title: A Rapid Method for Engineering Recombinant Polioviruses or Other Enteroviruses.
abstract: The cloning of large enterovirus RNA sequences is labor-intensive because of the frequent instability in bacteria of plasmidic vectors containing the corresponding cDNAs. In order to circumvent this issue we have developed a PCR-based method that allows the generation of highly modified or chimeric full-length enterovirus genomes. This method relies on fusion PCR which enables the concatenation of several overlapping cDNA amplicons produced separately. A T7 promoter sequence added upstream the fusion PCR products allows its transcription into infectious genomic RNAs directly in transfected cells constitutively expressing the phage T7 RNA polymerase. This method permits the rapid recovery of modified viruses that can be subsequently amplified on adequate cell-lines.
authors.affiliation: INSERM U994, Institut National de Santé et de La Recherche Médicale, Paris, France.|Institut Pasteur, Biologie des Virus Entériques, 25 rue du Dr Roux, 75015, Paris, France.
authors.forename: Maël
authors.initials: M
authors.lastname: Bessaud
authors.affiliation: INSERM U994, Institut National de Santé et de La Recherche Médicale, Paris, France.|Institut Pasteur, Biologie des Virus Entériques, 25 rue du Dr Roux, 75015, Paris, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Pelletier
authors.affiliation: INSERM U994, Institut National de Santé et de La Recherche Médicale, Paris, France.|Institut Pasteur, Biologie des Virus Entériques, 25 rue du Dr Roux, 75015, Paris, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Blondel
authors.affiliation: INSERM U994, Institut National de Santé et de La Recherche Médicale, Paris, France. francis.delpeyroux@pasteur.fr.|Institut Pasteur, Biologie des Virus Entériques, 25 rue du Dr Roux, 75015, Paris, France. francis.delpeyroux@pasteur.fr.
authors.forename: Francis
authors.initials: F
authors.lastname: Delpeyroux
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004770:Enterovirus / Q000235:genetics / Q000502:physiology; D004769:Enterovirus Infections / Q000821:virology; D005814:Genes, Viral; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011051:Poliomyelitis / Q000821:virology*; D017955:Poliovirus / Q000235:genetics* / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000235:genetics; D014162:Transfection / Q000379:methods; D014764:Viral Proteins / Q000235:genetics; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: Enterovirus; Genetic engineering; Poliovirus; Recombinant virus