(MOI)of0.1PFU/cell for1h in DMEM with10% FBS.After the cells were washed once with culture medium,a different concentration of NITD008was added to each well.Cell cultures were collected at48h postinfection(p.i.),and viral titers in supernatants were quantified by plaque assay.The incubations for each concentration treat-ment were performed in triplicate.Cytotoxicity assay.A3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay kit(a tetrazolium colorimetric assay; American Type Culture Collection)was used to determine the cytotoxic-ity of NITD008for Vero cells.We seeded Vero cells at1ϫ104per well(in 100␮l DMEM plus10%FBS)of a96-well plate,incubated them for6h, and then treated them with different concentrations of NITD008.After48 h of incubation,10␮l of MTT reagent was added and the plate was incu-bated for3.5h.Then100␮l of detergent reagents was added,and the plate was swirled gently and left in darkness at room temperature for4h.The absorbance of each well was measured with a microplate reader(Varios-kan Flash;Thermo Fisher)with a550-nmfilter.The cytotoxicity of NITD008was estimated by comparing the absorbance of the compound-treated cells with that of mock-treated cells.Generation of NITD008-resistant viruses.NITD008-resistant vi-ruses were selected by passaging WT EV71in Vero cells with increasing concentrations of NITD008.Two independent selections were carried out.For thefirst round of selection,Vero cells were infected with WT EV71at an MOI of0.1PFU/cell for1h,and then the infected cells were washed once and incubated with1␮M NITD008for another48h.The supernatants(designated P1)were collected and stored atϪ80°C.Viral titers from each selection were determined by plaque assay.The following selections were performed as described above.Different concentrations of NITD008are indicated for different passages(see Fig.2A).Sequencing of drug-resistant viruses.To identify mutations respon-sible for drug resistance,RNAs of selected viruses were extracted from virions in culture medium with RNeasy kits(Qiagen)according to the manufacturer’s instructions and amplified by reverse transcription(RT)-PCR with the SuperScript III one-step RT-PCR kit(Invitrogen).The com-plete viral genome was covered by four RT-PCRs with four pairs of prim-ers(22).The RT-PCR products were reclaimed by gel purification and sequenced.Preparation of recombinant EV71containing resistance mutations. EV71full-length cDNA clones with different mutations were constructed with the pACYC-EV71-FL infectious clone as the template(22).All mu-tations were introduced into the infectious clone by fusion PCR.Specifi-cally,fusion PCR products containing mutations within the2B/3A and 3D regions were introduced into pACYC-EV71-FL at the AatII-BsrGI and BsrGI-HindIII sites,respectively.All constructs were confirmed by DNA sequencing.To make recombinant viruses,WT and mutant genomic RNAs were in vitro transcribed from the HindIII-linearized cDNA plasmids with the MEGAscript T7kit(Ambion)according to the manufacturer’s protocols.The RNAs were transfected into Vero cells with DMRIE-C(Invitrogen).After transfection,supernatants were collected at different time points.The culture medium containing viruses was ali-quoted and stored atϪ80°C.Ethics statement.The animal experiments described here were ap-proved by the National University of Singapore Institutional Animal Care and Use Committee(protocol070/10).Nonterminal procedures were performed while animal were under anesthesia,and all efforts were made to minimize suffering.Mouse infection and drug treatment.Two-week-old AG129mice (B&K Universal)were bred and housed under specific-pathogen-free conditions in individual ventilated cages.Mice(nϭ8to10)were inocu-lated intraperitoneally(i.p.)with5865/SIN/000009(S41)(23)at a con-centration of107PFU/mouse as reported previously(24).Mice were treated orally with NITD008(6N HCl[1.5eq],1N NaOH[pH adjust-ment to3.5],and100mM citrate buffer[pH3.5]at a ratio of0.9:2.5:96.6) at a concentration of5mg/kg twice a day(b.i.d.)from the day of infection and for3consecutive days.Two control groups were included in the study design;one group was left untreated after infection,while the other was given the vehicle