West Nile virus (WNV) is an emerging mosquito-borne pathogen that was reported for the first time in the Western hemisphere in August 1999, when an encephalitis outbreak in New York City resulted in 62 clinical cases and 7 deaths. WNV, for which no antiviral therapy has been described, was recently recovered from a pool of mosquitoes collected in New York City. In anticipation of the recurrence of WNV during the summer of 2000, an analysis was made of the efficacy of the nucleoside analogue ribavirin, a broad-spectrum antiviral compound with activity against several RNA viruses, for treatment of WNV infection. High doses of ribavirin were found to inhibit WNV replication and cytopathogenicity in human neural cells in vitro

Briese, Thomas

Fischer, Nicole

Jordan, Ingo

Lau, Johnson Yiu-Nam

Lipkin, W. Ian

Columbia University Academic Commons

1214CONCISE COMMUNICATIONRibavirin Inhibits West Nile Virus Replication and Cytopathic Effectin Neural CellsIngo Jordan,1 Thomas Briese,1 Nicole Fischer,1Johnson Yiu-Nam Lau,2 and W. Ian Lipkin11Emerging Diseases Laboratory, Departments of Microbiologyand Molecular Genetics, Neurology, and Anatomy and Neurobiology,University of California, Irvine, and 2ICN Pharmaceuticals,Costa Mesa, CaliforniaWest Nile virus (WNV) is an emerging mosquito-borne pathogen that was reported for thefirst time in the Western hemisphere in August 1999, when an encephalitis outbreak in NewYork City resulted in 62 clinical cases and 7 deaths. WNV, for which no antiviral therapy hasbeen described, was recently recovered from a pool of mosquitoes collected in New York City.In anticipation of the recurrence of WNV during the summer of 2000, an analysis was madeof the efficacy of the nucleoside analogue ribavirin, a broad-spectrum antiviral compoundwith activity against several RNA viruses, for treatment of WNV infection. High doses ofribavirin were found to inhibit WNV replication and cytopathogenicity in human neural cellsin vitro.The Flaviviridae, a family of enveloped positive-strand RNAviruses, include West Nile virus (WNV), as well as other sig-nificant human pathogens, such as hepatitis C, yellow fever,dengue, Japanese encephalitis, and St. Louis encephalitis vi-ruses [1]. Whereas hepatitis C virus (genus Hepacivirus) maybe transmitted by blood products or sexual activity, most mem-bers of the Flavivirus genus, including WNV, are transmittedby arthropods [1]. After an incubation period of 3–6 days,WNV infections typically result in subclinical or nonspecific,mild febrile illnesses lasting 3–5 days [2]. Host factors, includingage and immune status, influence pathogenesis. Elderly indi-viduals are at higher risk of progressing to fatal neurologicaldisease, involving profound motor weakness and axonal neu-ropathy [3]. WNV is endemic in parts of Africa, Europe, theMiddle East, and Asia [2], and it has potential to cause largeepidemics in populations where background immunity is low.The largest African epidemic occurred in 1974 in the Capeprovince, South Africa, with 3000 clinical cases diagnosed afterunusually heavy rainfalls; a large outbreak in Romania in 1996resulted in 352 acute infections of the central nervous system,with a case-fatality rate of ∼4% [2]. WNV was recognized forthe first time in the Americas in August 1999, when an outbreakin New York City resulted in 62 cases of acute encephalitis.Seven patients died, and mortality among horses and birds wasReceived 7 June 2000; revised 12 July 2000; electronically published 22August 2000.Reprints or correspondence: Dr. W. Ian Lipkin, University of California,Emerging Diseases Laboratory, 3216 Gillespie Bldg., Irvine, CA 92697-4292(ilipkin@uci.edu).The Journal of Infectious Diseases 2000;182:1214–7q 2000 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2000/18204-0027$02.00substantial [3]. Genetic analysis of the envelope protein se-quence indicated that the viral strain most closely resembledan isolate from a goose in Israel in 1998, demonstrating thewide geographic distribution of this emerging pathogen [4, 5].Antiviral therapy for hepatitis C has been described [6]; how-ever, no specific treatment has been reported for WNV or otherflavivirus infections. We recently reported that the presence ofWNV RNA in cerebrospinal fluid may be associated with poorclinical outcome [7]. These findings, together with indicationsthat WNV has persisted in mosquitoes in New York City duringthe winter [8], emphasize the importance of rapid diagnosis ofWNV infections and of establishing antiviral treatment. Wetherefore tested a panel of nucleoside analogues for antiviralactivity against WNV.Materials and MethodsCells and virus. Human oligodendroglial (OL) cells [9] werecultured in a humidified atmosphere at 377C, 5% CO2 in Dulbecco’smodified Eagle medium/high glucose (DMEM; Irvine Scientific,Santa