Detection of dengue virus natural infection in Aedes aegypti from northern Peru by combined, portable real-time quantitative polymerase chain reaction and next generation sequencing

Aedes aegypti, vector of Yellow Fever, Dengue, Chikungunya, Zika and Mayaro, is one of the most important vector threats globally. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real time PCR (RT-qPCR) offer a reduced processing time and comparable sensitivity than virus isolation for arbovirus detection in Ae. aegypti. We compared two Dengue Virus (DENV) detection approaches in wild-caught Ae. aegypti: a Flavivirus RT-PCR and a field-deployable RT-qPCR (Biomeme Pan-Dengue Virus) combined with MinION sequencing.  Aedes aegypti (689 mosquitoes, 74 pools) collected in 2016-2019 from 12 Peruvian military bases and 22 civilian communities located in coastal and jungle regions in northern Peru were tested.  Aedes aegypti experimentally infected with DENV-2 and standard controls were used as positive controls. Four and eight DENV positive pools (Ct = 8.9 - 23.19) were detected by FU1/cFD2 RT-PCR and Biomeme RT-qPCR, respectively. All four Flavivirus RT-PCR positive pools corresponded to Biomeme DENV positive pools with fluorescence higher than 18.  RT-PCR amplicons (250 bp) were sequenced; BLASTn analysis showed 97-99% identity with DENV-3. Biomeme DENV positive pools were sequenced using MinION; 3 pools were negative and 5 were positive for DENV-3.  MinION sequencing results coincide with FU1/cFD2 amplicon sequencing for 4 pools.  These 4 DENV-3 mosquito positive pools belong to collections performed in 2017 from Piura coinciding with a dengue outbreak in this state during the coastal “El Niño”.  Our results indicate that our RT-PCR/sequencing approach and the combined portable Biomeme RT-qPCR/MInION sequencing provide comparable results for DENV detection in field-collected Ae. aegypti.