alone.Mice were monitored daily for20days for clinical illness and death.Clinical disease was scored as follows:0,healthy;1, ruffled hair and hunchbacked appearance;2,limb weakness;3,paralysis in one limb;4,paralysis in two limbs;5,death.To minimize animal suf-fering,mice were euthanized if they were paralyzed in two limbs.Determination of viral titers in organs from infected mice.Follow-ing systemic perfusion with50ml of sterile phosphate-buffered saline (PBS),organs and tissues were harvested from untreated infected mice and infected mice treated with NITD008or the vehicle at three different time points(days4,5,and6)p.i.and weighed.Samples were homoge-nized in DMEM,and the resulting homogenates were centrifuged at 14,000rpm for10min.Clarified supernatants werefiltered through a 0.22-␮m syringefilter(Millipore)before determination of the viral titer by plaque assay.Viral plaque assay.RD cells(ATCC CCL-136)were seeded into the wells of24-well plates(Nunc)at a density of105/well.The growth me-dium was removed,and the cells were infected with200-␮l volumes of suspensions containing10-fold serial virus dilutions ranging from10Ϫ1to 10Ϫ8.Infection was carried out for2h at37°C and5%CO2with rocking at half-hour intervals.One milliliter of1.2%Avicel in minimum essential medium(FMC BioPolymer)was added,and the cells were further incu-bated for3days.After the overlay medium was decanted,the cells were washed with PBS andfixed with4%paraformaldehyde.Crystal violet was used tofix and stain the cells.Plaques were then quantified via visual scoring.Deng et al. Journal of Virology on October 31, 2014 by Tsinghua University / Downloaded fromStatistical analyses.Differences between data on antiviral activity in cell culture were statistically evaluated with the open-source R-project software(Foundation for Statistical Computing,Vienna,Austria;http: ///).Antiviral activity was assessed with the Kruskal-Wallis rank sum test.Statistical analysis of the animal data was performed with GraphPad Prism,version5(GraphPad4Software).Data are ex-pressed as meansϮthe standard error of the means(SEM).Kaplan-Meier survival curves and clinical-score curves were analyzed with a log rank test and the Wilcoxon test,respectively.A two-tailed P value ofϽ0.05was considered statistically significant.RESULTSAntiviral activity of NITD008against EV71replication in cellculture.To facilitate antiviral activity discovery,we previouslydeveloped a stable EV71reporter virus containing an enhancedgreenfluorescent protein(eGFP)-encoding gene(eGFP-EV71)that could be used for high-throughput screening of inhibitors ofEV71(22).We tested NITD008in Vero cells infected with eGFP-EV71.The compound exhibited dose-dependent inhibition ofeGFP expression in infected cells(Fig.1A);under a phase-contrastmicroscope,equal numbers of cells from wells treated with differ-ent concentrations of the compound were observed(data notshown).To confirm the results from the reporter virus,we exam-ined the compound’s efficacy with WT EV71in a viral titer reduc-tion assay.As shown in Fig.1B,NITD008inhibited the WT virusat an EC50(50%effective concentration)of0.67␮M.At a concen-tration of4␮M,NITD008reduced viral titers by up to1,000-fold.Since infection with both EV71and CV-A16can cause HFMD,we tested NITD008for activity against CV-A16.The compoundshowed an EC50of0.64␮M(Fig.1C).To exclude the possibilitythat the inhibition of viral replication was due to NITD008-medi-ated cytotoxicity,we performed a cell proliferation-based cytotox-icity assay.As shown in Fig.1D,the compound showed a CC50(50%cytotoxic concentration)of119.97␮M in Vero cells.Even at16␮M NITD008,the cells retained around90%viability.Theresults allowed us to conclude that NITD008is a potent inhibitorof EV71in cell culture,with an estimated therapeutic index(TI[CC50/EC50])of179.It has been previously shown that rupintrivir(AG7088)effi-ciently blocked EV71replication as a potent3C proteinase inhib-itor(16,25,26).Therefore,we evaluated the antiviral effects ofdifferent concentrations of NITD008with or without rupintrivirby the Gluc-EV71infection assay(Fig.1E).The Gluc-EV71infec-tion assay was performed by infecting cells with complete EV71containing the gene