key: cord-0732005-wys8t1wi
authors: Günther, Stephan; Asper, Marcel; Röser, Christina; Luna, Luciano K.S.; Drosten, Christian; Becker-Ziaja, Beate; Borowski, Peter; Chen, Huan-Ming; Hosmane, Ramachandra S.
title: Application of real-time PCR for testing antiviral compounds against Lassa virus, SARS coronavirus and Ebola virus in vitro
date: 2004-06-15
journal: Antiviral Res
DOI: 10.1016/j.antiviral.2004.05.001
sha: 36dccf250d49da67c84d4c18c717c239d6fd7f73
doc_id: 732005
cord_uid: wys8t1wi

This report describes the application of real-time PCR for testing antivirals against highly pathogenic viruses such as Lassa virus, SARS coronavirus and Ebola virus. The test combines classical cell culture with a quantitative real-time PCR read-out. The assay for Lassa virus was validated with ribavirin, which showed an IC(50) of 9 μg/ml. Small-scale screening identified a class of imidazole nucleoside/nucleotide analogues with antiviral activity against Lassa virus. The analogues contained either dinitrile or diester groups at the imidazole 4,5-positions, and many of which possessed an acyclic sugar or sugar phosphonate moiety at the imidazole 1-position. The IC(50) values of the most active compounds ranged from 5 to 21 μg/ml. The compounds also inhibited replication of SARS coronavirus and Ebola virus in analogous assays, although to a lesser extent than Lassa virus.

tive only if administered early after infection . Several cases of Lassa fever recently imported into Europe have demonstrated that even state-of-the-art intensive care cannot prevent a fatal outcome if ribavirin treatment is initiated too late (Schmitz et al., 2002) . This report describes assays for testing potential antiviral compounds against Lassa virus and other highly pathogenic viruses, such as severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (Drosten et al., 2003) and Ebola hemorrhagic fever virus (Feldmann et al., 2003) . The test combines classical cell culture with real-time PCR. A class of imidazole nucleoside/nucleotide analogues was identified which showed inhibitory activity against Lassa virus and, to a lesser extent, against SARS-CoV and Ebola virus.

The flowchart of the test is shown in Fig. 1(A) . Vero cells were plated at a density of 4 × 10 4 /well of a 24-well plate. Twenty-four hours later, the cells were infected in the biosafety level 4 laboratory with Lassa virus strain AV at a multiplicity of infection (MOI) of 0.01, SARS-CoV at an MOI of 0.01, or Ebola virus subtype Zaire at an MOI of 0.1 in 100 l. The inoculum was removed after 1 h and replaced by fresh medium complemented with different concentrations of compound. None of the three viruses showed cytopathic effect under our culture conditions. Virus RNA concentration in supernatant was measured by real-time PCR during the exponential growth phase of the viruses (Fig. 1B) , i.e. after 2 days for Lassa virus and SARS-CoV, and after 3 days for Ebola virus. During this time, the viruses were growing by several orders of magnitudes if no inhibitor was added. For RNA preparation, a simple and inexpensive method (Boom et al., 1990 ) was adopted. A 140-l aliquot of supernatant was mixed with 560 l chaotropic lysis buffer AVL (Qiagen) and incubated at room temperature for 15 min. The lysate was added to 100 mg diatomaceous silica (Sigma-Aldrich) suspended in 560 l ethanol and incubated with agitation for 30 min at room temperature. The diatomaceous silica were pelleted by centrifugation and the pellet was washed with 500 l AW1 buffer (Qiagen), subsequently with 500 l AW2 buffer (Qiagen), and finally with 400 l acetone. The pellet was dried at 56 • C and the RNA eluted with 100 l water. Quantitative real-time PCR assays for Lassa virus, SARS-CoV, and Ebola virus Zaire were performed with the purified RNA based on previously published protocols (Gibb et al., 2001; Drosten et al., 2003; Asper et al., 2004) . The detailed reaction conditions are given in Table 1 . In vitro transcripts of the PCR target regions were amplified in the PCR to generate standard curves for quantification of the virus RNA in supernatant.

In selected experiments, infectious Lassa virus particles in the supernatant were quantified by immunofocus assay. Vero cells in 24-well plates were inoculated with serial 10-fold dilutions of supernatant. The inoculum was removed after 1 h and replaced by a 1% methylcellulose-medium overlay. After 5 days of incubation, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100, blocked with 10% fetal calf serum, and washed. Infected cell foci were detected with Lassa virus nucleoprotein (NP)-specific monoclonal antibody L2F1 diluted 1:50 (Hufert et al., 1989) . After washing and incubation with peroxidase-labelled anti-mouse IgG antibody 1:3000 (Dianova), foci were stained with 3,3 ,5,5 -tetramethylbenzidine (TMB) and counted.

Cell growth inhibition or cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) method. MTT solution (50 l, 5 mg/ml PBS) was added to the remaining supernatant of the cells from which an aliquot of supernatant was taken for measuring virus RNA or infectious particles concentration. The plate was incubated for 90 min at 37 • C. The supernatant was completely removed and the cells were fixed with 4% formaldehyde for 30 min. Tetrazolium crystals were dissolved in 1 ml ethanol for 10 min and the extinction was measured at 560 nm.

