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Viruses and Viral Diseases

# Blood donors as sentinels for genomic surveillance of West Nile virus in Germany using a sensitive amplicon-based sequencing approach

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Gábor Endre Tóth<sup>a</sup>, Marike Petersen<sup>a</sup>, Francois Chevenet<sup>b,c</sup>, Marcy Sikora<sup>a</sup>, Alexandru Tomazatos<sup>a</sup>, Alexandra Bialonski<sup>d</sup>, Heike Baum<sup>a,d</sup>, Balázs Horváth<sup>a</sup>, Padet Siriyasatien<sup>e,f</sup>, Anna Heitmann<sup>d</sup>, Stephanie Jansen<sup>d,i</sup>, Ruth Offergeld<sup>g</sup>, Raskit Lachmann<sup>g</sup>, Michael Schmidt<sup>h</sup>, Jonas Schmidt-Chanasit<sup>d,i</sup>, Dániel Cadar<sup>a,∗</sup>

## ARTICLE INFO

**Article history:**

Accepted 29 October 2025

Available online 4 November 2025

**Keywords:**

West Nile virus

Amplicon sequencing

Metagenomics

Genomic surveillance

Phylogeography

Virus evolution

Blood donor surveillance

Germany

## SUMMARY

**Background:** 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.

**Aim:** 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.

**Methods:** 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.

**Results:** 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.

**Conclusion:** 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.

© 2025 The Author(s). Published by Elsevier Ltd on behalf of British Infection Association. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

West Nile virus (WNV) or *Orthoflavivirus nilense* is a mosquito-borne orthoflavivirus that has become an emerging public health threat in the recent decades and a significant cause of viral encephalitis in humans and horses in many temperate regions.¹ Maintained in an enzootic transmission cycle between birds and *Culex* mosquitoes, WNV

<footnote><sup>a</sup>Virus Metagenomics and Evolution Group, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany</footnote>

<footnote><sup>b</sup>MIVEGEC, IRD, CNRS, Université de Montpellier, Montpellier, France</footnote>

<footnote><sup>c</sup>LIRMM, CNRS, Université de Montpellier, Montpellier, France</footnote>

<footnote><sup>d</sup>Department of Arbovirology and Entomology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany</footnote>

<footnote><sup>e</sup>Center of Excellence in Vector Biology and Vector-Borne Disease, Chulalongkorn University, Bangkok, Thailand</footnote>

<footnote><sup>f</sup>Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand</footnote>

<footnote><sup>g</sup>Department for Infectious Disease Epidemiology, Robert Koch Institute, Berlin, Germany</footnote>

<footnote><sup>h</sup>Institute of Transfusion Medicine and Immunohematology, German Red Cross Blood Transfusion Service Baden-Württemberg-Hessen, Frankfurt am Main, Germany</footnote>

<footnote><sup>i</sup>Faculty of Mathematics, Informatics and Natural Sciences, University of Hamburg, Hamburg, Germany</footnote>

<footnote>* Correspondence to: Bernhard Nocht Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Reference and Research, National Reference Centre for Tropical Infectious Diseases, Bernhard-Nocht-Straße 74, 20359 Hamburg, Germany.</footnote>

