Citation
- Authors: Choi WS. et al.
- Year: 2023
- Journal: Microbiol Spectr e0316722
- Applications: in vitro / DNA, mRNA / jetMESSENGER, jetPEI
- Cell types:
- Name: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T - Name: RD
- Name: VERO
Description: African green monkey kidney cells - Name: VERO E6
- Name: HEK-293T
Method
Enterovirus recovery from infectious clones.
To verify that the recombinant virus of an infectious clone of RNA form could be recovered, RNA was produced using a T7 MegaScript kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The concentration of in vitro-transcribed RNA was determined using a Qubit RNA High Sensitivity assay kit (Invitrogen). Using a JetMESSENGER kit (Polyplus Transfection, New York, NY, USA), 5 mg of the prepared RNA was transfected into cells placed into 6-well plates following the manufacturer’s protocol. To verify the possibility of directly recovering the recombinant virus from DNA plasmid-generated infectious clones, 3 mg of the DNA plasmid-based infectious clone was transfected into cells in a 6-well plate using the jetPEI transfection reagent (Polyplus Transfection). Each plate was prepared as follows: HEK293T+Vero E6 cells for EV-A71 clones, HEK293T+Vero cells for CV-B5 clones, and HEK293T+RD cells for ECHO6, ECHO30, CV-A24, and EV-D68. At 18 h posttransfection, each transfection supernatant was replaced with 2 mL of 2% DMEM and the cytopathic effect was monitored for the next 96 h. All recombinant viruses produced were titrated using TCID50 and plaque assays.
Abstract
Enteroviruses (EVs) have been associated with several human diseases. Due to their continuous emergence and divergence, EV species have generated more than 100 types and recombinant strains, increasing the public health threat caused by them. Hence, an efficient and universal cloning system for reverse genetics (RG) of highly divergent viruses is needed to understand the molecular mechanisms of viral pathology and evolution. In this study, we generated a versatile human EV whole-genome cDNA template by enhancing the template-switching method and designing universal primers capable of simultaneous cloning and rapid amplification of cDNA ends (RACE)-PCR of EVs. Moreover, by devising strategies to overcome limitations of previous cloning methods, we simplified significant cloning steps to be completed within a day. Of note, we successfully verified our efficient universal cloning system enabling RG of a broad range of human EVs, including EV-A (EV-A71), EV-B (CV-B5, ECHO6, and ECHO30), EV-C (CV-A24), and EV-D (EV-D68), with viral titers and phenotypes comparable to those of their wild types. This rapid and straightforward universal EV cloning strategy will help us elucidate molecular characteristics, pathogenesis, and applications of a broad range of EV serotypes for further development of genetic vaccines and delivery tools using various replication systems. IMPORTANCE Due to the broad spread, incidence, and genetic divergence of enteroviruses (EVs), it has been challenging to deal with this virus that causes severe human diseases, including aseptic meningitis, myocarditis, encephalitis, and poliomyelitis. Therefore, an efficient and universal cloning system for the reverse genetics of highly divergent EVs contributes to an understanding of the viral pathology and molecular mechanisms of evolution. We have simplified the important cloning steps, hereby enhancing the template-switching method and designing universal primers, which enable the important cloning steps to be completed in a day. We have also successfully demonstrated recovery of a broad range of human EVs, including EV-A to -D types, using this advanced universal cloning system. This rapid and robust universal EV cloning strategy will aid in elucidating the molecular characteristics, pathogenesis, and applications of a wide range of EVs for further development of genetic vaccines and antiviral screening using various replication systems.