Evaluation of luciferase and GFP-expressing Nipah viruses for rapid quantitative antiviral screening
Introduction
Nipah virus (NiV) is a highly pathogenic paramyxovirus in the genus Henipavirus of the subfamily Paramyxovirinae within the family Paramyxoviridae (Rota and Lo, 2012). Humans are infected upon spillover of NiV from its bat reservoir host or by person-to-person transmission (Luby, 2013). Due to its high pathogenicity, its potential use for bio/agro-terrorism, and to the current lack of approved therapeutics, NiV is designated as an overlap select agent requiring biosafety level-4 containment. Although several preventative measures against exposure to bat saliva or urine have been studied (Nahar et al., 2013), NiV continues to cause near-annual outbreaks of fatal encephalitis in Bangladesh. The development of therapeutic monoclonal antibodies and soluble subunit vaccines against henipaviruses have shown great promise (Broder et al., 2013), but screening available compound libraries for potentially efficacious therapeutics against NiV remains a high priority for investigation. Most high-throughput antiviral assays against NiV were developed to screen for inhibitors of virus entry and/or cell-to-cell fusion (Bossart et al., 2005, Porotto et al., 2009, Talekar et al., 2012, Tamin et al., 2009), while the NiV minigenome assay was used to screen for inhibitors of NiV replication (Freiberg et al., 2008). A chemiluminescent immunodetection assay for live henipavirus infection significantly streamlined antiviral screening when compared with cytopathic effect (CPE) or immune plaque-based assays, but had relatively limited signal-to-noise ratios (<100), and also required cell fixation and multiple incubation steps which are time and labor-intensive (Aljofan et al., 2008). Reverse genetic systems for henipaviruses have enabled the generation of recombinant viruses expressing fluorescent or luminescent proteins which allow for rapid detection of infection in vitro and in vivo (Lo et al., 2012, Marsh et al., 2013, Yoneda et al., 2006). We sought to determine whether NiVs expressing fluorescent or luminescent reporters could facilitate rapid quantitative antiviral screening. As a proof-of-concept study, we constructed and rescued NiVs expressing either Renilla luciferase (LUC2AM) or green fluorescent protein (GFP2AM), characterized their reporter signal kinetics in different human cell types, and tested them in parallel against cellular inhibitors of pyrimidine biosynthesis as well as innate immune agonists. We show that both reporter viruses can be used in the 96-well format and have excellent signal-to-noise ratios and Z′-factors. Furthermore, the 50% effective concentrations (EC50s) derived for inhibitors tested against both reporter viruses were not only within close range of one another (within 4-fold), but also of EC50s derived from virus yield-based dose–response assays against wild-type NiV (within 1 Log10), thus demonstrating that both reporter NiVs can serve as robust antiviral screening tools.
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Cells
Vero (African green monkey kidney), A549 (human lung epithelial carcinoma), HeLa (human cervical carcinoma), and HEK 293T (human embryonic kidney) cells were maintained at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 7.5% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine. Baby hamster kidney cells stably expressing T7 polymerase (BSRT7/5) (Buchholz et al., 1999) were incubated at every third passage in the presence of 1 mg/mL
Construction and characterization of luciferase and GFP expressing NiVs
To evaluate the use of luminescent reporters for antiviral screening against NiV, we constructed a Renilla luciferase-expressing NiV (LUC2AM) by employing previous methods used to generate a red fluorescent protein-expressing NiV (RED2AM) (Lo et al., 2012). We inserted the Renilla luciferase (LUC) gene into the NiV matrix (M) gene 5′ of the M ORF in the same reading frame, separated by the coding sequence of the Foot and Mouth Disease virus protease cleavage peptide (F2A) (Fig. 1A). We used the
Conclusions
The primary advantages to using viral reporter assays over viral antigen-based assays are the significant reduction in assay turnaround time and the minimal reagents required. Whereas a previously described henipavirus immunodetection assay required at least 2 h of post-infection processing (fixing, blocking, antibody staining) before the plate reading step, the LUC2AM assay required just 10 min of reagent incubation before plate reading, while the GFP2AM assay required even less time since no
Acknowledgements
We thank Mike Flint for helpful discussions and suggestions. The findings and conclusions in this report are those of the authors and do not necessarily represent those of the Centers for Disease Control and Prevention.
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