<footnote>E-mail address: cadar@bnitm.de (D. Cadar).</footnote>
occasionally spills over into humans and other mammals, which serve as dead-end hosts.² Most human infections are asymptomatic, while ~20% develop West Nile fever (WNF) and less than 1% progress to West Nile neuroinvasive disease (WNND), particularly elderly or immunocompromised individuals.¹,³ Over the past decade, Europe has witnessed a notable increase in WNV activity and an expanding geographic range of transmission. Following its establishment in Southeastern Europe in the late 1990s, the virus has expanded northward, potentially facilitated by climate change, land use patterns, and migratory bird movements.⁴,⁵ A record number of human cases in 2018 (n=2083) and another resurgence in 2022, with over 1300 cases reported across the EU/EEA.⁶,⁷ In Germany, the first local WNV detections occurred in 2018 in animals, with confirmed endemic transmission in birds, horses, and humans in subsequent years, particularly in Berlin and the Eastern federal states.⁸ Surveillance has shown that Germany's WNV activity stems from WNV lineage 2 strains introduced from Central Europe.⁵ Timely diagnosis and surveillance of WNV are challenged by the typically low and transient viremia in human infections. Nucleic acid amplification tests (NATs) are sensitive during the brief viraemic phase but may fail to detect infections later during disease.⁹ Serological testing is useful but limited by cross-reactivity with other orthoflaviviruses and by the prolonged persistence of IgM antibodies, as observed in patients with neuroinvasive WNV infection, both of which complicate result interpretation.¹⁰ These diagnostic challenges also impact the ability to recover viral genomes from clinical samples for molecular epidemiology. Genomic surveillance has become an essential component of arbovirus monitoring, as genome (> 70%) sequencing enables detailed tracking of viral lineages, mutations, and transmission pathways.⁵,¹¹ While metagenomic next-generation sequencing (mNGS) directly from samples can detect WNV without prior knowledge, in practice, it often lacks sufficient sensitivity when viral RNA levels are low or background host nucleic acid is abundant. Capture-based enrichment techniques offer higher sensitivity but are resource-intensive. In contrast, amplicon-based sequencing using virus-specific primers offers a practical and highly sensitive alternative. Several amplicon-based protocols have recently been developed for WNV genome recovery directly from clinical, veterinary, and mosquito samples.¹²⁻¹⁸ However, existing methods demonstrate sensitivity limitations: achieving substantially recovered genomes (≥70% coverage) becomes difficult in samples with high cycle threshold (Ct) values (> 31.5), a common status in asymptomatic infections and blood donors. These limitations are particularly problematic for blood donor surveillance, where early detection is critical but viral loads are typically very low. Blood donor screening represents a unique and valuable system for surveillance of WNV. Donations are systematically collected, geographically widespread, and subjected to highly sensitive NAT testing, making blood donors an effective early-warning group for WNV circulation. However, the low viral RNA concentrations in these samples have limitations in successful genome recovery using existing methods, restricting phylogenetic and epidemiological insights into circulating strains.¹⁹,²⁰ To address this gap, we developed a novel, highly sensitive amplicon-based WNV lineage 2 whole-genome sequencing protocol tailored for surveillance in samples with low viral load. In this study, we validate the newly developed method using blood donor samples collected during the 2020–2024 WNV transmission seasons in Germany and conduct a comprehensive genomic, epidemiological, and evolutionary analysis of the current WNV activity in Germany.

## Methods

### WNV-positive blood donor samples

In Germany, screening of blood donations for WNV using NAT has been required during the transmission season since 2000, as stipulated in a regulatory notification by the Paul-Ehrlich-Institute under

§28 of the Medicinal Products Act (Arzneimittelgesetz, AMG). Confirmatory testing is performed at reference laboratories using qRT-PCR and, when applicable, sequencing (metagenomic or amplicon-based). A donation is considered confirmed WNV-positive when reactivity is reproducible and viral RNA is detected by an independent method. Confirmed cases are reported by the blood establishment to the Paul-Ehrlich-Institute (PEI) and the Robert Koch Institute (RKI) under the Transfusion Act (TFG) and Infection Protection Act (IfSG) as part of the national haemovigilance system. This directive aims to minimize the risk of WNV transmission through blood components and stem cell preparations. Alternatively, a few blood establishments continue to apply a precautionary deferral of donors who have spent at least two days in endemic areas.¹⁹ Roughly 2.0–2.4 million donations were screened yearly between 2020 and 2024. A total of 43 blood donor samples that tested positive for WNV by NAT screening and were subsequently confirmed as WNV-positive via qRT-PCR/mNGS in our laboratory were included in this study. These samples were collected between 2020 and 2024 from various blood establishments across Germany and accounted for most confirmed WNV-positive donations. Sample metadata are provided in Table S1. The WNV transmission season in Germany refers to the period of increased transmission risk, defined by the Paul-Ehrlich-Institute, and currently set as June through November. Mandatory blood donor screening is implemented during this period. Travel history information was not systematically collected for all blood donors at the time of donation. For samples where travel information was documented during follow-up (n=5), this is explicitly indicated in Table S1. In the absence of documented travel history, sampling location (blood donation site/donor residence) was used as a proxy for likely infection location in phylogeographic analyses.

