2.8 Reverse transcription quantitative PCR (RT-qPCR)
RT-qPCR was performed in duplicate using 5 µl sample or RNA standards
and run on a QuantStudio 2.0 qPCR platform (Applied Biosystems).
Calculated virus amounts were adjusted to account for RNA copy number
per tissue. The following primers were used to quantitate RVFV RNA in
all samples: RVFL-2912fwdgg TGAAAATTCCTGAGACACATGG, RVFL-2971revAC
ACTTCCTTGCATCATCTGATG, RVFL-2950-Probe
(FAM)-CAATGTAAGGGGCCTGTGTGGACTTGTG-(BHQ1)(Bird, Bawiec, Ksiazek,
Shoemaker, & Nichol, 2007). TaqMan Fast Virus 1-Step Master Mix
(Applied Biosystems) was used with final primer concentrations of 500nM
and a probe concentration of 100nM. Samples and standards were loaded
into 96-well plates and run using fast cycling mode on an AB QuantStudio
machine, using the manufacturer’s recommended settings. The cycling
conditions were as follows: 50oC 5 min (1 cyc),
95oC 20 sec (1 cyc), (95oC 3 sec,
60oC 30 sec (40 cyc)).
RNA copy number standards were developed by amplifying a portion of the
L segment from 20 ng plasmid bearing the full length gene(Bird et al.,
2008). The RVFL2173_T7_F amplification forward primer
(TAATACGACTCACTATAGGGCAGGTGAGCCCTTCATTCT) contained a T7 promoter;
RVFL3542_R was the reverse primer (GAGGGGTAAATGGCAAGGTACA). 100 ng
input of PCR product was used in vitro transcription reactions
that incubated for 5 hours at 37oC using the
manufacturer’s recommendations. Transcription products were stored in 5
µl aliquots at -80°C; they were quantitated using a Qubit fluorometer
(ThermoFisher) using the manufacturer’s recommendations. For RT-qPCR,
fresh aliquots of in vitro transcription reactions were serially
diluted in 10-fold increments to generate standard curves to relate copy
number to raw cycle threshold (Ct value). One standards plate was run
for all samples screened on a given day. A representative standard curve
was y= -3.3111x + 36.655 R2= 0.9976, where y= Ct value
and x= log10 RNA copy number.
2.9 DDVax dose
response experiment
A dose response experiment was performed as a follow up to the mosquito
vector competence challenges, which were administered with only a single
high titer of over 8.0 log10 PFU/ml. The purpose of this
experiment was to test the hypothesis that Cx. tarsalis DDVax
infection rates vary as a function of virus titer in the artificial
blood meal. Cx tarsalis were exposed to oral bloodmeals at 6.2,
4.5, or 3.5 log10 PFU/ml and held for 14 days at
28oC, rH 80%. At 14 days-post-feeding legs/wings,
saliva and bodies were harvested into mosquito diluent as above in
individual tubes and stored at -80°C. Sample processing was performed as
described above.