Detection of Dengue Virus in Aedes aegypti Eggs Supports Vertical Transmission in Natural Settings

Dengue remains one of the most important mosquito-borne viral diseases worldwide, and vector control continues to be the cornerstone of disease prevention. Vertical transmission has been proposed as a mechanism contributing to the maintenance of dengue virus (DENV) during interepidemic periods. In this study, field-collected egg pools and second-generation eggs derived from field-collected females from the state of Rio de Janeiro were analyzed by RT-qPCR, Sanger sequencing, plaque assay, and transmission electron microscopy. The second-generation egg samples (E1-E4), corresponding to F2 eggs of the AEDES-RIO strain, were all DENV positive. Sample E1 was positive for DENV-1, whereas samples E2-E4 were positive for DENV-4, confirming transovarial vertical transmission. The detection of DENV-1 and DENV-2 in field-collected egg pools further supports the occurrence of vertical transmission. All PCR-amplified viral fragments were confirmed by Sanger sequencing. Furthermore, homogenized egg samples naturally infected with DENV successfully infected Vero cells, demonstrating that the virus remained infectious. Transmission electron microscopy revealed virus-like particles of approximately 50 nm in diameter within egg tissues, providing ultrastructural evidence that corroborated the molecular and virological findings. Collectively, these findings provide additional evidence supporting vertical transmission of DENV in naturally infected Aedes aegypti populations and highlight the potential of egg-based surveillance as a complementary tool for entomological monitoring. However, further longitudinal studies are needed to establish its epidemiological value.