Fig. 5. Expression of ROP2 antisense RNA compromises parasite invasion, replication
and virulence in mice. ROP2AS clones and control RH parasites were
inoculated into fresh human foreskin fibroblast (HFF) cells. After 2 hours,
parasites that did not infect were washed off, and the infection was continued
for 24 hours. (A) Invasion was monitored by counting the number of vacuoles
for control (wild type) and antisense clones (ROP2AS-1, ROP2AS-7,
ROP2AS-20). Data are derived by counting vacuoles in at least 50-100
randomly selected microscopic fields. Differences between values of the
control and all experimental groups were statistically significant
(P<0.005). (B) Parasite replication was monitored by
[3H]uracil incorporation assays using HFF cells infected for 24
hours. Data are expressed as mean±s.e.m. from 3-6 independent
experiments repeated at least four times. All ROP2AS clones over a
24-hour period post-infection reproducibly replicated more slowly than control
(P<0.005). (C) Replication of intracellular parasites was
determined at 24 hours after infection by counting the number of parasites
within each of 130 randomly selected vacuoles. The median vacuole in the
control contained 16 parasites (corresponding to four parasite doublings),
whereas in the antisense clones this was between 4-8 parasites (2-3
doublings), reflecting a delay in replication. Wild type (•);
ROP2AS-7 (
); ROP2AS-20 (
). (D) Survival curves
for mice (10 mice in each group) infected with 105 parasites of
control or ROP2AS clones. Wild type (
); ROP2AS-1
(
); ROP2AS-7 (); ROP2AS-20 ().