Ana, CA) supplemented with 10% heat-inactivated fetal calfserum (FCS), 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. Aedes albopictus C6/36 cells were cultured at257C in Leibovitz’s L-15 medium (Gibco-BRL, Gaithersburg, MD)supplemented with 10% heat-inactivated FCS, 10% tryptose phos-phate broth (Gibco-BRL), 100 U/mL penicillin, and 100 mg/mLstreptomycin. Virus was recovered after DMRIE-C (Gibco-BRL)liposome-mediated transfection of WNV-NY1999 RNA intoOL cells. Virus-maintenance medium was a 1:1 mixture of sup-plemented DMEM and OPTI-MEM I reduced-serum medium(Gibco-BRL) resulting in 5% final serum concentration. Virus waspassaged only once in either OL or C6/36 cells after recovery byRNA transfection; virus stocks were prepared as clarified OL cul-JID 2000;182 (October) Ribavirin Inhibits WNV Replication 1215ture supernatant when ∼80% of the cells had cytopathic effect oras C6/36 culture supernatant 4 days after infection with the initialOL virus isolate. Plaque-forming units were determined on con-fluent OL cell layers by serial dilution of viral stock in duplicatesafter overlay with 1% low-melting agarose in DMEM.Antiviral drug evaluation. Nucleoside analogues (3-deazaguan-ine; ICN 10169; ICN 10776; ICN 15100; 2′,3′-dideoxyinosine; ri-bavirin; ribavirin analogues ICN 17261 and ICN 17377; FTC[b-L-2′,3′-dideoxy-5-fluoro-3′-thiacytidine]; and pyrazofurin) wereprovided by ICN Pharmaceuticals (Costa Mesa, CA). The drugswere dissolved in dimethyl sulfoxide to 100 mM and in OPTI-MEM to working concentrations. Two hours before treatment withdrug at various concentrations, OL cells were seeded in virus-main-tenance medium to 80% confluence on microtiter plates and wereinfected at a multiplicity of 0.1. Half the wells of each plate weretreated with drug but not infected. After 3 days, 50 mL of super-natant were transferred onto fresh microtiter plates for cytotoxicityassays (release of lactate dehydrogenase from lysed cells; CytoTox96; Promega, Madison, WI); 50 mL of supernatant was extractedwith 150 mL Tri-Reagent LS (Molecular Research, Cincinnati, OH)for real-time reverse transcriptase (RT)–polymerase chain reaction(PCR) analysis [7], using diagnostic primers directed against theNS5 region; the remaining 100 mL of supernatant and the under-lying cell layer were supplemented with XTT labeling mix to assaycell proliferation (mitochondrial metabolism of XTT tetrazoliumsalt; Roche, Basel, Switzerland). The cytotoxicity and cell prolif-eration assays were measured with a vmax plate reader (MolecularDevices, Sunnyvale, CA) at 490 nm, with 650 nm as referencewavelength.ResultsA panel of 10 nucleoside analogues was screened for antiviralactivity in WNV-NY1999–infected human OL cells at 20 mMconcentrations. This initial screening was performed by sizefractionation and Northern blot analysis of total RNA fol-lowed by hybridization with probes against the NS5 poly-merase gene and host-cell glyceraldehyde-3-phosphate-dehy-drogenase (GAPDH) as reference (data not shown). Twocompounds, pyrazofurin and ribavirin, had antiviral activity.Pyrazofurin affects activity of orotate monophosphate decar-boxylase in pyrimidine biosynthesis. Because of its high cyto-toxicity, pyrazofurin was not tested further. However, the drug’sability to reduce WNV genomic RNA to levels !1% ofGAPDH-normalized infected untreated control values suggeststhat further drug screening using less toxic derivatives may beuseful.For a detailed evaluation of the activity of ribavirin, we mea-sured 3 parameters (figure 1): (1) cell lysis due to cytopathiceffect and drug-induced cytotoxicity was measured via the re-lease of a stable cytoplasmic enzyme (lactate dehydrogenase)into the culture supernatant; (2) viral replication was measuredwith real-time PCR, using diagnostic primers that amplified a69-bp fragment from the NS5 region, detected by a linearfluorochrome/quencher probe annealing within that region; and(3) inhibition of cellular proliferation and viability due to in-fection or drug side effects was measured at the end of treatmentby mitochondrial metabolic activity.Ribavirin reduced extracellular WNV RNA by ∼50% at 60-mM (ED50) and by ∼90% at 190-mM (ED90) concentrations (figure1A); the reference value of 100% extracellular viral RNA wasdetermined by real-time RT-PCR, using RNA isolated from thesupernatant of infected untreated cells. At the ED50 and ED90concentrations, cytopathogenicity decreased from 90% to 20%of the reference value obtained with infected untreated controls(figure 1A). Cell proliferation of infected untreated cells was∼55% of the noninfected untreated controls, and it recovered to95% at 200 mM ribavirin (figure 1A, 1C). The titer of WNV wasreduced by 4 orders of magnitude (from 107 to 103 pfu/mL) inthe presence of 200 mM ribavirin (figure 1D). The greater re-duction in plaque-forming units