Infectious cDNA clones of Langat tick-borne flavivirus that differ from their parent in peripheral neurovirulence

Tick-borne flavivirus strain Langat TP21 (LGT TP21) recovered from ticks, i s naturally attenuated for humans but retains demonstrable neurovirulence a nd peripheral virulence ("neuroinvasiveness'') for mice. Previously a mutan t, strain E5, less virulent for mice was derived from LGT TP21. Multiple at tempts ta prepare a full-length infectious TP21 cDNA from cDNA fragments cl oned in E. coli were uniformly unsuccessful. A more informative sequence th an that obtained from these cloned cDNA fragments and similar E5 cDNA fragm ents 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 di fference in virulence of these strains for mice. Eleven independent infecti ous cDNA clones of TP21 were recovered using two overlapping long RT-PCR fr agments, importantly low-titered virus used to prepare cDNA as template for PCR was harvested early in the growth cycle to minimize the frequency of d eletion mutants that accumulated late in infection. The four analyzed rescu ed clones exhibited clone-specific minimal divergence from the consensus se quence but this limited variation was associated with diminished peripheral virulence for immunocompetent mice. Manipulation of these clones should fa cilitate elucidation of LGT virulence. (C) 2000 Academic Press.