Application of real-time PCR for testing antiviral compounds against Lassa virus, SARS coronavirus and Ebola virus in vitro

Günther, Stephan; Asper, Marcel; Röser, Christina; Luna, L.; Drosten, Christian; Becker-Ziaja, Beate; Borowski, Peter; Chen, Huanming; Hosmane, Ramachandra S.

doi:10.1016/j.antiviral.2004.05.001

Cited by 53 publications

(35 citation statements)

References 39 publications

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“…Our data ex-tend these findings to Lassa virus and indicate that the drug directly acts on the replicon because inhibition of vaccinia virus by ribavirin (29) cannot play a role. In an in vitro infection system, ribavirin inhibited replication of Lassa virus strain AV with a 50% inhibitory concentration of 9 g/ml and a 90% inhibitory concentration of 14 g/ml (25). Both values are well in agreement with the inhibition kinetics that were observed with the strain AV-based replicon system, suggesting that the replicon system indeed mirrors the effect of the drug on infectious virus.…”

Section: Discussionsupporting

confidence: 74%

Replicon System for Lassa Virus

Haß

Gölnitz

Müller

et al. 2004

J Virol

124

122

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Lassa virus is endemic to West Africa and causes hemorrhagic fever in humans. To facilitate the functional analysis of this virus, a replicon system was developed based on Lassa virus strain AV. Genomic and antigenomic minigenomes (MG) were constructed consisting of the intergenic region of S RNA and a reporter gene (Renilla luciferase) in antisense orientation, flanked by the 5 and 3 untranslated regions of S RNA. MGs were expressed under the control of the T7 promoter. Nucleoprotein (NP), L protein, and Z protein were expressed from plasmids containing the T7 promoter and internal ribosomal entry site. Transfection of cells stably expressing T7 RNA polymerase (BSR T7/5) with MG in the form of DNA or RNA and plasmids for the expression of NP and L protein resulted in high levels of Renilla luciferase expression. The replicon system was optimized with respect to the ratio of the transfected constructs and by modifying the 5 end of the MG. Maximum activity was observed 24 to 36 h after transfection with a signal-to-noise ratio of 2 to 3 log units. Northern blot analysis provided evidence for replication and transcription of the MG. Z protein downregulated replicon activity close to background levels. Treatment with ribavirin and alpha interferon inhibited replicon activity, suggesting that both act on the level of RNA replication, transcription, or ribonucleoprotein assembly. In conclusion, this study describes the first replicon system for a highly pathogenic arenavirus. It is a tool for investigating the mechanisms of replication and transcription of Lassa virus and may facilitate the testing of antivirals outside a biosafety level 4 laboratory.

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Section: Discussionsupporting

confidence: 74%

Replicon System for Lassa Virus

Haß

Gölnitz

Müller

et al. 2004

J Virol

124

122

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“…4A). They had no effect on cell viability as tested by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay (8) (Fig. 4B).…”

mentioning

confidence: 99%

“…Vero cells in a well of a 24-well plate were transfected with 50 pmol of siRNA using X-tremeGENE and infected 4 h later at a multiplicity of infection of 0.01. The virus titer in cell culture supernatant was determined after 48 h by immunological focus assay (8). Both siRNAs inhibited replication of all virus strains tested by up to 1 log unit (Fig.…”

mentioning

confidence: 99%

Broad-Spectrum Antiviral Activity of Small Interfering RNA Targeting the Conserved RNA Termini of Lassa Virus

Müller

Günther

2007

Antimicrob Agents Chemother

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“…In vitro testing demonstrated antiviral activity against LF virus in Vero cell cultures at ribavirin concentrations as low as 5 µg/ ml to 50 µg/ml (IC50 of 9 µg/ml) (Günther et al, 2004;Jarhling et al, 1980;Stephen et al, 1980). The mean inhibitory concentrations (MIC) have ranged from 1 µg/ ml to 10 µg/ml (Connor et al, 1984).…”

Section: Lassa Fever In Vitro and Animal Ribavirin Studiesmentioning

confidence: 95%

Experience with Ribavirin for Treatment and Postexposure Prophylaxis of Hemorrhagic Fever Viruses: Crimean Congo Hemorrhagic Fever, Lassa Fever, and Hantaviruses

Rusnak

2011

Appl Biosaf.

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The increase in research laboratories with biosafety level-4 (BSL-4) capabilities may be associated with increased research with hemorrhagic fever (HF) viruses. The yellow fever vaccine and an investigational formalin-inactivated Rift Valley fever vaccine are the only vaccines for at-risk laboratory workers available in the United States against HF viruses. Ribavirin is a nucleoside analogue that has been demonstrated to have antiviral activity against a wide range of viruses, including many HF viruses. While intravenous (IV) ribavirin is not FDA-approved and oral ribavirin is approved only for treatment of hepatitis C in the United States, ribavirin has been used for treatment and postexposure prophylaxis for Crimean Congo hemorrhagic fever (CCHF), Lassa fever (LF), and hantaviruses, based mainly on in vitro sensitivity testing and efficacy studies in animals, but only on a limited number of studies and/or anecdotal experience in humans.The literature on both IV and oral ribavirin for treatment and postexposure prophylaxis for CCHF, LF, and hantaviruses is reviewed to aid occupational healthcare providers in the evaluation and recommendation of ribavirin to laboratory workers with exposures or illness from these HF viruses, which are associated with a significant morbidity and mortality.

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Application of real-time PCR for testing antiviral compounds against Lassa virus, SARS coronavirus and Ebola virus in vitro

Cited by 53 publications

References 39 publications

Replicon System for Lassa Virus

Replicon System for Lassa Virus

Broad-Spectrum Antiviral Activity of Small Interfering RNA Targeting the Conserved RNA Termini of Lassa Virus

Experience with Ribavirin for Treatment and Postexposure Prophylaxis of Hemorrhagic Fever Viruses: Crimean Congo Hemorrhagic Fever, Lassa Fever, and Hantaviruses

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