Detection and sequencing of Usutu virus during mosquito surveillance: Use of multiple assays and techniques for...

Usutu virus (USUV) is a medically important flavivirus linked to significant mortalities within bird populations and an increasing association with human infections, particularly within immunocompromised individuals. It's genetic and etiological similarity with West Nile virus (WNV) means that it is often detected within the same ecological niche, consequently the detection of USUV is a good indicator that the conditions are suitable for the introduction of WNV.

Surveillance approaches often require detection of low viral loads within arthropod vectors. Although useful for straightforward detection, strain and genetic details can be missed which provide important information on autochthonous survival and/or reintroduction. We evaluated a pan-flavivirus screening process followed by tiled amplicon sequencing for the detection and analysis of USUV within mosquito pools.

RT-PCR screening of 270 mosquito pools from a range of species revealed one positive (0.37%) in a pool of Culex modestus. Further analysis using pan-flavivirus end-point PCR produced DNA amplicons which sequencing confirmed to differ from the positive reference control. Tiled amplicon-based whole genome amplification using USUV specific primers followed by GridION sequencing produced a near full-length sequence which phylogenetic analysis placed within the African 3.2 clade.

Using targeted mosquito surveillance followed by an array of molecular techniques we were able to detect and phylogenetically analyse USUV from low viral loads within infected mosquito pools. This system is useful for the initial screening and subsequent taxonomy of viruses in samples with low viral loads and high host genomic background, providing an important tool for surveillance and the analysis of circulation dynamics.