Construction and Rescue of a DNA-Launched DENV2 Infectious Clone

Abstract

Flaviviruses represent a large group of globally significant, insect-borne pathogens. For many of these viruses, there is a lack of antivirals and vaccines. Thus, there is a need to continue the development of tools to further advance our efforts to combat these pathogens, including reverse genetics techniques. Traditionally, reverse genetics methods for flaviviruses rely on producing infectious RNA from in vitro transcription reactions followed by electroporation or transfection into permissive cell lines. However, the production of Zika virus has been successful from CMV promoter-driven expression plasmids, which provides cost and time advantages. In this report, we describe the design and construction of a DNA-launched infectious clone for dengue virus (DENV) serotype 2 strain 16681. An artificial intron was introduced in the nonstructural protein 1 segment of the viral genome to promote stability in bacteria. We found that rescued viruses maintained the ability to form plaques and replicate efficiently in commonly used cell lines. Thus, we present a rapid and cost-effective method for producing DENV2 strain 16681 from plasmid DNA. This construct will be a useful platform for the continued development of anti-DENV therapeutics and vaccines.

Keywords

• positive-strand RNA virus
• flavivirus
• dengue virus
• infectious clone
• reverse genetics