Flavivirus reverse genetic systems, construction techniques and applications: a historical perspective

Abstract

The study of flaviviruses, which cause some of the most important emerging tropical and sub-tropical human arbovirus diseases, has greatly benefited from the use of reverse genetic systems since its first development for yellow fever virus in 1989. Reverse genetics technology has completely revolutionized the study of these viruses, making it possible to manipulate their genomes and evaluate the direct effects of these changes on their biology and pathogenesis. The most commonly used reverse genetics system is the infectious clone technology. Whilst flavivirus infectious clones provide a powerful tool, their construction as full-length cDNA molecules in bacterial vectors can be problematic, laborious and time consuming, because they are often unstable, contain unwanted induced substitutions and may be toxic for bacteria due to viral protein expression. The incredible technological advances that have been made during the past 30years, such as the use of PCR or new sequencing methods, have allowed the development of new approaches to improve preexisting systems or elaborate new strategies that overcome these problems. This review summarizes the evolution and major technical breakthroughs in the development of flavivirus reverse genetics technologies and their application to the further understanding and control of these viruses and their diseases.

Keywords: Flavivirus; Infectious clone; Reverse genetics.

Fig. 1

Generic procedure for infectious clone construction. A double stranded cDNA copy of an RNA virus genome is stably incorporated into a vector and amplified into a host. After purification of the construction, infectious viruses are obtained either by direct transfection of permissive cells when an eukaryotic promoter is used or by transfection of genomic RNA obtained by in vitro transcription when a bacteriophage promoter is used.

Fig. 2

Bacteria-free approach, generic procedure for production of TBEV using Long PCR. Two Long PCR products were either ligated using restriction enzymes or merged by fusion PCR. After in vitro transcription and inoculation of the full-length RNA transcripts intracerebrally into young mice, infectious viruses were recovered.

Fig. 3

Bacteria-free approach, generic procedure for production of KUNV using CPEC reaction. Phusion high-fidelity DNA polymerase was used to assemble and incorporate multiple RT-PCR amplicons into a vector. The generated circular product was then transfected directly into competent cells to recover infectious viruses.

Fig. 4

Bacteria-free approach, generic procedure for production of DENV using Gibson assembly method. Using a combination of three enzymes (exonuclease, DNA polymerase and DNA ligase), multiple RT-PCR amplicons of both virus and vector are assembled during an isothermal and single-reaction. The generated circular product was then transfected directly into competent cells to recover infectious viruses.

Fig. 5

Bacteria-free approach, generic procedure for production of flaviviruses using the ISA method. The entire viral genome, flanked at the 5′ extremity by the human cytomegalovirus promoter (pCMV) and at the 3′ extremity by the hepatitis delta ribozyme followed by the simian virus 40 polyadenylation signal (HDR/SV40pA), was amplified by PCR in three overlapping cDNA fragments. Direct transfection of PCR products into competent cells enabled the recovery of infectious viruses (successfully applied to DENV, YFV, JEV and TBEV).

Fig. 6

Schematic representation of the general approach for the design of YFV 17D-based chimeric vaccine candidates. The sequences encoding the prM and E proteins of the vaccine target virus (e.g., JEV, WNV, DENV or SLEV) were engineered into the cDNA genome of the YFV 17D vaccine strain to replace the authentic prM and E sequences. Chimeric viral RNA were obtained by in vitro transcription and were transfected into permissive cells, resulting in the production of infectious chimeric virus particles that are covered by the vaccine target virus envelope proteins.