4.3.5. Serum Viral Load

Serum viral load was assessed via plaque assay and qRT-PCR. Plaque assays were performed as described above for the virus challenge suspensions. For qRT-PCR analysis, 50 µL of serum was added to 250 µL of TRIzol LS (Life Technologies, Carlsbad, CA, USA) and stored at ≤ −65 °C until used for RNA extraction. RNA extraction and qRT-PCR were performed as described previously [23]. In brief, samples in TRIzol LS were processed to RNA using Zymo Direct-zol™ RNA Mini Prep (Zymo Research, Irvine, CA, USA) kits per manufacturer instructions. RNA samples were analyzed via qRT-PCR targeting the EBOV glycoprotein gene (GeneBank accession {"type":"entrez-nucleotide","attrs":{"text":"AF086833","term_id":"10141003","term_text":"AF086833"}}AF086833). Primers were obtained from Invitrogen and probe was obtained from Integrated DNA Technologies. Probe was labeled as the 5′-end with fluorophore 9-carboxyfluroescein (6-FAM) and included a 3′-end nonfluorescent quencher/Minor Groove Binder (MGB). Master Mix was prepared by combining forward primer (1000 nM, 5′-TTTTCAATCCTCAACCGTAAGGC-3′), reverse primer (1000 nM, 5′-CAGTCCGGTCCCAGAATGTG-3′), and probe (100 nM, 5′-6FAM-CATGTGCCGCCCCATCGCTGC-MGBNFQ-3′) with 12.5 µL of 2X QuantiFast Probe Mix (QIAGEN, Hilden, Germany), 0.25 µL of 2X QuantiFast RT Mix (QIAGEN), 1.5 µL of 50 mM MgSO₄ and PCR-grade water (fill to 20 µL) for each planned reaction. To the Master Mix, test sample (5 µL) was added resulting in a final volume of 25 µL per reaction. Real-time analysis was performed using the Bio-Rad CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Thermocycling conditions were as follows: Step 1, 1 cycle, 50 °C for 15 min; Step 2, 1 cycle, 95 °C for 5 min; Steps 3–5, 45 cycles, 95 °C for 10 s, 60 °C for 30 s, single read. For quantification purposes, a HPLC-purified synthetic EBOV RNA standard containing the conserved EBOV glycoprotein sequence was used (5′-CAGUCCGGUCCCAGAAUGUGGCAUGUGCCGCCCCAUCGCUGCAGCAAGAAAUCAAUUGCCUUACGGUUGAGGAUUGAAAA-3′).