### Development of an amplicon-based protocol for WNV genome sequencing

We collected all complete (> 80%) WNV lineage 2 sequences from NCBI GenBank (2023-12-01). A total of 495 genomes were selected, and primer schemes were generated using PrimalScheme v1.4.1.¹⁷ The scheme targets approximately 10,815 bp of the WNV genome with 59 overlapping amplicons. To capture known lineage 2 diversity, alternative primers were included, resulting in two primer pools with 189 primers total (Pool_1: 99, Pool_2: 90). Amplicon sizes ranged from 252 to 280 bp (mean: 268.3 bp, SD ± 5.8) (Fig. S1). All primers were evaluated in silico using Primer3 software to assess potential cross-reactivity with related orthoflaviviruses (Usutu virus, Japanese encephalitis virus, Tick-borne encephalitis virus, yellow fever virus, Dengue virus serotypes 1–4). Analysis confirmed that the primer scheme does not support amplification of non-WNV flavivirus genomes. In silico analysis predicted that three genomic regions of WNV lineage 1 could potentially be co-amplified with this lineage 2-optimized primer set. This was validated experimentally using two WNV lineage 1-positive blood donor samples, which confirmed partial amplification only in the three predicted regions, demonstrating primer specificity for WNV with optimal performance for lineage 2. Primer details, source sequences, as well as a detailed description of the whole protocol, are available at protocols.io (https://www.protocols.io/view/west-nile-virus-orthoflavivirus-nilense-lineage-2-q26g71q98gwz/v1) and as Supplementary material S2. Protocol optimization was performed using three well-characterized WNV-2 isolates (UG37, B956, BNI-129, spanning both historical African diversity and the contemporary European/German outbreak clades) in serial dilution to ensure reproducibility and primer coverage across divergent genomes. Subsequently, the assay was validated on clinical blood donor samples. The protocol was developed and validated on the Illumina iSeq100 platform. It was intentionally designed to be compatible with any Illumina sequencer that supports 2x150 bp runs, as the amplicon size
(~270 bp) fits within this read structure. No platform-specific adjustments are expected to be necessary, and the only anticipated differences across platforms would be related to sequencing throughput and multiplexing capacity, which could further facilitate large-scale or cohort-level studies.

## Metagenomic next-generation sequencing of WNV-positive blood donors

WNV-positive blood donor samples were also subjected to unbiased metagenomic next-generation sequencing (mNGS) to further investigate NAT-reactive cases. RNA extracted and purified from plasma or serum samples was processed using an in-house mNGS pipeline established for virus discovery²¹ and sequenced on a NextSeq 2000 platform (Illumina).

## Genome assembly and data acquisition

Raw reads generated by the amplicon-based approach were checked using FastQC v0.12.1²² for quality assessment. Paired-end reads were merged with BBMerge v38.84,²³ and size-selected between 240 and 290 bp to match expected amplicon lengths. Primer trimming and quality filtering were performed using BBDuk v38.84. Clean reads were aligned to the WNV reference genome (GenBank: MN794937.1) using Bowtie2 v2.4.5²⁴ and consensus sequences were generated with SAMtools v1.18.²⁵ Mapping accuracy was visually inspected using Geneious Prime v2024.0.7. The complete workflow is summarized in Fig. S2. For this study, we define genome recovery thresholds as follows: "complete genome" indicates > 99% coverage; "near-complete genome" indicates 80-99% coverage; "substantially recovered genome" indicates ≥70% coverage. We selected ≥70% coverage as the primary threshold for phylogenomic inclusion based on prior orthoflavivirus genomic epidemiology studies⁵,¹¹ demonstrating that this level of genome recovery provides sufficient phylogenetic signal for reliable subclade assignment and spatiotemporal inference, while remaining achievable for low-viremia clinical samples. This threshold specifically enables: (1) coverage of multiple structural and non-structural protein-coding regions required for robust phylogenetic placement, (2) sufficient genetic variation for molecular clock calibration, and (3) adequate sequence length for continuous phylogeographic modeling. For comparative analyses, all publicly available nucleotide sequences of European WNV strains were retrieved from the NCBI Nucleotide database (https://www.ncbi.nlm.nih.gov) as of February 2025. The final compiled dataset consisted of 520 substantially recovered (≥70% coverage) genome sequences, spanning the years 2004 to 2024. Geospatial coordinates for all samples included in the dataset were standardized and integrated (Table S2).

## Phylodynamic reconstruction of the WNV spread pattern

To infer evolutionary timelines and characterize both cross-border and intra-national viral transmission patterns, we reconstructed time-scaled phylogenetic trees focusing on the dominant European WNV lineage 2a. Two analytical scenarios were considered: (i) a full European WNV dataset and (ii) a subset restricted to WNV transmission dynamics for Germany. Bayesian time-scaled phylogenies were reconstructed using BEAST v1.10.5,²⁶ applying an uncorrelated relaxed molecular clock model and a Skygrid coalescent prior. Model selection was informed by stepping-stone sampling. For the full dataset, MCMC chains were run for 400 million steps, and for the German subset, 200 million steps, with parameters sampled every 10,000 steps.

## Phylogeography and spatiotemporal dynamics of WNV in Germany

To reconstruct viral migration patterns in Germany, all publicly available substantially recovered WNV sequences (≥70% coverage) from both human and animal cases were included in the phylogeographic analyses, alongside the newly generated blood donor sequences from this study. We employed an asymmetric discrete trait phylogeographic model combined with the Bayesian Stochastic Search Variable Selection (BSSVS) procedure to identify statistically supported transition routes. In addition, a continuous phylogeographic model based on the Relaxed Random Walk (RRW) framework was implemented to visualize the spatial diffusion of WNV in continuous geographic space.²⁷ These analyses also provided estimates for the time to the most recent common ancestor (tMRCA). Maximum clade credibility (MCC) trees were generated and visualized using FigTree v1.4.1 (http://tree.bio.ed.ac.uk/software/figtree/). The spatiotemporal dynamics of WNV were further explored and visualized through EvoLaps 2 (www.evolaps.org), enabling advanced phylogeographic reconstruction and representation.²⁸

