# Persistent and Long-Term Infectivity of Dengue Virus in Mosquito Cells Revealed Reduced Replication in Vector Host and Human Endothelial Cells

## TL;DR

This study shows that dengue virus can persist in mosquito cells for over 175 days, with reduced replication and infectivity over time.

## Contribution

The study reveals the long-term persistence and reduced replication of DENV2 in mosquito and human cells.

## Key findings

- DENV2 infectivity and viral titers gradually decline in mosquito cells over 175 days.
- Extracellular vesicles from infected cells maintain infection ability despite decreasing viral load.
- The viral M protein is present in vesicles early but disappears at later timepoints.

## Abstract

Understanding the intrinsic potential of persistent dengue virus (DENV) replication and survival in vector host cells is critically important. In this study, we investigated to what extent DENV can replicate within the vector host Aedes albopictus C6/36 mosquito cells (cell line routinely used for propagation of DENV in research laboratories). We detected DENV serotype 2 (DENV2) loads in cell culture supernatants collected at different days post infection (3, 19, 33, 60, 90, 120 and 175) and found the presence of capsid transcripts and protein levels in these virus supernatants. Tissue culture infectious dose 50 (TCID50) assay revealed a gradual reduction in viral titers and infectivity from days 19 to 175 post DENV2 infection. Furthermore, infection kinetics with these virus supernatants collected at different days post DENV2 infection demonstrated declining viral replication in naïve C6/36 cells and human endothelial recipient cells. These results provided information on viral replication competence and the persistency of DENV2 infection from days 19 to 175 in mosquito cells. Extracellular vesicles (EVs) isolated from DENV2-infected C6/36 cell culture supernatants showed a progressive increase in EV concentration from day 33 to day 175. While DENV2 loads within these EVs declined over time, their ability to mediate infection in naïve C6/36 and endothelial cells remained constant. Notably, the viral membrane (M) protein was detected in EVs at days 3, 19, and 33 but was absent at later timepoints (days 60, 90, 120, and 175). The prM protein was not detected in any of the samples analyzed. In conclusion, DENV2 exhibits the capacity for persistent infection in mosquito cells, thereby potentially serving as a model for investigating the mechanisms that govern years of long-term and sustained viral infections within the vector host.