Rapid Generation of Recombinant Flaviviruses Using Circular Polymerase Extension Reaction

Abstract

The genus Flavivirus is a group of arthropod-borne single-stranded RNA viruses, which includes important human and animal pathogens such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), Dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Tick-borne encephalitis virus (TBEV). Reverse genetics has been a useful tool for understanding biological properties and the pathogenesis of flaviviruses. However, the conventional construction of full-length infectious clones for flavivirus is time-consuming and difficult due to the toxicity of the flavivirus genome to E. coli. Herein, we applied a simple, rapid, and bacterium-free circular polymerase extension reaction (CPER) method to synthesize recombinant flaviviruses in vertebrate cells as well as insect cells. We started with the de novo synthesis of the JEV vaccine strain SA-14-14-2 in Vero cells using CPER, and then modified the CPER method to recover insect-specific flaviviruses (ISFs) in mosquito C6/36 cells. Chimeric Zika virus (ChinZIKV) based on the Chaoyang virus (CYV) backbone and the Culex flavivirus reporter virus expressing green fluorescent protein (CxFV-GFP) were subsequently rescued in C6/36 cells. CPER is a simple method for the rapid generation of flaviviruses and other potential RNA viruses. A CPER-based recovery system for flaviviruses of different host ranges was established, which would facilitate the development of countermeasures against flavivirus outbreaks in the future.

The Chimeric Chaoyang-Zika Vaccine Candidate Is Safe and Protective in Mice

“…Previously, we generated ChinZIKV by replacing the prM-E of CYV with that of ZIKV GZ01 and established that ChinZIKV did not replicate in Vero and BHK-21 cell lines, which were derived from vertebrates ( Figure 1 A) [ 60 ]. However, it is unknown whether ChinZIKV infects and causes disease in mice.…”

“…There are some limitations in our study. Limited types of cell lines derived from vertebrates were tested for ChinZIKV [ 60 ]. More cell types from humans are needed to fully characterize the host tropism and safety of ChinZIKV.…”

“…Both mediums were supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Gibco, Waltham, MA, USA), and 1% penicillin streptomycin (P/S, Gibco, USA). The ZIKV GZ01 strain and ChinZIKV were described in previous works [ 60 , 62 , 63 ]. Briefly, the GZ01 strain of ZIKV was isolated from the urine sample of a ZIKV-infected patient.…”

“…Briefly, 10-fold dilutions of virus supernatants were seeded on confluent C6/36 cells in a 48-well plate for 1 h. Then, the supernatants were replaced with maintenance medium supplemented with 20 mM NH 4 Cl, which was used to inhibit the secondary infection of flaviviruses. Indirect immunofluorescence assay was performed on the cells after incubation at 28 °C for 3 days, as previously described [ 60 ]. Virus foci were stained with a mouse monoclonal antibody (1:500 diluted) (GeneTex, Irvine, CA, USA, Cat No.…”

“…One of the insect-specific flaviviruses, CYV was initially isolated in 2008 in Liaoning province, China [ 58 ]. We have previously generated ChinZIKV based on CYV by replacing the prME protein of CYV with that of a clinical isolate of ZIKV GZ01 [ 60 ]. Herein, we evaluated the safety and immunogenicity of ChinZIKV as a vaccine candidate against ZIKV in a type I interferon receptor knockout (IFNAR−/−) mouse model [ 61 ].…”