Detection of Oropouche-like viruses in mosquitoes

The collected females were grouped in pools of up to 10 mosquitoes by identified species. The pools were processed for RNA extraction using 1 mL of TRIzol^® Reagent, and the RNA was extracted according to the manufacturer’s instructions. The viral RNA was detected using TaqMan^® Fast Virus 1-Step Master Mix in a StepOnePlus Real-Time PCR System (Applied Biosystems) using MAYV and OROV primers and probes [29]. All mosquitoes were subjected to a sensitive and specific method (RT-qPCR) for the detection of MAYV, OROV, and other OROV-like viruses carrying the OROV S segment [9, 10, 29, 30].

The RT-qPCR conditions were 50°C for 5 min, 95°C for 20 s, then 45 cycles of 95°C for 3 s, and 60°C for 30 s with fluorescence acquisition. For all RT-qPCR assays, the MS2 RNA bacteriophage was spiked prior to RNA extraction to track false-negative reactions due to PCR inhibition, as described elsewhere [29]. The RT-qPCR reactions were analyzed based on the cycle threshold (CT) values described by Naveca et al. [29], who considered a Ct value lower than 38 as positive (mean Ct values, 34.9–38.3 and 34.8–38.1 for MAYV and OROV, respectively). Positive MAYV and OROV samples were used for positive control. Data were analyzed using the QuantStudio 5 Real-Time PCR System.