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Several studies have shown Dengue Virus (DENV) nucleic acids and/or antibodies present in Neotropical wildlife including bats, suggesting that some bat species may be susceptible to DENV infection. Here we aim to elucidate the role of house-roosting bats in the DENV transmission cycle. Bats were sampled in households located in high and low dengue incidence regions during rainy and dry seasons in Costa Rica. We captured 318 bats from 12 different species in 29 households. Necropsies were performed in 205 bats to analyze virus presence in heart, lung, spleen, liver, intestine, kidney, and brain tissue. Histopathology studies from all organs showed no significant findings of disease or infection. Sera were analyzed by PRNT90 for a seroprevalence of 21.2% (51/241), and by PCR for 8.8% (28/318) positive bats for DENV RNA. From these 28 bats, 11 intestine samples were analyzed by RT-PCR. Two intestines were DENV RNA positive for the same dengue serotype detected in blood. Viral isolation from all positive organs or blood was unsuccessful. Additionally, viral load analyses in positive blood samples by qRT-PCR showed virus concentrations under the minimal dose required for mosquito infection. Simultaneously, 651 mosquitoes were collected using EVS-CO2 traps and analyzed for DENV and feeding preferences (bat cytochrome b). Only three mosquitoes were found DENV positive and none was positive for bat cytochrome b. Our results suggest an accidental presence of DENV in bats probably caused from oral ingestion of infected mosquitoes. Phylogenetic analyses suggest also a spillover event from humans to bats. Therefore, we conclude that bats in these urban environments do not sustain DENV amplification, they do not have a role as reservoirs, but function as epidemiological dead end hosts for this virus. [ABSTRACT FROM AUTHOR]