than in extracellular viral RNAat 200 mM ribavirin suggests that not all RNA measured by RT-PCR is present as infectious units. In noninfected cells, ribavirindid not inhibit cell growth or produce cytotoxic effects at con-centrations as high as 400 mM (figure 1B). All studies were per-formed in duplicate. Results were similar for virus stocks gen-erated on OL or C6/36 cells and for 2 batches of ribavirin. Onthe basis of these experiments, we estimate that ribavirin has anin vitro selectivity index of ∼7.DiscussionRibavirin is a guanosine analogue with in vitro antiviral ac-tivity against a broad spectrum of RNA and DNA viruses,including members of the Arenaviridae, Bunyaviridae, Flavi-viridae, Herpesviridae, Orthomyxoviridae, Paramoyxoviridae,and Bornaviridae families. Within the Flaviviridae, in vitro ac-tivity has been demonstrated for yellow fever, dengue types 1–4,banzi, Japanese encephalitis [10, 11], and hepatitis C viruses.Ribavirin has been used to treat human patients infected withrespiratory syncytial [12], Lassa [13], Hantaan [14], La Crosse[15], and (in combination with interferon-a) hepatitis C [6] vi-ruses. Ribavirin is phosphorylated by cellular enzymes and ispostulated to exert antiviral effects by several mechanisms: (1)depletion of the intracellular GTP pool by interference withhost-cell inosine monophosphate (IMP) dehydrogenase, the en-zyme that converts IMP to xanthosine monophosphate, a pre-cursor molecule in the biosynthesis of GTP and dGTP; (2)interference with mRNA capping guanylylation; (3) specific in-hibition of some viral polymerases; and (4) enhancement of theTh1 antiviral immune response [11, 16].After oral administration, ribavirin concentrations in cere-brospinal fluid are ∼70% of those in serum [17]. We are notaware of published data concerning levels of ribavirin in thebrain after intravenous administration; however, on the basisof results from the intravenous 7-day regimens employed for1216 Jordan et al. JID 2000;182 (October)Figure 1. Concentration-dependent effect of ribavirin on viral replication, cell proliferation, and cytopathogenicity in oligodendroglial (OL)cells. Two hours before treatment with ribavirin at various concentrations, cells were infected with WNV-NY1999 at a multiplicity of 0.1. Cellswere assayed 3 days later for host cell proliferation (v), cytopathogenicity (m), and amount of viral RNA in the culture supernatant (m; A).Noninfected cells were assayed in parallel for cell proliferation (V) and drug-induced cytotoxic effect (M; B). Results are expressed as percentageof control values. One hundred percent cellular proliferation values were obtained from untreated uninfected cells. One hundred percent viralreplication and cytopathogenicity values were obtained from untreated infected cells. Reduction of cytopathogenicity (magnification, 3200; C)and virus load (plaque-forming units per millimeter; D) after 3 days treatment with 200 mM ribavirin are also shown.treatment of La Crosse encephalitis and Hantaan fever with aloading dose of 25–33 mg/kg of body weight followed by 24–64mg/kg of body weight per day administered intravenously [14,15], we extrapolate that intravenous administration of ribavirinwill probably result in ∼100 mM concentrations in the cerebro-spinal fluid. This concentration exceeds our measured ED50 of60 mM for reduction of extracellular viral RNA. Although ahigher concentration of ribavirin in cerebrospinal fluid may bemore desirable, the efficacy:benefit ratio will be revealed onlythrough clinical studies. It is important to note that reversiblehemolytic anemia can occur at these doses and may requiretransfusion [18].One pool of mosquitoes collected in New York City in Feb-ruary 2000 yielded live WNV [8], indicating that this emergingpathogen has established its presence in the Western hemi-sphere. WNV is not yet endemic in the Americas, and the shortperiod since its introduction, in August 1999, may aggravateoutbreaks until background immunity has developed in thehuman and peridomestic animal population. It is clearly im-portant to identify drugs for treatment of WNV that will havebetter selectivity and therapeutic indices than ribavirin. Fur-thermore, the efficacy of ribavirin in animal models of WNVencephalitis remains to be addressed. Nonetheless, our findingssuggest that ribavirin at high doses may improve prognosis inindividuals with WNV encephalitis.AcknowledgmentsWe thank Charlie Calisher for helpful comments and Stanley Lipkinfor research support.References1. 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Ribavirin inhibits West Nile virus replication and cytopathic effect in neural cells

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Ribavirin inhibits West Nile virus replication and cytopathic effect in neural cells

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