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.