## Results

### Validation of the WNV amplicon-based sequencing protocol

For our initial validation, we first tested 3 in vitro WNV isolates (UG37, B956, BNI-129) and sequenced them across 10-fold serial dilutions. The corresponding Ct values ranged from 19.45 to 40.52, reflecting viral RNA concentrations from 667,225 to 0.13 copies/µL. Sequencing yielded a mean depth of 309,295 reads (SD ± 66,297.3). A marked decline in genome recovery was noted at concentrations below 8.1 copies/µL. At higher input levels (> 8.1 copies/µL), over 70% of the WNV genome from all three genetically diverse WNV isolates was recovered at ≥10× coverage (Fig. S3; Table S3). To assess its performance, we validated the protocol using 43 WNV-positive blood donor samples. Ct values ranged from 26.92 to 43.63, corresponding to viral loads between 2797.97 and 0.01 WNV RNA copies/µL (Fig. 1a). The mean sequencing depth was 771,805.7 single reads per sample (SD ± 589,487.5), while the median was 561,238. Only 6 samples (13%) exceeded 1 million reads in total sequencing depth (Fig. S4; Table S4). Altogether, 30 out of 43 blood donor samples achieved substantially recovered genomes (≥70% coverage). All samples meeting this threshold were successfully characterized up to a Ct value of 35.12, with two additional samples exceeding this threshold despite lower viral loads (Ct 35.62 and 36.35). We applied a generalized linear model (GLM) to estimate the WNV RNA copy number required to surpass the 70% genome recovery threshold. Based on our dataset, a concentration of 11.2 copies/µL was sufficient to recover usable genomes at ≥10× depth, while at ≥1× depth, the threshold decreased to 6.9 copies/µL (Figs. 1b, S4). The efficiency of certain amplicons (e.g., amplicons 23 and 58) was reduced, likely due to chimeric read formation, which contributed to discrepancies in genome recovery between 1× and 10× coverage thresholds (Fig. 1). While the proportion of WNV-specific reads generally correlated with viral concentration, a marked drop was observed at Ct values around 35. Moreover, several outliers deviated from this trend, underscoring the impact of sample quality on sequencing success (Figs. S4, S5). Unsuccessful samples (n=13, Table S1) were defined as those in which genomic recovery was limited to short, fragmented regions or restricted to highly conserved loci, which precluded confident clade/subclade assignment. To improve recovery in low-input cases, we allowed for limited protocol flexibility, including increasing the number of PCR cycles, re-amplification of weak libraries when material permitted, and repeating sequencing runs. These modifications did not alter the primer scheme or downstream analysis pipeline. Overall, these results indicate that our protocol performs reliably across a broad range of viral loads and sample conditions, achieving comparable efficiency to that observed during in vitro validation with serially diluted virus isolates.
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Performance of WNV amplicon-based sequencing across blood donor samples. **Panel a** assesses performance by ≥70% genome recovery at 1× coverage, while **Panel b** shows complete genome recovery at both 1× and 10× coverage. The x- and y-axes of **Panel a** display Ct values and viral RNA copies for each analyzed blood donor sample. Panel b presents viral RNA copies and percentage genome coverage at 1x and 10x read depth. The performance of the sequencing protocol was evaluated using R v4.5.0 and the packages dplyr v1.1.4, ggplot2 v3.5.2, readxl v1.4.5, scales v1.4.0, ggpubr v0.6.0, gggenes v0.5.1, gridExtra v2.3, and patchwork v1.3.0.</figcaption></figure>

*Metagenomics versus targeted sequencing for WNV genome recovery*

We compared metagenomic and amplicon-based sequencing approaches for WNV genome recovery using the same panel of WNV-positive blood donor samples with varying viral loads. Both methods demonstrated high sensitivity, reliably detecting WNV across all samples. However, significant differences in genome recovery were observed. With the metagenomic approach, full genome recovery at 1× coverage was achieved from samples with ≥10² RNA copies/µL, and at 10× coverage from ≥10³ copies/µL (Fig. S6). In contrast, the amplicon-based approach outperformed the metagenomics approach, enabling appropriate genome recovery at 1× coverage from less than 10 RNA copies/µL, and 10× from ~10¹ copies/µL (Fig. S6). These findings highlight the increased sensitivity and genome completeness of the targeted amplicon strategy, particularly for low-copy clinical samples.

