Construction of Stable Reporter Flaviviruses and Their Applications

Abstract

Flaviviruses are significant human pathogens that cause frequent emerging and reemerging epidemics around the world. Better molecular tools for studying, diagnosing, and treating these diseases are needed. Reporter viruses represent potent tools to fill this gap but have been hindered by genetic instability. Recent advances have overcome these hurdles, opening the way for increased use of stable reporter flaviviruses to diagnose infections, screen and study antiviral compounds, and serve as potential vaccine vectors.

Reporter Flaviviruses as Tools to Demonstrate Homologous and Heterologous Superinfection Exclusion

“…Initial studies on the construction of reporter flaviviruses used various approaches for the insertion of reporter genes, primarily internal ribosome entry site (IRES)-based systems (reviewed by [ 28 ]). However, these initial construction methods typically resulted in instability of the reporter genes after serial passaging and virus attenuation due to recombination occurring at the insertion site [ 28 ].…”

“…The first infectious flavivirus reporter virus was engineered for Japanese encephalitis virus (JEV) in 2003 and contained GFP and luciferase in the 3′ UTR [ 27 ]. Unfortunately, early reporter viruses were plagued with genetic instability and spontaneous loss of the insert (Reviewed by [ 28 ]). A YFV reporter virus described in 2007 [ 29 ] improved stability by fusing the first 101-aa capsid sequence with GFP, followed by a foot-and-mouth disease virus 2A ribosome stuttering peptide (FMDV 2A), codon-optimised capsid gene, and the rest of YFV polyprotein [ 29 ].…”

“…A YFV reporter virus described in 2007 [ 29 ] improved stability by fusing the first 101-aa capsid sequence with GFP, followed by a foot-and-mouth disease virus 2A ribosome stuttering peptide (FMDV 2A), codon-optimised capsid gene, and the rest of YFV polyprotein [ 29 ]. Since these early studies, many reporter viruses for the VIF flaviviruses have been constructed [ 28 ]. In contrast, only one report has demonstrated reporter viruses’ construction for the Lineage I ISF Niénokoué virus [ 30 ].…”

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

“…Further improvements to achieve long-term stability (i.e., stable for ten passages in cell culture) have been developed. Besides using Viruses 2022, 14, 1253 2 of 13 split reporter systems, codon optimization of the capsid gene to reduce homology and optimization of capsid duplication length can increase reporter stability [19]. Although these new approaches can improve reporter virus construction, the insertion of reporter genes into the viral genome as exogenous genes can reduce the virulence of engineered flaviviruses.…”

“…Although a reporter DENV, based on a conventional luciferase construct, could be visualized in vivo in mice [13], a stable reporter virus still only allows the insertion of small exogenous genes. The insertion of reporter genes of proteins with large molecular weight into flaviviruses can attenuate the pathogenicity and affect the virulence mechanisms of these viruses in relevant applications [19]. In this study, we used the gene of NanoLuc luciferase, which has a low molecular weight and high luminescence intensity, to construct a reporter DENV.…”