3.1 DDVax variant analysis
We used sequencing to track the genetic stability of DDVax over 5
passages in Vero cell culture (P1 through P5, MOI 0.0005). The P5
preparation was used for goat inoculations. Total RNA from virus
preparations (supernatant: P1-P4, or filtered supernatant: P5) was
converted into shotgun Illumina libraries and sequenced on an Illumina
NextSeq 500 instrument to produce a median of 1.2x107single end 150 nucleotide (nt) reads per sample. After removing low
quality and adapter-derived bases, a median of 1.1x107reads (88%) remained per sample. After removing reads mapping to the
host cell genome (Chlorocebus sabeus ), a median of
3.4x106 reads (28%) remained. A median of
4.4x105 reads mapped to the DDVax reference sequence,
producing a median coverage of 6055x across all viral segments (Figure
S2). SNVs and short insertion and deletions were quantified using
lofreq, and larger structural variants, including possible DVGs, were
quantified using DI-tector (Beauclair et al., 2018; Wilm et al., 2012).
The virus remained generally stable over passage in Vero cells. We
report variants with ≥ 3% frequency in coding sequences (Table 1).
Three single nucleotide variants in the glycoprotein precursor gene rose
to above 50% frequency by P5 (Table 1). A variant at position 31
(predicted to produce the amino acid change G3E in the DDVax NSm-deleted
glycoprotein, equivalent to Gly 133 in the RVFV NSm/Gn/Gc polyprotein,
NC_014396) rose to 54% frequency by P5. A variant at position 499
(G159D, equivalent to Gly 289 in ZH501) rose to 55% by
P5. And a variant at position 926 (N301K, equivalent to Asn 431 in
ZH501) rose to 90% frequency by P5.The highest frequency L segment
variant was a synonymous variant at position 4665 that rose to 16% by
P5. No variants on the S segment rose above 3% frequency in any sample.
Lofreq did not identify any short insertion or deletion variants above
3%. Similarly, DI-tector did not identify any structural variants
(larger insertions, deletions, incomplete transcripts consistent with
DVGs or copy-back variants) with a frequency ≥ 3%.
3.2 Mosquito vector
competence
To measure differences in viral infection kinetics, Ae. aegyptiand Cx. tarsalis were challenged with 1:1 mixtures of blood and
freshly grown DDVax and then compared against those infected with MP-12
or the ZH501 parental strain. Because of the need to use freshly-grown
virus for infections, it was not possible to control for differences in
bloodmeal titers. Mean bloodmeal titers ranged from ~8.1
logs/ml with DDVax to 6.5 or 6.8 log10 PFU/ml in MP-12
and ZH501, respectively (Figure 1A). Thus, DDVax titers were
significantly higher than that of the other two virus strains (ANOVA,
p=1.8e-5). Virus infection phenotypes were measured by detection of
viral RNA in Cx. tarsalis bodies, legs/wings and saliva at 14
days post-infection (Figure 1B, Table S1). All saliva samples were also
subjected to plaque assay for detection of infectious virus.
The percentage of Culex mosquito DDVax viral RNA positive bodies
was not statistically different from MP-12 or ZH501 infections (Figure
1B, Table S1). However, the RNA genome copy number in Culex orAedes infected with DDVax was at least two log10values lower than those infected with either MP-12 or ZH501 strains,
despite exposure of mosquitoes to a DDVax titer over one
log10 PFU greater than controls (Figure 2).
Dissemination of DDVax viral RNA to Culex legs/wings was also
significantly reduced compared to MP-12 (χ2 test,p = 2.078e-07). Moreover, infectious DDVax was detected in only
one of 140 Culex saliva samples at 14 dpi, whereas 96% and 82%
of MP-12 and ZH501 infected saliva samples, respectively, showed CPE
consistent with the presence of infectious virus (Table 2,
χ2 test, p = 2.2e-16 vs MP-12, 2.2e-16, vs
ZH501). To rule out the possibility that sample freeze-thaw compromised
virus viability, an additional subset of saliva samples from 14 dpi
DDVax exposed mosquitoes were assessed for the presence of infectious
virus; still, none was detected (Table 2).