*Genomic characterization and phylogenetic placement of WNV from blood donors*

A total of 43 WNV-positive blood donor samples collected between 2020 and 2024 were characterized based on substantially recovered (≥70% coverage) or partial genome sequences. These

donors originated from both known endemic regions (e.g., Brandenburg, Saxony, Saxony-Anhalt) and newly affected federal states, including Schleswig-Holstein and Lower Saxony, reflecting the expanding geographic distribution of WNV circulation in Germany (Table S1; Fig. 2). Five samples had documented domestic travel within Germany during the probable exposure period (Table S1 and Fig. 2B), introducing uncertainty in the precise geographic origin of infection for these cases. Importantly, phylogenetic analysis confirms that all travel-associated samples cluster within German WNV subclades (2A-2F) alongside samples without documented travel history, demonstrating that these represent acquisition of domestically circulating strains at different locations within Germany rather than importation from outside the country. The genetic variation of German WNV strains across the viral genome was relatively heterogeneous, exhibiting at least 94 synonymous and 23 non-synonymous mutations (Fig. S7). Despite this variation, the virus has maintained a highly conserved genome since its first detection in Germany in 2018. The genome-wide and gene-specific identity matrices were consistently greater than 99% at both the nucleotide and amino acid levels. Bayesian maximum clade credibility (MCC) phylogenetic analysis of the European WNV dataset (substantially recovered genomes, ≥70% coverage) revealed that all German strains, including those derived from blood donors,
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Geographical distribution of West Nile virus in Germany (2019–2024). **Panel a** shows the location of all WNV genome sequences from Germany that are publicly available in GenBank, covering both human and animal cases. This comprises previously published sequences (from birds, horses, and symptomatic human infections) as well as the blood donor sequences generated in our current study, while **panel b** displays only locations of WNV-positive blood donors identified and sequenced in the present study. Samples with documented domestic travel during the exposure period are marked with red asterisks. Colored circles denote the genetic clustering of detected WNV strains within subclades 2A–2F, based on phylogenetic reconstruction. Map created with Datawrapper.</figcaption></figure>

clustered within a single major clade of the WNV lineage 2 phylogeny (Fig. S8). A more detailed analysis of this clade indicated that the German sequences could be classified into six distinct subclades, designated as subclades 2A–2F based on monophyletic clustering (posterior probability ≥0.9, bootstrap ≥70%), geographic coherence, and characteristic amino acid substitutions (Fig. S7). This working classification was introduced to systematically describe the regional diversity of German WNV lineage 2 strains. Subclades 2A, 2D, 2E, and 2F exhibited monophyletic structures, suggesting single introduction events into Germany for these groups. In contrast, subclades 2B and 2C were polyphyletic, indicating that German strains within these groups evolved from multiple ancestral Austrian strains (Fig. 3). Blood donor-derived WNV genomes were distributed across five of the six subclades, except for subclade 2B, from which no blood donor sequences were identified (Fig. 3). Among the identified subclades, subclade 2F appears to be the most frequently sampled variant in our dataset, suggesting it may represent a dominant circulating group within the German WNV population (comprising sequences from human, avian, and equine hosts) based on current surveillance data. This observation, based on temporal persistence, is consistent with the potential local establishment of subclade 2F. Analysis of the complete polyprotein sequences identified subclade-defining and/or geographically associated point mutations (Fig. S7). Notably, all members of subclade 2F, which has emerged as the dominant group in Germany, harbor a unique amino acid substitution (K609R) within the helicase domain of the NS3 protein (Fig. S7).

### Evolutionary dynamics and phylogeographic reconstruction of the WNV spread pattern in Germany

To investigate the emergence, introduction routes, and dispersal dynamics of WNV in Germany, we applied both discrete-trait and