The concentrations required to inhibit virus replication by 50% (IC 50 ) or 90% (IC 90 ) were calculated by fitting a sigmoidal curve to the data following logarithmic transformation of the drug concentration. Curve fitting was performed by probit regression analysis using Statgraphics plus 5.0 software (Statistical Graphics Inc.).

Initial experiments were performed with the Lassa virus assay. It was validated using ribavirin as a positive control (Fig. 1C ). In agreement with published data on related arenaviruses (Huggins et al., 1984; Rodriguez et al., 1986; Andrei and De Clercq, 1990) , ribavirin inhibited Lassa virus replication with an IC 50 of 9 g/ml and an IC 90 of 14 g/ml. Furthermore, the inhibition kinetics measured with real-time PCR perfectly corresponded to the kinetics measured with (Demby et al., 1994; Asper et al., 2004) 20-l reaction based on Brilliant single-step quantitative RT-PCR kit (Stratagene): 2 l RNA, 1× buffer, 2.5 mM MgCl 2 , 800 M dNTP, 0.8 g bovine serum albumin, 5 M SybrGreen-binding molecule SGS (Drosten, 2003) , SybrGreen 1:10,000 (Molecular Probes), 1.25 U StrataScript RT, 1 U SureStart Taq the immunofocus assay (Fig. 1C) , indicating that real-time PCR can be used to measure the effect of compounds on Lassa virus replication. About 100 nucleoside analogues of different chemical classes available at the Institute were tested using this assay. For screening, three different concentrations in the range between 10 and 50 g/ml were tested. A class of imidazole nucleoside/nucleotide analogues was identified that showed inhibitory effects on Lassa virus without severely affecting cell growth and viability. Eighteen compounds of this class were tested ( Fig. 2 and Table 2 ). Particularly active compounds (3, 8, 23, 24, 34, 35, 56 , and 64) were analysed in detail using real-time PCR and immunofocus assay as a read-out (Fig. 3A) . Each of these compounds showed a similar effect on Lassa virus RNA and infectious particles, again demonstrating the validity of the PCR-based assay. IC 50 values as determined with the RNA concentration data ranged from 5 to 21 g/ml and IC 90 values ranged from 27 to 58 g/ml (Table 2 ). In the 48-h assay, compounds 8, 23, 24, 35, and 56 showed no significant influence on the rapidly growing Vero cells in concentrations up to 100 g/ml (Fig. 3A) . In order to test whether this class of nucleoside analogues shows broad-spectrum activ-ity, the complete set of 18 compounds was also tested against SARS-CoV and Ebola virus (Table 2) . Virus-specific effects were observed with the less toxic compounds 8, 10, 13, 23, 24, and 35 ( Fig. 3B and C) . However, the inhibiting effects against SARS-CoV and Ebola virus were considerably lower than observed for Lassa virus and overlapped with cell toxicity at higher concentrations. There was no evidence for a SARS-CoV-or Ebola virus-specific effect with compound 3 due to its toxicity. This study shows that real-time PCR is a useful approach to determine the antiviral effect of compounds. The assay provides an impartial and quantitative value of virus growth in cells in the presence of a compound over a broad dynamic range (i.e. several orders of magnitudes), with the effect of the compound on cell growth and viability being measured with the same cells. However, it is more laborious than classical methods, such as plaque reduction tests, immunofluorescence tests, or tests based on inhibition of virus-induced cytopathogenicity.

The nucleoside analogues identified as inhibitors of Lassa virus replication contained a 5-membered heterocyclic ring, namely an imidazole. A number of nucleoside analogues containing 5-membered heterocyclic rings, such as imidazole, pyrazole, and triazole, have since long been documented as potent antiviral, antibacterial, and antitumor agents (De Clercq and Luczak, 1975; Srivastava et al., 1976; Korbukh et al., 1978; Smith and Kirkpatrick, 1980; Revankar et al., 1981) . These include, but are not limited to, bredinin (Gerber et al., 1998) , a potent nucleoside antibiotic containing an imidazole ring, and ribavirin Witkowski et al., 1972; Smith and Kirkpatrick, 1980; Tam et al., 2001; Crotty et al., 2002; Lau et al., 2002) , a broad-spectrum antiviral agent containing a triazole nucleus. In addition to possessing a 5-membered heterocyclic ring, the target compounds also contain acyclic sugar or sugar phosphonate groups attached to the heterocyclic ring, and thus mimic part or whole of the natural sugar or sugar phosphate of nucleosides and nucleotides. In this context, they are analogues of the widely known potent, broad-spectrum antivirals, namely acyclovir [9-(2hydroxyethoxymethyl)guanine] (Schaeffer et al., 1978; Kaplan and Bain, 1999; Emmert, 2000; De Clercq et al., 2001) and PMEA/PMEG [9-(2-phosphonylmethoxyethyl)adenine/guanine] Kramata et al., 1998; Compton et al., 1999; Noble and Goa, 1999) . The observation that SARS-CoV and Ebola virus were also inhibited to a certain extent suggests that this class of analogues potentially has broad-spectrum antiviral activity. The mode of action is completely speculative and may be different depending on the chemical characteristics of a given compound. One possibility is direct inhibition of the virus replication machinery. Alternatively, the analogues might affect cellular pathways of nucleotide metabolism. The RNA synthesis of a highly replicating virus like Lassa virus probably is more susceptible to inhibition of nucleotide metabolism than the corresponding cellular processes. Inhibition of nucleotide metabolism could also account for the toxicity associated with some of the compounds.

