A rapid and convenient variant of fusion-PCR to construct chimeric flaviviruses.
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.
Charlier
Nathalie
N
Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000, Leuven, Belgium.
Molenkamp
Richard
R
Leyssen
Pieter
P
Vandamme
Anne Mieke
AM
De Clercq
Erik
E
Bredenbeek
Peter
P
Neyts
Johan
J
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
D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D014759:Viral Envelope Proteins; C074932:prM protein, Flavivirus; C078400:yellow fever virus envelope protein E