for Gaussia luciferase(Gluc),and Gluc activ-ity was quantified to monitor antiviral activity.Our results indi-cated that the luciferase activities that resulted from dual treat-ments(NITD008plus0.25␮M rupintrivir and NITD008plus0.5␮M rupintrivir)were lower than when NITD008was used alone (P values were0.02and0.004,respectively),which was also thecase for treatment with rupintrivir alone(P values were both0.03).It is suggested that the combination of rupintrivir andNITD008had a more inhibitory effect on EV71replication than either NITD008or rupintrivir alone.Selection of resistant viruses.NITD008-resistant viruses were selected by continuously culturing EV71for18rounds in the pres-ence of gradually increasing concentrations of NITD008(Fig.2A), i.e.,1␮M for eight passages,1.5␮M for one passage,2␮M for four passages,and2.5␮M forfive passages.Two independent selec-tions(SelA and SelB)were performed to test the reproducibility of the resistance results.For each passage,viral titers in the mock-treated infection and the NITD008-treated infection were deter-mined;the viruses were subjected to resistance analysis with a viral titer reduction assay.Figure2B and C show two representative data from such resistance assays for passage8(P8)and P18,re-spectively.At P8,the selected viruses exhibited marginal resistance to1␮M NITD008compared with the WT virus.In contrast,at P18,both the SelA and SelB viruses exhibited significant resistance to NITD008.Specifically,at a2.5␮M inhibitor concentration, SelA and SelB viral titers were reduced by approximately50-and FIG1Antiviral and cytotoxic analyses of NITD008.(A)Antiviral activities of NITD008were analyzed by the eGFP-EV71infection assay.Different concen-trations of NITD008were added to Vero cells infected with eGFP-EV71at an MOI of0.1PFU/cell.At48h p.i.,eGFP expression was observed under a fluorescence microscope.Antiviral activity was indicated by the reduction of the number of eGFP-positive cells upon NITD008treatment.(B)Antiviral activity of NITD008analyzed by WT EV71viral titer reduction assay.Different concentrations of NITD008were added to Vero cells after1h of incubation with EV71at an MOI of0.1PFU/cell.At48h p.i.,culture medium was col-lected and viral titers were measured with plaque assays.Antiviral activity was indicated by the reduction of viral titers upon NITD008treatment.(C)Anti-viral activities of NITD008against CV-A16.The antiviral activity assay was performed the same way as that with EV71.(D)Cytotoxicity was evaluated by incubation of Vero cells with the indicated concentration of NITD008.At48h posttreatment,the viability of treated cells was measured by an MTT assay and presented as a percentage of that of untreated cells(see details in Materials and Methods).(E)Inhibition of EV71replication by a combination of rupintrivir and NITD008.The antiviral activities of different concentrations of NITD008, alone or in combination with rupintrivir,against EV71were tested.Vero cells were infected with Gluc-EV71at an MOI of0.1PFU/cell.The construction of Gluc-EV71will be reported elsewhere.At48h p.i.,infected cells were collected for the luciferase assay.Antiviral activity was indicated by a reduction of lucif-erase activity.Statistical significance(PϽ0.01)was detected for the antiviral activity of four treatments.NITD008as an Inhibitor of Enterovirus7111917 on October 31, 2014 by Tsinghua University / Downloaded from10-fold,respectively,whereas WT viral titers were suppressed by almost1,000-fold.These results demonstrate that resistant EV71 virus can be reproducibly obtained in cell culture and that the SelB virus is more resistant than the SelA virus to NITD008inhibition.As shown in Fig.2B and C,in the absence of NITD008,the WT and selected resistant viruses grew to comparable titers.In agree-ment with this result,similar plaque morphologies of the WT, SelA,and SelB viruses were observed(Fig.2D).The results indi-cate that the mutations that occurred in the resistant viruses did not reduce the magnitudes of replication in virus-infected cell cultures.Identification of resistance mutations.To identify mutations responsible for drug resistance,we sequenced the complete ge-nomes of P18SelA and SelB viruses,as well as the P18WT virus. No mutation was found in the P18WT virus.In contrast,several sense and nonsense mutations were found in the P18SelA and SelB viruses(Fig.3A).The nonsense mutations occurred at VP2, VP3,3B,and3C.The sense mutations in the SelA and SelB viruses were located in three regions.