title: A rapid and convenient variant of fusion-PCR to construct chimeric flaviviruses.
abstract: So far, full-length cDNAs of chimeric flaviviruses have been constructed by restriction-enzyme cleavage of the gene(s) to be exchanged or by fusion-PCR of two amplified PCR fragments. The construction of a chimeric flavivirus by a faster and more convenient variant of the standard fusion-PCR is reported. A Modoc/yellow fever chimeric virus was engineered in which the structural prM and E genes of yellow fever virus 17D were replaced by the homologous genes of Modoc virus. In two PCR steps, a fusion was made between the 3' end of the C gene of yellow fever virus and the 5' end of the prM gene of Modoc virus, and between the 3' end of the E gene of Modoc virus and the 5' end of the NS1 gene of yellow fever virus. For each of the two fusions between yellow fever and Modoc virus, a standard PCR was performed to amplify a short fragment with one overlapping end that could be used as one of the primers in the subsequent (fusion) PCR.
authors.affiliation: Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000, Leuven, Belgium.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Charlier
authors.forename: Richard
authors.initials: R
authors.lastname: Molenkamp
authors.forename: Pieter
authors.initials: P
authors.lastname: Leyssen
authors.forename: Anne Mieke
authors.initials: AM
authors.lastname: Vandamme
authors.forename: Erik
authors.initials: E
authors.lastname: De Clercq
authors.forename: Peter
authors.initials: P
authors.lastname: Bredenbeek
authors.forename: Johan
authors.initials: J
authors.lastname: Neyts
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D001483:Base Sequence; D002460:Cell Line; D002678:Chimera / Q000235:genetics; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005416:Flavivirus / Q000235:genetics*; D005814:Genes, Viral; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*; D015005:Yellow fever virus / Q000235:genetics
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D014759:Viral Envelope Proteins; C074932:prM protein, Flavivirus; C078400:yellow fever virus envelope protein E