title: A stable full-length yellow fever virus cDNA clone and the role of conserved RNA elements in flavivirus replication.
abstract: Yellow fever virus (YF) is the prototype member of the Flavivirus genus. Here, we report the successful construction of a full-length infectious cDNA clone of the vaccine strain YF-17D. YF cDNA was cloned into a low-copy-number plasmid backbone and stably maintained in several E. coli strains. Transcribed RNAs had a specific infectivity of 10(5)-10(6) p.f.u. ( micro g RNA)(-1), and the resulting virus exhibited growth kinetics, plaque morphology and proteolytic processing similar to the parental virus in cell culture. This clone was used to analyse the importance of conserved flavivirus RNA sequences and the 3' stem-loop structure in virus replication. The conserved sequences 5'CS and CS1, as well as the 3' stem-loop structure, were found to be essential for virus replication in cell culture, whereas the conserved sequence CS2 and the region containing YF-specific repeated sequences were dispensable. This infectious clone will aid future studies on YF replication and pathogenesis, as well as facilitate the development of YF-17D-based recombinant vaccines.
authors.affiliation: Department of Medical Microbiology, Center of Infectious Disease, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Bredenbeek
authors.affiliation: Department of Medical Microbiology, Center of Infectious Disease, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Engbert A
authors.initials: EA
authors.lastname: Kooi
authors.affiliation: Department of Molecular Microbiology, Washington University Medical School, St Louis, MO, USA.
authors.forename: Brett
authors.initials: B
authors.lastname: Lindenbach
authors.affiliation: Department of Medical Microbiology, Center of Infectious Disease, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Nicolette
authors.initials: N
authors.lastname: Huijkman
authors.affiliation: Department of Molecular Microbiology, Washington University Medical School, St Louis, MO, USA.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Rice
authors.affiliation: Department of Medical Microbiology, Center of Infectious Disease, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Willy J M
authors.initials: WJM
authors.lastname: Spaan
mesh_terms: D020413:3' Untranslated Regions / Q000737:chemistry / Q000235:genetics; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006224:Cricetinae; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D010948:Viral Plaque Assay; D014779:Virus Replication; D015005:Yellow fever virus / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology
chemical_list: D020413:3' Untranslated Regions; D018076:DNA, Complementary; D012367:RNA, Viral