3.5 Viral growth curves in mosquito cell lines
To further characterize DDVax replication kinetics compared to MP-12 and
ZH501 strains, growth curves were performed in three insect cell lines.
Aag2 (Ae. aegypti, embryonic), ATC10 (Ae. aegypti, larval)
and Ct (Cx. tarsalis, embryonic) cells were infected with DDVax,
MP-12 or ZH501 over 6-day time courses. The Aedes aegypti larval
cell line ATC10 was not susceptible to infection with any virus strain.
DDVax replicated in Aag2 cells to lower peak titers than did MP-12 or
ZH501 strains (Figure S5) (random effects mixed model ANOVA, p =
8.0e-4). Similarly, DDVax also attained lower titers than control
viruses in Ct cells (random effects mixed model ANOVA, p =
3.5e-4). MP-12 grew to similar peak titers in Ct and Aag2 cells, at 9.1
and 9.5 log10 PFU/ml, respectively. Peak ZH501 titers
were 8.0 and 6.9 log10 PFU/ml, in Ct and Aag2
cells, respectively. The virulent strain caused syncytial
formation and lifting of cell monolayers, consistent with
pathogenicity(Turell, Gargan, & Bailey, 1984), which could have
affected final titers. Lastly, mean peak DDVax titers were 7.1 and 6.3
log10 PFU/ml, in Ct and Aag2 cells, respectively,
which are lower than peak titers for MP-12. DDVax grew better in Ct
cells than in Aag2 cells (Two way ANOVA, p = 4.5e-5), consistent
with the mosquito data. While the calculated DDVax MOI was 0.01 for all
cell lines, the actual MOI was 0.0052 for Aag2, 0.0127 for Ct, and
0.0153 for ATC-10 cells. The difference observed in replication kinetics
of DDVax in the Ct cells as compared with the Aag2 cells may be due in
part to the actual MOI being half the calculated value for Aag2 cells
(Figure S5).