First Detection and Molecular Characterization of Usutu Virus in Culex pipiens Mosquitoes Collected in Romania

Abstract

Usutu virus (USUV) is an emergent arbovirus in Europe causing mortality in bird populations. Similar to West Nile virus (WNV), USUV is maintained in sylvatic cycles between mosquito vectors and bird reservoirs. Spillover events may result in human neurological infection cases. Apart from indirect evidence provided by a recent serological study in wild birds, the circulation of USUV in Romania was not assessed. We aimed to detect and molecular characterize USUV circulating in mosquito vectors collected in South-Eastern Romania-a well-known WNV endemic region-during four transmission seasons. Mosquitoes were collected from Bucharest metropolitan area and Danube Delta, pooled, and screened by real-time RT-PCR for USUV. Partial genomic sequences were obtained and used for phylogeny. USUV was detected in Culex pipiens s.l. female mosquitoes collected in Bucharest, in 2019. The virus belonged to Europe 2 lineage, sub-lineage EU2-A. Phylogenetic analysis revealed high similarity with isolates infecting mosquito vectors, birds, and humans in Europe starting with 2009, all sharing common origin in Northern Italy. To our knowledge, this is the first study characterizing a strain of USUV circulating in Romania.

Keywords: BG-Sentinel trap; CDC gravid trap; CDC light trap; Culex pipiens mosquitoes; Europe 2 lineage; Romania; Usutu virus; phylogenetic analysis.

Figure 1

Mosquito sampling sites, South-Eastern Romania, 2012–2013 and 2019–2020. Sampling sites where wild birds tested positive for USUV antibodies were reported by Coroian et al. [16].

Figure 2

Phylogenetic tree of Usutu virus Europe 1–4 lineages (EU1-EU4) showing the Romanian sequence clustering into sub-lineage EU2-A. White dot: sequence obtained in this study. Lineages were assigned according to [13,14,22,26] and similar sequences retrieved by NCBI BLAST were also included in the analysis. The evolutionary history was inferred from a preM-M-E fragment (nucleotides 591–2447, reference sequence GenBank acc. no. NC_006551) by using the Maximum Likelihood method and Kimura 2-parameter model, 1000 bootstrap replicates. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.1000)). Numbers at nodes represent the bootstrap percentages (values <70% are not shown).

Figure 3

Phylogenetic tree of Usutu virus Europe 1–4 lineages (EU1-EU4) showing the Romanian sequence clustering into sub-lineage EU2-A. White dot: sequence obtained in this study. Lineages were assigned according to [13,14,22,26] and similar sequences retrieved by NCBI BLAST were also included in the analysis. The evolutionary history was inferred from a NS4B-NS5 fragment (nucleotides 7372–10,398, reference sequence GenBank acc. no. NC_006551) by using the Maximum Likelihood method and Tamura–Nei model, 1000 bootstrap replicates. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.1000)). Numbers at nodes represent the bootstrap percentages (values <70% are not shown).