continuous phylogeographic models. This integrative framework enabled reconstruction of WNV evolutionary history, identification of geographic transmission hubs, and quantification of viral migration patterns. Time-scaled Bayesian phylogenies placed the most recent common ancestor (TMRCA) of the German WNV-associated subclades around June 2015 (95% HPD: July 2014–March 2016), suggesting that the viruses seeding Germany shared a common ancestor with strains circulating in Hungary and Austria (Fig. S8). Discrete and continuous phylogeographic analysis and Evolaps reconstruction (Fig. 4) suggest that Germany likely experienced multiple independent introductions of closely related viruses from Central European source populations rather than a single introduction followed by in-country diversification (Figs. 4–6; Fig. S9). This interpretation is supported by the existence of six distinct WNV subclades (2A–2F), which consist of German strains, each with its introduction timing and geographic dispersal pattern (Fig. 5). Phylogeographic modeling estimates subclade 2A introduction around late August 2018, potentially from Austria. Repeated detection across multiple years in Brandenburg, Lower Saxony, Schleswig-Holstein, and Saxony-Anhalt, with a total diffusion range of ~185 km and an estimated migration rate of ~25.7 km/year for the whole subclade (Figs. 5 and 6), suggests possible sustained transmission. Subclade 2B, with an estimated introduction around September 2017, was detected only in 2018 and 2019. The absence of subsequent detections may indicate failure to establish sustained local transmission, though surveillance gaps cannot be excluded. Based on our models, subclade 2C appeared in September 2015, with persistent detections in Berlin, Brandenburg, Thuringia, and Saxony since 2019. The subclade exhibits a moderate diffusion (~240 km) and a migration rate of ~36.9 km/year, consistent with stable local expansion. Subclade 2D, introduced probably around July 2018 in Bavaria, remained limited in distribution and lacked evidence of establishment or
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Bayesian maximum clade credibility (MCC) tree showing the time-scaled phylogeny of West Nile virus. This tree is based on substantially recovered (≥70% coverage) genome sequences, including the German sequences (subclades 2A–2F). The MCC tree was inferred using BEAST v1.10.5 with an uncorrelated relaxed molecular clock and a Skygrid coalescent tree prior, with model selection guided by stepping-stone sampling. Markov chain Monte Carlo (MCMC) chains were run for 200 million steps, sampling every 10,000 steps. Branch colors indicate the most probable geographic origin of descendant nodes (see color code). Subclades containing German WNV strains are displayed to the right of the tree. The German WNV strains from blood donors sequenced in this study are highlighted. The time scale is shown along the x-axis and represents years before the last sampling date (2024).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** The phylogeographic reconstruction of West Nile virus origin and dispersal in Germany. The time-scaled maximum clade credibility (MCC) tree (top right) is based on substantially recovered genome sequences (≥70% coverage, n = 170) of the subclades 2A–F. The phylogeny was inferred using a continuous Bayesian phylogeographic model based on 2000 posterior trees. Viral dispersal is depicted on a geographic map, with branches representing inferred migration pathways and superimposed over the 80% highest posterior density (HPD) regions that reflect phylogeographic uncertainty. Branch colors indicate the time scale, ranging from black (time to the most recent common ancestor, TMRCA) to red (most recent sampling time). Green shaded areas represent the HPD regions, highlighting zones of concentrated viral activity and inferred transmission hubs. The inset panel (top left) displays the inferred geographic spread of WNV-2a as of December 2024.</figcaption></figure>

geographic spread beyond its origin. Subclade 2E, with an estimated introduction date near July 2016, has remained geographically restricted to Saxony-Anhalt and Brandenburg, with a modest spatial range (~110 km) and migration rate of ~18.9 km/year, suggesting localized persistence. Subclade 2F shows the widest geographic distribution and greatest genetic diversity in our dataset, with an estimated introduction in July 2016. It is frequently detected across Saxony, Saxony-Anhalt, Thuringia, and likely imported cases in Bavaria and North Rhine-Westphalia. This subclade has diffused over ~380 km and shows the highest migration rate at ~62.3 km/year (Figs. 5 and 6). Combined data from continuous diffusion models and k-means clustering (Figs. 4–6) identify Brandenburg, Berlin, Saxony, and Saxony-Anhalt as potential epidemiological centers, with connectivity patterns consistent with regional virus dispersal. Brandenburg appears as a central node in phylogeographic reconstructions, with sequences from multiple subclades (2A, 2C, 2E) and inferred migration pathways suggesting bidirectional viral movement with surrounding regions. Berlin sequences, particularly in subclade 2C, show strong phylogenetic clustering with sequences from Brandenburg and Thuringia, consistent with a potential role in facilitating viral connectivity between eastern and central Germany. The repeated detection of genetically related virus strains in Berlin

across multiple seasons, along with its central position in phylo-
geographic models, is consistent with potential urban virus main-
tenance. Phylogeographic analysis suggests possible westward
dispersal patterns from Saxony and Saxony-Anhalt toward Thuringia,
particularly for subclades 2F and 2E. In contrast, federal states such
as Schleswig-Holstein and Lower Saxony showed sporadic detec-
tions with no evidence of sustained circulation or onward spread yet.