(i)An Ala¡Val substitution at amino acid position38of the2B protein(A38V2B)was found in SelB;(ii)two mutations in the3A protein,M62T3A and V75A3A, were recovered from SelA and SelB,respectively;and(iii)a com-mon mutation,V63A3D,in the3D polymerase was recovered from both selection viruses,while an additional M393L3D mutation in the3D protein was recovered from SelB.A close examination of the sequence chromatograms showed that the two mutations from SelA—M62T3A and V63A3D—contained pure adaptive se-quences(Fig.3B),whereas the two mutations from SelB—V63A3D and M393L3D—had adaptive sequences in a mixture with the WT sequence(Fig.3C).In summary,all of the amino acid changes were mapped to the3A and3D regions of both resistant viruses,except that an additional A38V2B mutation was found in SelB.Mutations in both3A and3D confer resistance to NITD008. Using an infectious cDNA clone of EV71,we examined the roles of the mutations recovered from the selection isolates in drug resis-tance.Individual or combined mutations were engineered into the infectious cDNA clone to generate recombinant viruses.The resistance phenotype of each recombinant virus was analyzed by comparing the viral titers after the virus was cultured in the pres-ence or absence of NITD008.The WT and selected(SelA and SelB) P18viruses were included as controls.Figure4A shows the resis-tance analysis of mutations recovered from a SelA isolate.Muta-tion M62T3A or V63A3D alone was sufficient to confer resistance to a level close to that of the SelA isolate.The M62T3A V63A3D double mutation slightly improved the resistance to a level equiv-alent to that of the original SelA isolate.The results demonstrate that both M62T3A and V63A3D play a role in resistance.Figure4B summarizes the resistance analysis of mutations re-covered from a SelB isolate.A mutant virus containing A38V2B alone did not show any resistance,whereas viruses containing the single mutation V75A3A,V63A3D,or M393L3D showed partial re-sistance.The V75A3A V63A3D,V75A3A M393L3D,and V63A3D M393L3D double mutations slightly increased resistance in com-parison with that of the single mutant viruses.The V75A3A V63A3D M393L3D triple mutation further increased resistance to a level similar to that of the original SelB isolate.Addition of A38V2B to the double mutation V75A3A V63A3D or to the triple mutation V75A3A V63A3D M393L3D did not change the level of resistance. These results demonstrate that(i)the mutation in2B(A38V2B) does not contribute to resistance,(ii)the mutations in3A (V75A3A)and3D(V63A3D and M393L3D)confer resistance,and (iii)combining two and three mutations in3A and3D additively increases resistance.Furthermore,Fig.4A and B clearly indicate that three mutations in SelB(V75A3A V63A3D M393L3D)account for the higher resistance than the two mutations in SelA(M62T3A V63A3D).NITD008treatment completely protected EV71-infected mice from lethal disease.The in vivo efficacy of NITD008was assessed in an AG129mouse model of EV71infection(24).Two-week-old AG129mice were infected i.p.with107PFU of EV71 strain41(S41).On the day of infection and for3consecutive days, the animals were administered NITD008at5mg/kg orally twice daily.Control groups consisted of untreated infected animals and infected animals given the vehicle alone.The animals were ob-served daily for20days p.i.,and clinical manifestations were scored.When two-limb paralysis was observed,the animals were euthanized.Untreated infected mice and infected animals treated with the vehicle displayed a100%mortality rate,with some deaths observed as early as6days p.i.(Fig.5A).These mice displayed a characteristic progression of severity of clinical symptoms,includ-ing ruffled hair,a hunched back,limb weakness,and one-limb paralysis,followed by two-limb paralysis,at which point the ani-mals were euthanized(Fig.5B).Although there was a delay in the manifestation of clinical symptoms in the vehicle-treated infected mice compared to that of the untreated infected group,treatment with the vehicle alone did not result in significant protection,as evidenced by the100%mortality rate observed by day11p.i.