An advanced sequence clustering and designation workflow reveals the enzootic maintenance of a dominant West Nile virus subclade in Germany

Abstract

West Nile virus (WNV) is the most widespread arthropod-borne (arbo) virus and the primary cause of arboviral encephalitis globally. Members of WNV species genetically diverged and are classified into different hierarchical groups below species rank. However, the demarcation criteria for allocating WNV sequences into these groups remain individual, inconsistent, and the use of names for different levels of the hierarchical levels is unstructured. In order to have an objective and comprehensible grouping of WNV sequences, we developed an advanced grouping workflow using the ″affinity propagation clustering″-algorithm and newly included the ″agglomerative hierarchical clustering″-algorithm for the allocation of WNV sequences into different groups below species rank. In addition, we propose to use a fixed set of terms for the hierarchical naming of WNV below species level and a clear decimal numbering system to label the determined groups. For validation, we applied the refined workflow to WNV sequences that have been previously grouped into various lineages, clades, and clusters in other studies. Although our workflow regrouped some WNV sequences, overall, it generally corresponds with previous groupings. We employed our novel approach to the sequences from the WNV circulation in Germany 2020, primarily from WNV-infected birds and horses. Besides two newly defined minor (sub)clusters comprising only of three sequences each, subcluster 2.5.3.4.3c was the predominant WNV sequence group detected in Germany from 2018-20. This predominant subcluster was also associated with at least five human WNV-infections in 2019-20. In summary, our analyses imply that the genetic diversity of the WNV population in Germany is shaped by enzootic maintenance of the dominant WNV subcluster accompanied by sporadic incursions of other rare clusters and subclusters. Moreover, we show that our refined approach for sequence grouping yields meaningful results. Although we primarily aimed at a more detailed WNV classification, the presented workflow can also be applied to the objective genotyping of other virus species.

Vaccination of Zoo Birds against West Nile Virus—A Field Study

“…19) tested positive for WNV-RNA by WNV-specific RT-qPCR (Cycle threshold (Ct) value 27.68) on 19 May 2021. Whole genome sequencing (Accession number: OQ326499) was performed and revealed WNV lineage 2 of Eastern German clade (new classification as subcluster 2.5.3.4.3c), which also circulated in the zoo in 2020 [ 56 ]. The bird showed no clinical signs.…”

“…Beside the direct reaction to the vaccine, a wide variety of factors could explain the different immune responses following vaccination. As the virus is known to have circulated in the zoo the year before vaccination [ 46 , 56 ], natural exposure to the virus may explain a rise in antibody titres, which limits interpretation of titres. Accordingly, it cannot be ruled out that the virus was also circulating in the zoo in early 2021, although there is no evidence from virus isolation from birds or mosquitos in 2021 (personal communication by N. Becker).…”