Developing and Benchmarking One Health Genomic Surveillance Tools for Influenza A Virus in Wastewater

Area of Science:

• Environmental microbiology
• Virology
• Genomic surveillance

Background:

• Influenza A virus (IAV) poses a significant One Health threat.
• Wastewater surveillance offers a promising avenue for IAV monitoring.
• IAV is often present at low concentrations in wastewater, complicating genomic sequencing.

Purpose of the Study:

• To benchmark different targeted enrichment methods for whole-genome sequencing of IAV in wastewater.
• To evaluate the performance of amplicon-based and probe-based methods.
• To assess the compatibility of sequencing methods with upstream virus concentration techniques.

Main Methods:

• Benchmarking of four targeted enrichment methods: custom and off-the-shelf amplicon panels, and probe-based methods.
• Evaluation of custom HA tiled-amplicon panel for sensitivity, speed, and cost-effectiveness.
• Comparison of ultrafiltration-based virus concentration with large-volume direct extraction.
• Assessment of method compatibility with RNA degradation and mismatches.

Main Results:

• The custom HA tiled-amplicon panel demonstrated sensitivity, speed, and cost-effectiveness for known IAV subtypes.
• Probe-capture methods showed resilience to RNA degradation and mismatches, aiding broader surveillance.
• A universal amplicon method performed poorly due to reliance on intact genome segments.
• Ultrafiltration-based virus concentration outperformed direct extraction across all tested sequencing methods.

Conclusions:

• Targeted enrichment methods vary in suitability for IAV wastewater surveillance.
• Custom amplicon panels are effective for seasonal variants, while probe-capture methods offer broader potential for emerging strains.
• Ultrafiltration is a superior method for concentrating viruses from wastewater for genomic sequencing.
• These findings provide critical data for establishing routine IAV genomic surveillance in wastewater.