Development, Characterization, and Application of Two Reporter-Expressing Recombinant Zika Viruses

Abstract

Zika virus (ZIKV), a mosquito-borne transplacentally transmissible flavivirus, is an enveloped virus with an ~10.8 kb plus-strand RNA genome that can cause neurological disease. To facilitate the identification of potential antivirals, we developed two reporter-expressing ZIKVs, each capable of expressing an enhanced green fluorescent protein or an improved luminescent NanoLuc luciferase. First, a full-length functional ZIKV cDNA clone was engineered as a bacterial artificial chromosome, with each reporter gene under the cap-independent translational control of a cardiovirus-derived internal ribosome entry site inserted downstream of the single open reading frame of the viral genome. Two reporter-expressing ZIKVs were then generated by transfection of ZIKV-susceptible BHK-21 cells with infectious RNAs derived by in vitro run-off transcription from the respective cDNAs. As compared to the parental virus, the two reporter-expressing ZIKVs grew to lower titers with slower growth kinetics and formed smaller foci; however, they displayed a genome-wide viral protein expression profile identical to that of the parental virus, except for two previously unrecognized larger forms of the C and NS1 proteins. We then used the NanoLuc-expressing ZIKV to assess the in vitro antiviral activity of three inhibitors (T-705, NITD-008, and ribavirin). Altogether, our reporter-expressing ZIKVs represent an excellent molecular tool for the discovery of novel antivirals.

The development and characterization of a stable Coxsackievirus A16 infectious clone with Nanoluc reporter gene

“…Besides, eGFP-EV71 and DsRed-EV71 infectious clones have also been reported ( Shang et al, 2013 ). When compared, the recombinant viruses with fluorescent protein reporters are not as suitable as those with luciferase reporters in quantitative and high-throughput analyses, Yun et al reported two recombinant Zika viruses expressing eGFP-or Nanoluc-reporter respectively, nevertheless only the ZIKA-Nluc was adopted to further evaluate antivirals ( Yun et al, 2020 ). Nanoluc luciferase has a higher luminescence intensity, a lower background, and is more stable in variable pH and temperatures than Firefly luciferase and Renilla luciferase ( Hall et al, 2012 ).…”

Construction of a Dengue NanoLuc Reporter Virus for In Vivo Live Imaging in Mice

“…Therefore, NanoLuc, a luciferase recently developed by Promega, was used in this study. With a low molecular weight (19.1 kDa, 171 amino acids), NanoLuc can be used for enhanced viral delivery and protein fusion, is easily secreted by cells, and is widely used in reporter flavivirus construction [7,23,[26][27][28][29]. Moreover, the NanoLuc system displayed a specific activity over 150-fold higher than both North American Firefly (FLuc) and Renilla (RLuc) luciferases, providing better performance in cells that are difficult to transfect [30,31].…”

Construction of Stable Reporter Flaviviruses and Their Applications

“…These results corroborated those from WNV-GFP and established RT-PCR on passaged viral RNA as the traditional assay to assess genetic stability. Similar versions of other reporter flaviviruses, such as DENV [ 19 , 24 ] and ZIKV [ 38 ], using an IRES in the 3′ UTR have been made, though not every report details the instability of the reporter gene.…”