Tick-borne flaviviruses are maintained in nature in an enzootic cycle involving 
a tick vector and a vertebrate host. Thus, the virus replicates in two disparate 
hosts, each providing selective pressures that can influence virus replication 
and pathogenicity. To identify viral determinants associated with replication in 
the individual hosts, plaque purified Langat virus (TP21pp) was adapted to 
growth in mouse or tick cell lines to generate two virus variants, MNBp20 and 
ISEp20, respectively. Virus adaptation to mouse cells resulted in four amino 
acid changes in MNBp20 relative to TP21pp, occurring in E, NS4A and NS4B. A 
comparison between TP21pp and ISEp20 revealed three amino acid modifications in 
M, NS3 and NS4A of ISEp20. ISEp20, but not MNBp20, was attenuated following 
intraperitoneal inoculation of mice. Following isolation from mice brains, 
additional mutations reproducibly emerged in E and NS3 of ISEp20 that were 
possibly compensatory for the initial adaptation to tick cells. Thus, our data 
implicate a role for E, M, NS3, NS4A and NS4B in host adaptation and 
pathogenicity of tick-borne flaviviruses.