title: Infectious transcripts of tick-borne encephalitis virus, generated in days by RT-PCR.
abstract: Construction of infectious clones of flaviviruses can be problematic owing to instability, toxicity, and recombination events occurring while cloning cDNA in the bacterial vectors. To overcome these difficulties we have devised a rapid and simple method for producing an infectious genetically engineered tick-borne encephalitis virus in less than 10 days using viral RNA from an unpurified virus suspension. The experimental protocol utilized the high fidelity reverse transcription-polymerase chain reaction to produce two long (5.7 and 5.2 kb) overlapping cDNA segments. To produce full-length cDNA the two overlapping segments were either ligated or fused by polymerase chain reaction. The cDNA was then transcribed and the derived full-length RNA was injected intracerebrally into young mice which reproduced the infectious virus within 8-20 days. To differentiate the engineered virus from parent virus, a Sunl restriction site was introduced by substituting nucleotides at positions 5688 and 5691 of the viral genome. This restriction site was present in the engineered virus recovered from infected mice. Antigenic and electrophoretic analysis of the proteins recovered from the engineered virus confirmed that it was indistinguishable from parent virus. In addition to its applicability as a rapid method of producing infectious engineered virus, this protocol offers the opportunity to introduce changes by site-directed mutagenesis without needing to clone the viral DNA. The method should be applicable to most viruses possessing an infectious RNA molecule and reduces the time required to produce a genetically engineered virus from years to days. When appropriate, the choice of mice for transfection of RNA has the advantage of being extremely simple, very sensitive, and producing high titers of stable virus.
authors.affiliation: Institute of Virology and Environmental Microbiology, Oxford, United Kingdom.
authors.forename: T S
authors.initials: TS
authors.lastname: Gritsun
authors.forename: E A
authors.initials: EA
authors.lastname: Gould
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D004195:Disease Models, Animal; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D004675:Encephalitis, Tick-Borne / Q000821:virology*; D005814:Genes, Viral; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods*; D020365:RNA Helicases; D012367:RNA, Viral / Q000235:genetics / Q000502:physiology*; D012194:RNA-Directed DNA Polymerase; D012697:Serine Endopeptidases; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D020365:RNA Helicases; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases