Elsevier

Antiviral Research

Volume 126, February 2016, Pages 117-124
Antiviral Research

Discovery of berberine, abamectin and ivermectin as antivirals against chikungunya and other alphaviruses

https://doi.org/10.1016/j.antiviral.2015.12.012Get rights and content

Highlights

  • We screened 3000 bioactive compounds using a chikungunya virus replicon cell line.

  • Berberine, abamectin and ivermectin were broadly antiviral against alphaviruses.

  • They inhibited viral RNA and protein accumulation, likely affecting replication.

Abstract

Chikungunya virus (CHIKV) is an arthritogenic arbovirus of the Alphavirus genus, which has infected millions of people after its re-emergence in the last decade. In this study, a BHK cell line containing a stable CHIKV replicon with a luciferase reporter was used in a high-throughput platform to screen approximately 3000 compounds. Following initial validation, 25 compounds were chosen as primary hits for secondary validation with wild type and reporter CHIKV infection, which identified three promising compounds. Abamectin (EC50 = 1.5 μM) and ivermectin (EC50 = 0.6 μM) are fermentation products generated by a soil dwelling actinomycete, Streptomyces avermitilis, whereas berberine (EC50 = 1.8 μM) is a plant-derived isoquinoline alkaloid. They inhibited CHIKV replication in a dose-dependent manner and had broad antiviral activity against other alphaviruses - Semliki Forest virus and Sindbis virus. Abamectin and ivermectin were also active against yellow fever virus, a flavivirus. These compounds caused reduced synthesis of CHIKV genomic and antigenomic viral RNA as well as downregulation of viral protein expression. Time of addition experiments also suggested that they act on the replication phase of the viral infectious cycle.

Introduction

Alphaviruses are vector-borne, enveloped, positive-strand RNA viruses of the Togaviridae family. Chikungunya virus (CHIKV) causes an acute febrile illness, characterized chiefly by myalgia, nausea, headache, and maculopapular rash, which can progress to chronic arthralgia in a large fraction of the patients and persist for several months or even years, causing considerable suffering and economic loss (Burt et al., 2012). Hitherto restricted to tropical areas of Africa, the Indian sub-continent and South-East Asia, CHIKV recently invaded the western hemisphere, causing more than 1 million suspected infections in the Caribbean, as well as in Central, South and North America (Fischer et al., 2014, Leparc-Goffart et al., 2014, Weaver and Forrester, 2015).

Currently, since there are no licensed antivirals, the treatment for CHIKV is based on symptomatic management (Thiberville et al., 2013). Since the re-emergence of CHIKV, there has been an increasing interest in identifying inhibitory targets in the CHIKV replication cycle as well as investigating different therapeutic or vaccination strategies to combat the virus (Ahola et al., 2015). Many different bio-active compounds, directed either towards a viral component or a host factor, with low to moderate antiviral activity have been described (Abdelnabi et al., 2015). Although chloroquine and ribavirin treatment showed promising results in vitro, testing in patients has yielded inconclusive or negative results, due to discrepancies and study limitations (Kaur and Chu, 2013), and lack of an added advantage over other drugs used for alleviating musculo-skeletal pain (Chopra et al., 2014).

The alphavirus genome of ∼11.5 kb is divided into regions coding for non-structural and structural proteins (Kääriäinen and Ahola, 2002). Assays using replicons, lacking the structural proteins, target the intracellular steps of virus RNA replication, and stable viral RNA replicon containing cell lines have been frequently used as an important tool for high throughput antiviral screening (Hsu et al., 2012, Kim et al., 2013). A screen based on Western equine encephalitis virus replicon identified thieno [3,2-b] pyrrole derivatives as potent inhibitors for virus replication (Peng et al., 2009). We earlier developed a CHIKV-replicon cell line-based assay, which was used for a small screen of 356 natural compounds (Pohjala et al., 2011). In this work we have used a fully automated assay to screen approximately 3000 compounds, notably bioactive compounds including drug molecules approved for use as well as those in clinical trials. The screen and subsequent experiments identified three novel inhibitors of CHIKV replication, which in addition to potently reducing virus production, also inhibited viral RNA and protein expression.

Section snippets

Cells and viruses

Baby hamster kidney BHK-21 cell line and a CHIKV replicon containing cell line (BHK–CHIKV) were obtained and grown as described (Pohjala et al., 2011). Human hepatocellular Huh-7.5 cells were kindly provided by Prof. Ralf Bartenschlager (University of Heidelberg, Germany). Wild type CHIKV (CHIKV; strain LR2006 OPY1) stock was produced from the infectious clone SP6-ICRES (Pohjala et al., 2011). CHIKV-Rluc is a CHIKV derivative containing Renilla reniformis luciferase (Rluc) gene insertion within

Screening for CHIKV replication inhibitors

The development of a stable CHIKV replicon-based bioassay with Renilla luciferase reporter in BHK-21 cells was described earlier (Pohjala et al., 2011). The assay was now miniaturized to a 384-well microplate format in an automated robotic system, and optimized for cell number, assay medium and volume of detection reagents. Z′ value of the replication test was 0.7 and coefficients of variation were 11% for the positive control 6-azauridine and 7% for the negative control of 0.1% DMSO,

Discussion

CHIKV has spread world-wide in tropical and sub-tropical regions, but no approved antiviral treatment exists for CHIKV to date. In the present study, we used a CHIKV replicon-based assay to screen bioactive compounds – including drugs already in clinical use or in clinical trials to discover novel inhibitors impeding CHIKV replication. Six candidate compounds (abamectin, berberine, bromocriptine, cerivastatin, fenretinide and ivermectin) obtained from the entire screening procedure inhibited

Acknowledgments

We thank Päivi Tammela for help with the EC50 calculations, Ilkka Harri for help in antiviral assays, and Janett Wieseler for excellent technical assistance. Anna Lehto and Laura Turunen from the High Throughput Biomedicine Unit of the Institute for Molecular Medicine Finland are thanked for support with automated chemical screening. This study was funded by the EU 7th framework project ICRES (grant agreement no. 261202) and Academy of Finland grant 265997 (to TA). FSV is a fellow of the

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    F.S.V. and P.K. contributed equally to this work.

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