Discussion

The diagnostic and epidemiological challenges associated with
WNV infections, particularly among asymptomatic individuals, re-
quire sensitive and scalable genome sequencing methods that can be
integrated into routine public health surveillance and transfusion
safety systems. These challenges are particularly pronounced in
blood donor surveillance, where viremia is typically low, often re-
sulting in suboptimal genome recovery for molecular epidemiolo-
gical analyses.⁹,²⁰ To address these limitations, we developed and
validated a highly sensitive amplicon-based next-generation se-
quencing (NGS) protocol, optimized for low RNA input and high Ct
value samples. This approach refines previously developed am-
plicon-based protocols for WNV genome recovery and other
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Spatial reconstruction of WNV subclades 2A-F based on continuous phylogeographic analysis. The time-scaled maximum clade credibility (MCC) tree (top left) is based on substantially recovered genome sequences (≥70% coverage). Each dot represents a node from the maximum clade credibility (MCC) tree, while curved lines represent phylogenetic branches and illustrate the directionality of viral movement across geographic space. The left panel shows the estimated time of introduction (year/month) into Germany, while the right panels display the geographic distribution of each subclade.</figcaption></figure>

orthoflaviviruses,¹²⁻¹⁸ and our results show it performs well even with blood donor samples carrying Ct values up to 35 (less than 10 copies/µL). However, we observed that successful genomic recovery from blood donor samples, as well as clinical samples, is strongly influenced by the overall quality of the sample material. Even among samples with similar Ct values, differences in storage conditions, RNA integrity, and sample handling impacted sequencing success. This reinforces the need for standardized sample processing protocols when applying genomic tools in routine surveillance or diagnostic workflows. Low-viremia samples occasionally produced

partial genome coverage, as expected, but these did not assemble into spurious genomes. Nevertheless, we recognize that even low-level contamination could in theory generate false-positive assemblies or mislead phylogenetic inference. We therefore emphasize that the combined use of rigorous laboratory separation, batch-specific negative controls, UDIs, and bioinformatic filtering provides high confidence that the reported genomic sequences reflect true biological signal rather than contamination artifacts. We also note that while all sequencing approaches have inherent strengths and limitations, our rationale for employing the amplicon-based method
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** (continued)</figcaption></figure>

was driven by its demonstrated sensitivity for low-viremia samples, reproducibility across laboratories, and cost-effectiveness for regional genomic surveillance. Although we used the smallest Illumina sequencing platform (iSeq100) for our experiments, the protocol is compatible with any Illumina sequencing platform that supports 300-cycle runs (2 × 150 bp), as the designed amplicon lengths fall well within this read range. Using this method, we sequenced a large panel of WNV genomes collected from blood donors between 2020 and 2024. While our blood donor samples represent only human infections, phylogenetic analyses incorporated published sequences from avian and equine hosts to contextualize human cases within the broader enzootic transmission cycle. The phylogenetic analyses

revealed six distinct subclades circulating in Germany (2A-2F). These represent separate introduction events, primarily originating from Central Europe, especially Austria and Slovakia, as supported by discrete trait phylogeography and continuous diffusion models. Several of these subclades, including 2F and 2C, show evidence of temporal persistence and geographic expansion consistent with potential regional establishment, particularly in Brandenburg, Berlin, Saxony, and Saxony-Anhalt. Our models suggest these regions may function as epidemiological centers, with phylogenetic patterns consistent with sustained local circulation and potential onward dispersal to northwestern regions. Berlin emerged as a particularly important urban transmission node with reported focal
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Spatial clustering analysis of WNV subclades 2A–F in Germany. (a) Locations associated with each node of the MCC tree are subjected to K-means clustering (k = 9) to define geographic zones and improve the visualization of the phylogeographic scenario. Localities are colored according to their assigned cluster, and paths are colored based on the cluster of origin. (b) The transition graph displays source-to-destination relationships between and within clusters, with nodes labeled by country names. Node size is proportional to the number of localities in each cluster. (c) The migration distance curve shows the distribution of geographic distances associated with dispersal events, highlighting the relative contributions of local versus long-distance spread in shaping the population structure of WNV in Germany.</figcaption></figure>

establishment of WNV,²⁹ showing repeated seasonal detections and strong phylogenetic linkage to nearby federal states. Our findings include the detection of WNV-positive blood donor samples in federal states not previously considered endemic, such as North Rhine-Westphalia, Bavaria, and Schleswig-Holstein. Five samples had documented domestic travel within Germany during the probable exposure period. Importantly, phylogenetic analysis demonstrates that all five travel-associated samples cluster within German WNV subclades (2A-2F) alongside samples from donors without documented travel, confirming these represent domestically

