SPONTANEOUS AND ENGINEERED DELETIONS IN THE 3'-NONCODING REGION OF TICK-BORNE ENCEPHALITIS-VIRUS - CONSTRUCTION OF HIGHLY ATTENUATED MUTANTS OF A FLAVIVIRUS

The flavivirus genome is a positive-strand RNA molecule containing a s ingle long open reading frame flanked by noncoding regions (NCR) that mediate crucial processes of the viral life cycle. The 3' NCR of tick- borne encephalitis (TEE) virus can be divided into a variable region t hat is highly heterogeneous in length among strains of TEE virus and i n cel tain cases includes an internal poly(A) tract and a 3'-terminal conserved core element that is believed to fold as a whole into a well -defined secondary structure, We hale now investigated the genetic sta bility of the TBE virus 3' NCR and its influence on viral growth prope l ties and virulence. We observed spontaneous deletions in the variabl e region during growth of TEE vir us in cell culture and in mice, Thes e deletions varied in size and location but always included the intern al poly(A) element of the TEE virus 3' NCR and never extended into the conserved 3'-terminal core element, Subsequently, we constructed spec ific deletion mutants by using infectious cDNA clones with the entire variable region and increasing segments of the core element removed. A virus mutant lacking the entire variable region was indistinguishable from wild-type virus with respect to cell culture growth properties a nd virulence in the mouse model, In contrast, even small extensions of the deletion into the core element led to significant biological effe cts, Deletions extending to nucleotides 10826, 10847, and 10870 caused distinct attenuation in mice without measurable reduction of cell cul ture growth properties, which, however, were significantly restricted when the deletion was extended to nucleotide 10919. An even larger del etion (to nucleotide 10994) abolished viral viability. In spite of the ir high degree of attenuation, these mutants efficiently induced prote ctive immune responses even at low inoculation doses, Thus, 3'-NCR del etions represent a useful technique for achieving stable attenuation o f flaviviruses that can be included in the rational design of nov el f lavivirus live vaccines.