Inhibitory effect of selected antiviral compounds on arenavirus replication in vitro

Molecular approaches for the treatment of hemorrhagic fever virus infections

Inhibition of different Lassa virus strains by alpha and gamma interferons and comparison with a less pathogenic arenavirus

phosphonylmethoxyethyl)adenine, 9-(2-phosphonylmethoxyethyl)-guanine, (R)-9-(2-phosphonylmethoxypropyl)adenine, and MDL 74,968] in cell cultures and murine sarcoma virus-infected newborn NMRI mice

Effect of fatty acids on arenavirus replication: inhibition of virus production by lauric acid

Rapid and simple method for purification of nucleic acids

A newly developed immunofluorescent assay for determining the Pichinde virus-inhibitory effects of selected nucleoside analogues

Inhibition of arenavirus multiplication in vitro by phenotiazines

-Phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cpr-PMEDAP) as a prodrug of 9

Ribavirin's antiviral mechanism of action: lethal mutagenesis?

Fluoroimidazoles as antiviral agents and inhibitors of polynucleotide biosynthesis

Antiviral activities of 5-ethynyl-1-beta-d-ribofuranosylimidazole-4-carboxamide and related compounds

Acyclic/carbocyclic guanosine analogues as anti-herpesvirus agents

Early diagnosis of Lassa fever by reverse transcription-PCR

Identification of a novel coronavirus in patients with severe acute respiratory syndrome

The therapeutic efficacy of ribamidil and virazole in experimental Lassa fever in monkeys

Treatment of common cutaneous herpes simplex virus infections

Ebola virus: from discovery to vaccine

Antiviral and virucidal activities against arenaviruses of zinc-finger active compounds

Immunosuppressive agents: recent developments in molecular action and clinical application

Development and evaluation of a fluorogenic 5 nuclease assay to detect and differentiate between Ebola virus subtypes Zaire and Sudan

Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies

Synergistic antiviral effects of ribavirin and the C-nucleoside analogs tiazofurin and selenazofurin against togaviruses, bunyaviruses, and arenaviruses. Antimicrob

Lassa virus infection of rhesus monkeys: pathogenesis and treatment with ribavirin

Cognitive outcome after emergent treatment of acute herpes simplex encephalitis with acyclovir

4-Amino-1-beta-d-ribofuranosylpyrazole-3-carboxamide: synthesis of pyrazole nucleosides by the fusion method without a catalyst

-phosphonylmethoxyethyl)guanine (PMEG) by DNA polymerase delta and epsilon in vitro

Mechanism of action of ribavirin in the combination treatment of chronic HCV infection

Lassa fever. Effective therapy with ribavirin

Therapeutic potential of HPMPC (cidofovir), PMEA (adefovir) and related acyclic nucleoside phosphonate analogues as broad-spectrum antiviral agents

PMEA (adefovir) and related acyclic nucleoside phosphonate analogues: a review of their pharmacology and clinical potential in the treatment of viral infections

New hypoxanthine nucleosides with RNA antiviral activity

Adefovir dipivoxil

Synthesis and biological activity of certain 1,2,3-triazole carboxamide nucleosides related to bredinin and pyrazofurin

Antiviral effect of ribavirin on Junin virus replication in vitro

-hydroxyethoxymethyl) guanine activity against viruses of the herpes group

Monitoring of clinical and laboratory data in two cases of imported Lassa fever

Broad-spectrum antiviral activity of Virazole: 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide

Selective inhibition of arthropod-borne and arenaviruses in vitro by 3 -fluoro-3 -deoxyadenosine

Ribavirin: a Broad Spectrum Antiviral Agent

Synthesis and antiviral and antimicrobial activity of certain 1-beta-d-ribofuranosyl-4,5-disubstituted imidazoles

Experimental Lassa fever virus infection successfully treated with ribavirin

Mechanisms of action of ribavirin in antiviral therapies

Antiviral effect of brassinosteroids against herpes virus and arenaviruses

Design, synthesis, and broad spectrum antiviral activity of 1--d-ribofuranosyl-1,2,4-triazole-3-carboxamide and related nucleosides

The research was supported by a grant # 9RO1 AI55452 from the United States National Institute of Allergy and Infectious Diseases (NIAID/NIH) (to R.S.H.), grant E/B41G/1G309/1A403 from the Bundesamt für Wehrtechnik und Beschaffung (to S.G.), and a grant from the Concelho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil (to L.K.S.L.). The Bernhard-Nocht-Institut is supported by the Bundesministerium für Gesundheit and the Freie und Hansestadt Hamburg.