Multi-color fluorescent reporter dengue viruses with improved stability for analysis of a multi-virus infection

Abstract

Reporter virus is a versatile tool to visualize and to analyze virus infections. However, for flaviviruses, it is difficult to maintain the inserted reporter genes on the viral genome, limiting its use in several studies that require homogeneous virus particles and several rounds of virus replication. Here, we showed that flanking inserted GFP genes on both sides with ribosome-skipping 2A sequences improved the stability and the consistency of their fluorescent signals for dengue-virus-serotype 2 (DENV2) reporter viruses. The reporter viruses can infect known susceptible mammalian cell lines and primary CD14+ human monocytes. This design can accommodate several fluorescent protein genes, enabling the generation of multi-color DENV2-16681 reporter viruses with comparable replication capabilities, as demonstrated by their abilities to maintain their fluorescent intensities during co-infections and to exclude superinfections regardless of the fluorescent tags. The reported design of multi-color DENV2 should be useful for high-throughput analyses, single-cell analysis, and characterizations of interference and superinfection in animal models.

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

“…Interestingly, however, it appears that our eGFP-and nLUC-expressing ZIKVs described in the present study showed greater levels of genetic instability than have the similar GFP-and luciferase-expressing JEVs previously developed by our laboratory [60,104]. An alternative approach commonly employed in the development of reporter-expressing flaviviruses is to place a reporter gene upstream of the viral full-length ORF, such as with WNV [109], DENV [99,[110][111][112], and ZIKV [67,86,87,97]. In all these cases, reporter-expressing flaviviruses are genetically unstable, eventually losing the ability to express the reporter gene.…”

Zikavirus prME Envelope Pseudotyped Human Immunodeficiency Virus Type-1 as a Novel Tool for Glioblastoma-Directed Virotherapy

“…With the emergence of the new neurotropic ZIKV with its unexpected preference for infecting GSCs, a new research area for glioma virotherapy opened up [26,[28][29][30]43,44]. In regard to Flaviviridae, recombinant reporter viruses, for example, for West Nile and Dengue virus, were constructed using the original flavivirus genome as a packaging template [45,46]. Moreover, pseudotypes between yellow fever and West Nile viruses were constructed to study antibody responses against yellow fever [47].…”

Construction of Stable Reporter Flaviviruses and Their Applications

“…As a rule, reporter viruses made without reducing the homology between duplicated capsids show marked instability, similar to the 3′ UTR reporter viruses [ 23 , 29 , 30 , 33 , 35 ]. On the other hand, reducing the homology increases stability, reported usually out to five passages in cell culture [ 25 , 26 , 32 , 35 ]. Case in point, Reporter WNV and JEV with no homology reduction were initially shown to be unstable.…”

“…The capsid duplication strategy has been successfully applied to many other flaviviruses, with slight variations giving different results in stability and utility, though addition of a reporter gene causes general attenuation in all cases [ 22 , 23 , 25 , 26 , 28 , 29 , 30 , 32 , 33 , 35 , 36 , 39 ]. As a rule, reporter viruses made without reducing the homology between duplicated capsids show marked instability, similar to the 3′ UTR reporter viruses [ 23 , 29 , 30 , 33 , 35 ]. On the other hand, reducing the homology increases stability, reported usually out to five passages in cell culture [ 25 , 26 , 32 , 35 ].…”

“…Engineering of similar mutations in other flaviviruses, on the other hand, has not led to similar levels of stability [ 26 ]. Lastly, a report using DENV indicated that doubling the 2A sequence immediately downstream of the reporter gene could increase genetic stability by up to two or three passages [ 35 ].…”