Identification of insect-specific viruses in mosquitoes collected from wildlife and rural areas in north-eastern parts of South Africa using a metagenomic RNA sequencing approach

Next generation sequencing (NGS) has expanded virus detection capabilities beyond the limitations of sequence-specific methods. While mosquito collections in South Africa (SA) have been investigated for the major arbovirus genera (Alpha-, Orthobunya-, and Orthoflavivirus), research on insect-specific viruses (ISVs) is limited. Here we used an RNA-sequencing viral-metagenomics approach to investigate arboviruses and ISVs in mosquitoes collected in SA. Ten archived mosquito pools, representing up to 50 mosquitoes per pool, previously tested for the major arbovirus genera were investigated for the presence of additional viruses using NGS. These pools included known arbovirus mosquito vectors from the Aedes, Anopheles, Culex, and Mansonia genera, collected from wildlife areas in the Kruger National Park (Mpumalanga/Limpopo) and rural areas of Jozini (KwaZulu-Natal). Extracted RNA was DNA-depleted, subjected to Sequence Independent Single Primer Amplification (SISPA), and sequenced on the Illumina MiSeq platform. Although no mammalian arboviruses were detected, bioinformatic analysis detected several ISVs in pools of Aedes aegypti, Ae.ochraceus, and Anopheles squamosus mosquitoes. These ISVs included the cell fusing agent-, Tesano aedes-, Fako-, formosus-, verdadero-, Aedes partiti-like virus 1 and the Kwale mosquito virus and several novel ISVs within the Iflavivirus genus as well as some unclassified viruses. This study confirms the value of sequence independent metagenomics approaches to detect novel viruses in mosquito pools. Such studies provide insight into viral diversity and mosquito-virus interactions, supporting the future evaluation of ISVs as biological control agents. This in turn can contribute to protecting the health of animals and humans within a One Health approach.