(Fig. 5A).In contrast,mice treated with NITD008remained healthyFIG2Selection and characterization of NITD008-resistant EV71.(A)Scheme used for selection of NITD008-resistant EV71.The concentrations of NITD008corresponding to different passages during selection are indicated. (B,C)Characterization of resistance phenotypes of P8(B)and P18(C)EV71. The indicated concentrations of NITD008were added to Vero cells after1h of incubation with EV71at an MOI of0.1PFU/cell.The viral titers in culture fluids in the presence or absence of NITD008were determined at48h p.i.byplaque assay.Two independent selections were performed(SelA and SelB).(D)Plaque morphologies of WT and selected resistant viruses.Deng et al. Journal of Virology on October 31, 2014 by Tsinghua University / Downloaded fromthroughout the course of the experiment and did not manifest any clinical signs of illness associated with severe EV71infection(Fig. 5A and B).These data thus demonstrate the protective efficacy of NITD008in a mouse model of severe EV71infection.Viral titers in organs from drug-treated infected mice were significantly reduced compared to those of the control groups. To further assess the in vivo protective efficacy of NITD008,at4,5, and6days p.i.,the viral titers in the front limbs,hind limbs,spines, and brains harvested from mice infected with EV71and treated with NITD008were quantified.These viral titers were compared to those measured in the control groups(untreated infected mice and infected mice treated with the vehicle).Infectious viruses were detected in the untreated infected mice and infected mice treated with the vehicle in all of the organs assessed(Fig.6A to D)accord-ing to a typical kinetic whereby the viruses accumulatedfirst in the limb muscles before reaching the spine and eventually accumu-lated in the brain,as reported previously(24).Interestingly,the viral titers measured in the infected mice treated with the vehicle were significantly lower than those measured in the untreated in-fected mice,suggesting that oral administration of the vehicle alone affects the infectivity of the virus.These lower viral titers correlate with the delayed appearance of the clinical manifesta-tions observed in this mouse group(Fig.5A and B).However, since eventually all of the mice in the vehicle-treated group dis-played the full range of clinical manifestations with a100%mor-tality rate,we conclude that the vehicle-mediated reduction in viral titers is not sufficient to protect the animals from EV71in-fection effectively.In contrast,significantly lower virus titers in all of the organs assessed,including the central nervous system,were measured in infected mice treated with NITD008(Fig.6A to D).Compared to those of vehicle-treated mice,reductions of average virus titers in the organs of NITD008-treated mice ranged from2-fold to a com-plete absence of viral detection(order of magnitude of5)by plaque assay.The results thus strongly support the in vivo protec-tive efficacy of NITD008against EV71.DISCUSSIONThe increase in EV71-associated morbidity and mortality in hu-mans has underlined the urgent need to develop an effective therapy for EV71infection.In this study,we demonstrated the in vitro and in vivo efficacy of NITD008,an adenosine analog,as anFIG3Mutations identified in NITD008-resistant EV71.(A)Mutations recovered from resistant viruses were tabulated according to the amino acid positions and the corresponding plete-genome sequencing of P18viruses from SelA and SelB was performed.Sense and nonsense mutations are in red and black,respectively.(B,C)Sequence chromatograms of mutations from resistant viruses.Representative sequencing chromatograms are presented for SelA(B) and SelB(C)viruses for P18selection.Original mutations are indicated at the top of each panel.Amino acid changes and positions in the corresponding proteins are indicated.NITD008as an Inhibitor of Enterovirus71 11919 on October 31, 2014 by Tsinghua University / Downloaded from。