Reconstruction of the molecular evolution of Usutu virus in Germany: Insights into virus emersion and circulation

Abstract

Usutu virus (USUV) is a mosquito-borne flavivirus that is widely distributed in southern and central Europe. The zoonotic virus circulates primarily between birds and mosquitoes, can, however, in rare cases infect other mammals including humans. In the past, USUV has been repeatedly associated with mass mortalities in birds, primarily blackbirds and owls. Birds commonly succumb either due to the peracute nature of the infection or due to severe encephalitis. In Germany, USUV has spread rapidly since its first detection in 2010 in mosquitoes under the presence of susceptible host and vector species. Nonetheless, there is to date limited access to whole genome sequences resulting in the absence of in-depth phylogenetic and phylodynamic analyses. In this study, 118 wild and captive birds were sequenced using a nanopore sequencing platform with prior target enrichment via amplicons. Due to the high abundancy of Europe 3 and Africa 3 in Germany an ample quantity of associated whole genome sequences was generated and the most recent common ancestor could be determined for each lineage. The corresponding clock phylogeny revealed an introduction of USUV Europe 3 and Africa 3 into Germany three years prior to their first isolation in the avifauna in 2011 and 2014, respectively. Based on the clustering and temporal history of the lineages, evidence exists for the genetic evolution of USUV within Germany as well as new introductions thereof into the country.

Fig 1

(A) Geographic distribution of USUV-RNA detection throughout Europe. The first detected USUV-positive case per country is depicted in the figure. In addition, the icons for mosquito, bird, horse, bat, and human indicate in which species USUV-RNA has been detected so far and whether a corresponding whole genome sequence is available (in orange). (B) The graph shows all USUV-positive birds detected in Germany since 2011, highlighting the first occurrence of each lineage in the following years, with the exception of USUV Europe 3, which was first detected in 2010 in a mosquito pool. *Results not finalized and only based on dead bird surveillance (updated on 21th February 2023). Created with BioRender.com (Agreement number: NB25GJXN1B).

Fig 2. Phylogeny of the sequenced USUV isolates from 2017–2021.

(A) Samples were numbered consecutively and coloured according to the lineages. Reference genomes are marked as EU3 (Europe 3), EU2 (Europe 2), A3 (Africa 3), and A2 (Africa 2). Detailed information to each sample number can be found in S1 Table including GenBank accession numbers and the years in which the samples were detected. Scale bars indicate the mean number of nucleotide substitutions per site. (B. 1) Cluster analyses suggesting three subclusters belonging to lineage Europe 3 and (B. 2) two subclusters belonging to lineage Africa 3. (The supplementary file (S1 Table) provides comprehensive details, including sequence assignments, for the identified subclusters).

Fig 3. Geographic distribution of whole genome sequences of USUV in Germany from 2017 to 2021.

The different USUV lineages are depicted as coloured triangles: pink = Europe 2, red = Europe 3, and light blue = Africa 3 with the appropriate sample number (detailed information to each sample in S1 Table). GIS analysis was performed using ArcGIS ArcMap 10.8.1, license number: ESU748788721 (ESRI, Redlands, CA, USA) and open data from GeoBasis-DE/BKG 2022 [84] using the following link: https://daten.gdz.bkg.bund.de/produkte/vg/vg250_ebenen_1231/aktuell/vg250_12-31.utm32s.shape.ebenen.zip.

Fig 4. Overview of the distribution of the different USUV lineages in Germany (2017–2021) (depicted in blue, red, and pink with the total number per lineage) with regard to the total number of live and dead birds molecularly tested (depicted with black diamonds) and those tested RT-qPCR positive for USUV (depicted in grey).

* Number of tested birds not recorded in 2017 and 2018; ^† Results not finalized and only based on dead bird surveillance (updated on 21st February 2023). Bird samples with USUV lineage Europe 5 or Africa 2 were not available for this study.

Fig 5

Molecular clock phylogeny of the complete coding sequences of USUV lineages, (A) Europe 3 and (B) Africa 3 detected in Germany. The blue bars indicate 95% confidence intervals of node dates. The branches are colored according to the sampling location of their nodes.