circulating strains rather than external introductions. Three samples from non-endemic states (two from North Rhine-Westphalia and one from Bavaria) documented travel to established endemic regions within Germany. The phylogenetic placement of these samples within established German subclades supports the interpretation that these likely represent infections acquired during domestic travel rather than evidence of local establishment in these non-endemic states. A standardized approach to collecting all travel histories during the follow-up of WNV-positive blood donors would substantially improve the resolution of future surveillance and
epidemiological analyses. Phylogeographic reconstructions indicate that multiple WNV lineage 2 subclades were introduced into Germany as early as 2016–2018, predating previous estimates. This suggests that undetected transmission occurred before the first confirmed autochthonous cases, despite efforts to identify hidden WNV infections in at-risk populations.³⁰ This highlights the value of retrospective genomic analysis for tracing virus emergence. The presence of a unique mutation in the helicase domain of the NS3 protein in subclade 2F, which now dominates the German WNV landscape, suggests possible adaptive changes relevant to viral fitness, though functional studies are needed to confirm this. Our study also reinforces the role of blood donors as effective sentinels for monitoring WNV activity. Donors are systematically screened and provide geographically widespread, temporally dense sampling across the country. Prior studies have shown that blood donor screening can detect WNV circulation ahead of symptomatic cases, providing a sensitive early-warning signal for public health response.¹⁹,²⁰ Our findings extend this value by demonstrating that blood donor samples can also yield high-quality genomic data, enabling phylogenetic and phylogeographic inference, even with low viral load. Genomic data from blood donors thus serve not only diagnostic or confirmatory purposes, but also power surveillance systems to understand WNV introduction routes, local evolution, and the impact of environmental drivers such as climate and vector ecology.³¹ This is especially relevant considering the predicted northward expansion of mosquito habitats under climate change, which has already been associated with WNV spread into previously unaffected regions of Europe.³² Our findings are timely given the accelerating impact of climate change on the spread and intensity of mosquito-borne diseases in Europe.³¹ Warmer temperatures and extended vector seasons may expand the ecological niche of WNV, though *Culex pipiens*, the primary vector, is already established throughout Germany. Climate effects likely influence transmission dynamics through vector competence and viral replication rates rather than vector distribution *per se*. This study has some limitations that should be considered when interpreting the findings. Blood donors represent a healthy subset of the population and may not fully reflect community WNV circulation patterns. Our 43 samples account for a proportion of confirmed WNV-positive donations but exclude potential undetected cases with very low-viremia. Travel history was not systematically collected for all donors, with many entries marked "unknown". This could create uncertainty in distinguishing locally acquired infections from travel-associated cases, particularly for samples from federal states not previously considered endemic. Genome recovery was influenced not only by viral load but also by sample quality factors, including storage conditions and RNA integrity. Even among samples with similar Ct values, recovery rates varied substantially. Despite methodological improvements, 13 of 43 samples (30%) failed to achieve ≥70% genome coverage, and recovery remains challenging below approximately 7 copies/µL. Our analyses assumed that the sampling location represents the infection location for cases without documented travel history. Continuous diffusion models are sensitive to sampling density, and sparse sampling in some regions may result in uncertain phylogeographic inferences. Despite these limitations, this study provides a blueprint for future genomic surveillance of WNV in Germany and beyond. It demonstrates that even in settings with low virus titers and decentralized testing structures, whole-genome sequencing is feasible and epidemiologically valuable when supported by sensitive and flexible molecular protocols. The validated sequencing protocol and bioinformatic workflows are publicly available and can be adopted or adapted by other laboratories in the EU for harmonized surveillance efforts.

## Conclusion

This study demonstrates that integrating amplicon-based sequencing with blood donor screening enables early, accurate, and geographically extensive genomic surveillance of WNV. This

combined approach strengthens outbreak preparedness, supports timely detection of viral introductions, informs risk-based public health responses, and advances our understanding of WNV evolutionary dynamics across Europe.

## Ethical statement

This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki (revised 2013). All blood donor samples, and associated metadata used in this study were fully anonymized before analysis. The legal basis for the collection and analysis of these samples is established by Section 28 of the German Medicinal Products Act (Arzneimittelgesetz, AMG), Section 14 of the Transfusion Act (Transfusionsgesetz, TFG), and Section 4 and 7 of the Infection Protection Act (Infektionsschutzgesetz, IfSG). Section 7 IfSG mandates the reporting of WNV infections, while Section 4 IfSG provides the framework for the analysis of such data. Case reporting and laboratory-based surveillance are further governed by Sections 63i AMG and 19 TFG. As these activities are part of official public health responsibilities under German national legislation, no separate ethics committee approval was required, and no institutional ethics review was sought.

## Funding

The mNGS investigations were funded by the German Federal Ministry of Education and Research (BMBF) within the project PREPMedVet no. 13N15449 whereas the amplicon-based sequencing approach within Culifo3 project no. 2819107A22 by the German Federal Ministry of Food and Agriculture.

## Data availability

The genomic sequences obtained in this study are available in the GenBank under accession numbers PV220993-PV221022, and the corresponding raw sequencing data were deposited in the Sequence Read Archive (SRA) under Bioproject ID PRJNA1223181.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

The authors would like to thank all participating blood establishments for reporting cases and providing blood donor samples. We also acknowledge the regional public health authorities for their collaboration in case investigation, data provision, and support of surveillance activities during the West Nile virus transmission seasons in Germany. We are grateful to Unchana Lange for her excellent technical assistance.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jinf.2025.106647.

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