Dataset Information

SuPReMe: a rapid reverse genetics method to generate clonal populations of recombinant RNA viruses.

ABSTRACT: Reverse genetics systems enable the manipulation of viral genomes and are proving to be essential for studying RNA viruses. Methods for generating clonal virus populations are particularly useful for studying the impact of genomic modifications on viral properties. Here, by exploiting a chikungunya virus model, we compare viral populations and their replicative fitness when generated using either the rapid and user-friendly PCR-based ISA (Infectious Subgenomic Amplicons) method or classical infectious clone technology. As anticipated, the ISA method resulted in greater genetic diversity of the viral populations, but no significant difference in viral fitness in vitro was observed. On the basis of these results, a new ISA-derived reverse genetics procedure was developed. This method, designated 'SuPReMe' (Subgenomic Plasmids Recombination Method), in which digested plasmids containing subgenomic DNA fragments were directly transfected into permissive cells, retains the following major advantages of the ISA method: it is rapid, flexible and does not require the cloning of complete genomes. Moreover, SuPReMe has been shown to produce virus populations with genetic diversity and replicative fitness similar to those obtained using conventional infectious clone technology. SuPReMe, therefore, represents an effective and promising option for the rapid generation of clonal recombinant populations of single-stranded positive-sense RNA viruses.

SUBMITTER: Driouich JS

PROVIDER: S-EPMC5861084 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

SuPReMe: a rapid reverse genetics method to generate clonal populations of recombinant RNA viruses.

Driouich Jean-Sélim JS Ali Souand Mohamed SM Amroun Abdennour A Aubry Fabien F de Lamballerie Xavier X Nougairède Antoine A

Emerging microbes & infections 20180321 1

Reverse genetics systems enable the manipulation of viral genomes and are proving to be essential for studying RNA viruses. Methods for generating clonal virus populations are particularly useful for studying the impact of genomic modifications on viral properties. Here, by exploiting a chikungunya virus model, we compare viral populations and their replicative fitness when generated using either the rapid and user-friendly PCR-based ISA (Infectious Subgenomic Amplicons) method or classical infe ...[more]

PMID: 29559627

Similar Datasets

Project description:Reverse genetics systems have played a central role in developing recombinant viruses for a wide spectrum of virus research. The circular polymerase extension reaction (CPER) method has been applied to studying positive-strand RNA viruses, allowing researchers to bypass molecular cloning of viral cDNA clones and thus leading to the rapid generation of recombinant viruses. However, thus far, the CPER protocol has only been established using cap-dependent RNA viruses. Here, we demonstrate that a modified version of the CPER method can be successfully applied to positive-strand RNA viruses that use cap-independent, internal ribosomal entry site (IRES)-mediated translation. As a proof-of-concept, we employed mammalian viruses with different types (classes I, II, and III) of IRES to optimize the CPER method. Using the hepatitis C virus (HCV, class III), we found that inclusion in the CPER assembly of an RNA polymerase I promoter and terminator, instead of those from polymerase II, allowed greater viral production. This approach was also successful in generating recombinant bovine viral diarrhea virus (class III) following transfection of MDBK/293T co-cultures to overcome low transfection efficiency. In addition, we successfully generated the recombinant viruses from clinical specimens. Our modified CPER could be used for producing hepatitis A virus (HAV, type I) as well as de novo generation of encephalomyocarditis virus (type II). Finally, we generated recombinant HCV and HAV reporter viruses that exhibited replication comparable to that of the wild-type parental viruses. The recombinant HAV reporter virus helped evaluate antivirals. Taking the findings together, this study offers methodological advances in virology.ImportanceThe lack of versatility of reverse genetics systems remains a bottleneck in viral research. Especially when (re-)emerging viruses reach pandemic levels, rapid characterization and establishment of effective countermeasures using recombinant viruses are beneficial in disease control. Indeed, numerous studies have attempted to establish and improve the methods. The circular polymerase extension reaction (CPER) method has overcome major obstacles in generating recombinant viruses. However, this method has not yet been examined for positive-strand RNA viruses that use cap-independent, internal ribosome entry site-mediated translation. Here, we engineered a suitable gene cassette to expand the CPER method for all positive-strand RNA viruses. Furthermore, we overcame the difficulty of generating recombinant viruses because of low transfection efficiency. Using this modified method, we also successfully generated reporter viruses and recombinant viruses from a field sample without virus isolation. Taking these findings together, our adapted methodology is an innovative technology that could help advance virologic research.

Dataset Information

SuPReMe: a rapid reverse genetics method to generate clonal populations of recombinant RNA viruses.

Publications

SuPReMe: a rapid reverse genetics method to generate clonal populations of recombinant RNA viruses.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets