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.
Highlights
Flaviviruses are a constant threat to global public health, with re-emerging outbreaks of yellow fever [1], new threats from Zika [2], and recurrent outbreaks of dengue in various countries [3]
Our initial effort to generate a reporter GFP virus of dengue-virus-serotype 2 (DENV2) strain 16681 entailed the expression of enhanced green fluorescent protein fused to the first twenty-five amino acids of capsid (C25) at its N-terminus
The reporter protein cannot be expressed from the 50 terminus of the viral genome since C25 is needed for translation initiation of dengue virus [14]
Summary
Flaviviruses are a constant threat to global public health, with re-emerging outbreaks of yellow fever [1], new threats from Zika [2], and recurrent outbreaks of dengue in various countries [3]. Flaviviruses are positive-sense RNA viruses with non-segmented, single-stranded RNA genome with the size of approximately 10–12 kb [4]. Several flaviviruses are human pathogens transmitted by arthopods such as mosquitoes and ticks [5]. Many tools and innovative techniques have been employed to dissect flavivirus replication, transmission, and evolution. Reporter virus has been a versatile tool to visualize and analyze virus infection. Signal intensity from the reporter provides a convenient measurement of virus replication for highthroughput assays and screens. With advances in single-cell sequencing, fluorescent reporter virus in combination with fluorescent-activated cell sorting (FACS) can be used to isolate target cells for molecular profiling [6]. Bioluminescent reporter virus can serve as a sensitive probe to track virus infection in animal models [7].
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