97-12-098
Abstract:
Spontaneous and engineered deletions in the 3' noncoding region of
tick-borne encephalitis virus: construction of highly attenuated
mutants of a flavivirus.
Christian W. Mandl, Heidemarie Holzmann, Tamara Meixner,
Susanne Rauscher, Peter F. Stadler, Steven L. Allison,
Franz X. Heinz
The flavivirus genome is a positive-strand RNA molecule containing a
single 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 (TBE) virus can be divided into a variable
region that is highly heterogeneous in length among strains of TBE
virus and in certain 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 have now investigated the
genetic stability of the TBE virus 3' NCR and its influence on viral
growth properties and virulence. We observed spontaneous deletions in
the variable region during growth of TBE virus in cell culture and in
mice. These deletions varied in size and location but always included
the internal poly(A) element of the TBE virus 3' NCR and never
extended into the conserved 3'-terminal core element. Subsequently, we
constructed specific 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 and virulence in the mouse model. In
contrast, even small extensions of the deletion into the core element
led to significant biological effects. Deletions extending to
nucleotides 10826, 10847, and 10870 caused distinct attenuation in
mice without measurable reduction of cell culture growth properties,
which, however, were significantly restricted when the deletion was
extended to nucleotide 10919. An even larger deletion (to nucleotide
10994) abolished viral viability. In spite of their high degree of
attenuation, these mutants efficiently induced protective immune
responses even at low inoculation doses. Thus, 3'-NCR deletions
represent a useful technique for achieving stable attenuation of
flaviviruses that can be included in the rational design of novel
flavivirus live vaccines.
keywords:
J Virol 1998 Mar;72(3):2132-2140
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