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.

Keywords: CPER; Flavivirus; reporter virus; reverse genetic; vaccine.

Figure 1

Development of CPER for JEV in Vero cells. (A) Scheme for assembly of JEV genome using CPER and virus recovery. (B) CPE caused by recovered JEV in Vero cells and mock-infected Vero cells was included as negative control. Vero cells were infected culture supernatant from transfected cells (passage 0 JEV) and CPE was imaged at 3 days post infection (dpi). (C) Confirmation of successful generation of JEV by sequencing of the genetic marker. RT-PCR was performed on viral RNA extracted from passage 3 culture supernatant. Elimination of an SapI restriction site by an A-T synonymous mutation was identified as the designed genetic marker using Sanger sequencing on the resulting DNA fragments. (D) Plaque morphology of JEV in Vero cells. Confluent Vero cells infected with JEV were fixed with 4% formaldehyde at 5 dpi and stained with 0.1% crystal violet. (E) Growth kinetics of JEV in Vero cells. Cells were infected with JEV at an MOI of 0.1. Culture supernatants were harvested at the indicated time points. Viral RNA levels in the supernatants were determined using real-time PCR. Data represent an average of two experiments and error bars indicate the standard deviation.

Figure 2

Generation of ChinZIKV by CPER in C6/36 cells. (A) Scheme for assembly of ChinZIKV genome using CPER and recovery. (B) Confirmation of generation of ChinZIKV using IFA. E protein expressed by ChinZIKV was identified using IFA analysis with a monoclonal antibody against Flavivirus E protein (green). C6/36 cells infected with ChinZIKV were fixed and immunolabeled at 5 dpi. Cell nuclei were stained with DAPI (blue). ZIKV was included as a positive control. (C) Confirmation of generation of ChinZIKV using Sanger sequencing. RT-PCR was performed on ChinZIKV genome to amplify ZIKV-prME region. The resulting DNA fragments were analyzed using Sanger sequencing.

Figure 3

ChinZIKV fails to infect vertebrate cells. (A) ChinZIKV infected C6/36 cells and caused significant CPE but not Vero cells. As a control, ZIKV infected and caused significant CPE in both cells. Confluent C6/36 and Vero cells were inoculated with ChinZIKV or ZIKV at an MOI of 1. CPE was monitored daily and imaged at 5 dpi. (B) Confirmation of ChinZIKV’s inability to replicate in Vero and BHK-21 cells by IFA. ChinZIKV was inoculated onto Vero and BHK-21 cells and IFA was performed on both cells with the monoclonal antibody against Flavivirus E protein (green) at 5 dpi. Cell nuclei were stained with DAPI (blue). No ZIKV E protein was detected in cells inoculated with ChinZIKV. ZIKV was included as a positive control. (C) Growth kinetics of ChinZIKV in C6/36 cells. Cells were infected at an MOI of 0.1. Supernatants were harvested at the indicated time points. Viral RNA levels in the supernatants were determined using real-time PCR. Error bars represent the standard deviation from two independent replicates.

Figure 4

Generation of CxFV-GFP using CPER and its genetic stability. (A) Schematic diagram of design and construction of CxFV-GFP. The first 34 amino acid of capsid (C34) was duplicated and placed upstream of GFP. A self-cleaving FMDV 2A peptide was inserted downstream of GFP, followed by a codon scrambled C34 sequence (represented by slanted lines). (B) Detection of GFP in C6/36 cells infected with CxFV-GFP using fluorescence microscopy. (C) Genetic stability of CxFV-GFP. RT-PCR was performed on CxFV-GFP RNA extracted from serially passaged viruses to amplify the fragment containing the GFP gene. The fragment is 1045 bp in length and GFP is 720 bp. CxFV-GFP was passaged in C6/36 cells at an MOI of 0.1. Culture supernatant from each passage was harvested at 4 dpi.