Mosquitoes were divided into pools according to species (Ae. aegypti and Ae. albopictus) and collection site, each pool contained from 1 to 10 mosquitoes. Mosquitoes captured in 2016 were only tested for dengue virus, and in 2017 and 2018 samples were tested for dengue, chikungunya, and Zika. RNA was obtained with the RNeasy Mini Kit (Qiagen, Germantown, USA commercial kit. Each pool was mechanically macerated in 100 µL of lysis buffer (buffer RLT) and RNA was obtained with the RNeasy Mini Kit (Qiagen) commercial kit according to manufacturer instructions. Viral detection was performed with RT-PCR. Copy DNA (cDNA) was synthesized using 10 µL extracted RNA, 1× specific buffer for reverse transcriptase, 1 mM dNTPs, 10 µM direct primer specific for the dengue virus (DV1-5′-GGRACKTCAGGWTCTCC-3′) and 1 µL reverse transcriptase (Fermentas, Vilnius, Lithuania), as well as incubation at 42 °C for 1 h and enzyme inactivation at 94 °C for 10 min. The following was used for dengue virus amplification: 3 µL cDNA, 1× buffer, 2.0 mM MgCI2, 0.2 mM dNTPs, 0.2 µM of each reverse primer specific for the four virus serotypes (DSP1-5′-AGTTTCTTTTCCTAAACACCTCG-3′, DSP2-5′-CCGGTGTGCTCRGCYCTGAT-3′, DSP3-5′-TTAGAGTYCTTAAGCGTCTCTTG-3′ and DSP4-5′-CCTGGTTGATGACAAAAGTCTTG-3), and 0.5 U taq polymerase. PCR amplification was performed at an initial temperature of 95 °C for 2 min, followed by 35 cycles at 95 °C for 30 s, 55 °C for 1 min, and 72 °C for 40 s, with a final extension at 72 °C for 3 min. The size of the NS3 gene fragments obtained was 169 pb for DENV-1, 362 pb for DENV-2, 265 pb for DENV-3 and 426 pb for DENV-4. The protocol used by [25] was applied to detect Zika virus. For chikungunya amplification, the protocol proposed by [26] was used.

Amplification products were analyzed in 2% agarose gels dyed with ethidium bromide and observed under ultraviolet light. For each reaction, extraction and amplification negative controls were included, as well as positive controls comprising RNA of Zika and chikungunya viruses, and the four serotypes of dengue virus obtained from supernatant of infected cells. To confirm pool positivity, 15% of positive samples were randomly selected for bidirectional sequencing at Macrogen (Seoul, Korea).

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