Blood donors as sentinels for genomic surveillance of West Nile virus in Germany (2020–2024) using a sensitive amplicon-based sequencing approach

West Nile virus (WNV) has emerged as a public health concern in Germany since its first detection in 2018, with evidence of expanding geographic spread. Genomic surveillance is critical for tracking viral evolution, identifying introductions, and monitoring local transmission. However, genome recovery from low-viremia samples such as those obtained through blood donor screening remains technically challenging. To develop and validate a sensitive amplicon-based sequencing protocol optimized for WNV lineage 2 and apply it to low-titer samples to support genomic surveillance in Germany. A novel primer scheme was designed for WNV lineage 2 and applied to 43 nucleic acid testing (NAT)-positive blood donor samples collected between 2020 and 2024. Amplicon-based sequencing performance was benchmarked against metagenomic next-generation sequencing (mNGS). Recovered genomes were subjected to phylogenomic analysis to assess viral diversity and transmission dynamics. The amplicon protocol enabled genome recovery (>70% coverage) from samples with viral loads as low as ∼10¹ RNA copies/µL, outperforming metagenomic NGS (mNGS). Of the 43 samples, 30 yielded complete or near-complete genomes. Six distinct WNV subclades (2A–2F), including German strains, were identified, indicating multiple introductions into Germany from Central Europe. Subclade 2F emerged as the dominant and widely distributed group. Berlin, Brandenburg, Saxony, and Saxony-Anhalt were identified as persistent transmission hubs. This study highlights blood donors as valuable sentinels for WNV genomic surveillance. The validated amplicon-based sequencing approach enables sensitive, scalable genome recovery from low-viremia samples, and when integrated with routine blood donor screening, provides a robust framework for early detection, transmission dynamics, and public health preparedness.