title: Infectious cDNA clones of Langat tick-borne flavivirus that differ from their parent in peripheral neurovirulence.
abstract: Tick-borne flavivirus strain Langat TP21 (LGT TP21) recovered from ticks, is naturally attenuated for humans but retains demonstrable neurovirulence and peripheral virulence ("neuroinvasiveness") for mice. Previously a mutant, strain E5, less virulent for mice was derived from LGT TP21. Multiple attempts to prepare a full-length infectious TP21 cDNA from cDNA fragments cloned in E. coli were uniformly unsuccessful. A more informative sequence than that obtained from these cloned cDNA fragments and similar E5 cDNA fragments was derived from RT-PCR fragments that had not been cloned in E. coli. Comparison of the RT-PCR consensus sequence of TP21 and E5 identified only seven amino acid differences that might be responsible for the observed difference in virulence of these strains for mice. Eleven independent infectious cDNA clones of TP21 were recovered using two overlapping long RT-PCR fragments. Importantly, low-titered virus used to prepare cDNA as template for PCR was harvested early in the growth cycle to minimize the frequency of deletion mutants that accumulated late in infection. The four analyzed rescued clones exhibited clone-specific minimal divergence from the consensus sequence but this limited variation was associated with diminished peripheral virulence for immunocompetent mice. Manipulation of these clones should facilitate elucidation of LGT virulence.
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
authors.forename: M S
authors.initials: MS
authors.lastname: Campbell
authors.forename: A G
authors.initials: AG
authors.lastname: Pletnev
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D016384:Consensus Sequence / Q000235:genetics; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity* / Q000502:physiology; D004675:Encephalitis, Tick-Borne / Q000401:mortality / Q000473:pathology / Q000821:virology*; D014644:Genetic Variation / Q000235:genetics; D016679:Genome, Viral; D007928:Lethal Dose 50; D051379:Mice; D016513:Mice, SCID; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000473:pathology / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014774:Virulence / Q000235:genetics; D014779:Virus Replication / Q000502:physiology
chemical_list: D018076:DNA, Complementary