Vertical transmission of Dengue virus type-3 and metagenomic virome profiles of Aedes aegypti mosquitoes collected in Kisumu, Kenya

Abstract

Aedes aegypti is the main vector of several arboviruses including chikungunya, dengue, yellow fever and Zika. Beyond arboviruses, Aedes aegypti harbours insect-specific viruses (ISVs), which can modulate mosquito's ability to transmit diseases by interfering with viral processes and triggering immune responses. Both arboviruses and ISVs can be transmitted vertically, where viruses are passed from parent to offspring. The lack of systematic molecular and entomological surveillance, has left the diversity of viruses in local Aedes aegypti populations largely unexplored. This study aimed to characterize the viromes of Aedes aegypti mosquitoes from Kisumu, Kenya, focusing on viral diversity. Immature larvae and pupae were collected from Jua Kali area in Kisumu, reared into adults, and subjected to viral isolation by cell culture and metagenomic next-generation sequencing. RNA extraction, library preparation, and Illumina MiSeq sequencing were performed on CPE positive pools and metagenomic superpools. Initial data analysis was conducted using the CZ-ID platform, with quality control applied using PrinseqLite v0.20.4 to filter low-quality reads and remove adapters. De novo sequence assembly was performed with MEGAHIT v1.2.9, followed by BLAST analysis. Phylogenetic relationships were analyzed using the Maximum Likelihood method. A total of 2,142 female Aedes aegypti, grouped into 86 pools and 4 superpools, were analyzed using cell culture and metagenomic next-generation sequencing respectively. Dengue virus type-3 was detected in one of the 86 pool. Additionally, a variety of ISVs were identified, including Iflaviruses related to Tesano Aedes Iflavirus (TeAV), Armigeres Iflavirus, and Negeviruses related to Rabai Virus. An unclassified virus closely related to Korle-Bu Aedes virus was also detected. Our study provides insights into the viral diversity within Aedes aegypti mosquitoes in Kisumu and evidence of natural vertical transmission, specifically transovarial transmission of dengue virus type-3. Ongoing research is imperative to unravel vertical transmission mechanisms and subtleties governing ISV-arbovirus interactions across diverse environmental settings.

Fig 1. The overall map of Kenya and Kisumu County showing the site where sampling was conducted.

Base maps, boundaries and shape files of Kenyan map and administrative boundaries of the County and Sub-county were derived from GADM data version 4.1 (https://gadm.org) and the maps were generated using ArcGIS Version 10.2.2 (http://desktop.arcgis.com/en/arcmap) advanced license) courtesy of Samuel Owaka.

Fig 2. Temperature and Rainfall prevailing in Jua kali area at the time of mosquito sampling in November 2022 (Source: Kenya Meteorological Department, 2022).

Fig 3. A. Shows the innoculated cells; I before CPE and II with CPE, B. A Maximum Likelihood phylogenetic tree constructed with 1000 bootstrap replicates using the NS5 protein.

The bootstrap value threshold on the nodes was set at 70.

Fig 4. A hundred percent stacked column chart of relative abundance of the different viral families.

Fig 5. Maximum likelihood phylogenetic trees constructed using the conserved amino acid domains detected in this study.

A. Negevirus (Accession no. PP590325). B. Unclassified Virus (Accession no. PP590324). C. Iflavirus 1 (Accession no. PP590327). D. Iflavirus 2 (Accession no. PP590328). Bootstrap support values from 1000 bootstrap replicates are indicated on the branches of each tree. Bootstrap values >70% are displayed at important nodes.