2.9. Viral Genome Extraction and Quantitation

For the extraction of the viral genome, lung, heart and skeletal muscle fragments from 10 mice infected with DENV-2 lineages euthanized at 72 hpi were macerated in 500 µL of Leibovitz culture medium (Invitrogen, Waltham, MA, USA) and centrifuged for 15 min at 10,000 RPM at a temperature of 4 °C. RNA was extracted from 140 µL of the macerated organ supernatant using the QIAmp Viral RNA mini kit (Qiagen, Düsseldorf, Germany) according to the manufacturer’s recommendations. For the detection and quantification of the viral genome, a standard curve was constructed from a serial dilution of RNA extracted from a DENV-2 sample (strain {"type":"entrez-protein","attrs":{"text":"S16083","term_id":"109772","term_text":"pir||S16083"}}S16083), with a known titer (8.7 × 10⁶ PFU/mL). The protocol used was described by Johnson et al. [57], using the primers DENJ2-R (5′-CCATCTGCAGCAACACCATCTC-3′) and DENJ2-F (5′-CAGGTTATGGCACTGTCACGAT-3′), designed from a fragment of the 3′ non-coding region, and the probe DENJ2-P (CY5 5′-CTCTCCGAGAACAGGCCTCGACTTCAA-3′ BHQ-1). The reaction was performed according to the protocol of the commercial kit SuperScript III Platinum One-Step Quantitative RT-PCR (Invitrogen, USA), with ideal concentrations of the primers and probe determined by optimization assays. In a 96-microwell optical-bottom plate Waltham, MA, USA1 µL of each primer (50 µM) and 12.5 µL of the reaction mix (0.4 µM of each dNTP and 6 µM of MgSO₄) were added, followed by 0.5 µL SuperScript III RT enzyme, 3.5 µL DNase/RNase free water, 1 µL MgSO₄ (5 mM) and 0.75 µL probe (9 µM), for a total volume of 20 µL per well. Soon after, 5 mL of extracted RNA was added, obtaining a final volume of 25 mL/reaction. Each sample and control were applied in duplicate. The plates were placed on the 7500 FAST platform (Applied Biosystems, USA) for the qRT-PCR reaction, according to the following cycling parameters: reverse transcription (50 °C, 15′, 1 cycle), enzyme activation (95 °C, 2′, 1 cycle), denaturation (95 °C, 15″, 40 cycles) and annealing/extension (60 °C, 1′, 40 cycles).