INFECTIOUS TRANSCRIPTS OF TICK-BORNE ENCEPHALITIS-VIRUS, GENERATED INDAYS BY RT-PCR

Construction of infectious clones of flaviviruses can be problematic o wing to instability, toxicity, and recombination events occurring whil e cloning cDNA in the bacterial vectors. To overcome these difficultie s we have devised a rapid and simple method for producing an infectiou s genetically engineered tick-borne encephalitis virus in less than 10 days using viral RNA from an unpurified virus suspension. The experim ental protocol utilized the high fidelity reverse transcription-polyme rase chain reaction to produce two long (5.7 and 5.2 kb) overlapping c DNA segments. To produce full-length cDNA the two overlapping segments were either ligated or fused by polymerase chain reaction. The cDNA w as then transcribed and the derived full-length RNA was injected intra cerebrally into young mice which reproduced the infectious virus withi n 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. Antigen ic and electrophoretic analysis of the proteins recovered from the eng ineered virus confirmed that it was indistinguishable from parent viru s. In addition to its applicability as a rapid method of producing inf ectious engineered virus, this protocol offers the opportunity to intr oduce changes by site-directed mutagenesis without needing to clone th e Viral DNA. The method should be applicable to most viruses possessin g an infectious RNA molecule and reduces the time required to produce a genetically engineered virus from years to days. When appropriate, t he choice of mice for transfection of RNA has the advantage of being e xtremely simple, very sensitive, and producing high titers of Stable v irus. (C) 1995 Academic Press, Inc.