Screening for Orthoflavivirus RNA in Bats from Urban and Rural Landscapes of Guayas Province, Ecuador

Background: Orthoflaviviruses are arthropod-borne viruses of major importance for human and animal health. In Ecuador, surveillance has focused on human cases and mosquito vectors, and evidence from wildlife remains scarce. This study screened bats from Guayas Province for orthoflavivirus RNA and examined whether positivity was associated with host taxon, landscape, or sex. Materials and Methods: A cross-sectional study was conducted using liver tissue from bats captured during 19 nocturnal mist-netting sessions at 10 localities in Guayas Province between October 2020 and May 2024. Total RNA was extracted and analyzed by a generic nested RT-PCR assay targeting a conserved region of the NS5 gene, with β-actin amplification as an internal control. A total of 128 samples yielded valid results. Prevalence was reported with Wilson 95% confidence intervals (CIs), and associations were assessed by bivariate tests and multivariable logistic regression. Results: Twenty-five of 128 bats (19.5%; 95% CI: 13.6–27.2) were positive in the generic NS5 assay. In bivariate analysis, positivity was higher in urban than rural bats (40.0% vs. 13.3%; odds ratio [OR] 4.36, 95% CI: 1.71–11.10; p = 0.0029). However, all 30 urban bats were Molossus molossus , and after adjustment for host taxon the landscape association disappeared (adjusted OR: 0.99, 95% CI: 0.31–3.14), whereas M. molossus remained strongly associated with positivity (adjusted OR: 15.61, 95% CI: 3.79–64.21; p < 0.001). Within M. molossus , positivity was identical in rural and urban settings (40.0% vs. 40.0%; p = 1.000). Sequencing could not be completed because of low template yield. Conclusions: These findings provide presumptive evidence of flavivirus-like RNA in bats from coastal Ecuador, concentrated in one synanthropic insectivorous species rather than distributed along an urban–rural gradient. Because the products could not be sequenced, no specific orthoflavivirus can be identified. Wildlife merits inclusion in integrated arbovirus surveillance under a One Health framework.