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-based surveillance offers a promising avenue for monitoring IAV.
• Low viral abundance in wastewater presents a major challenge for whole-genome sequencing.

Purpose of the Study:

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

Main Methods:

• Benchmarking of four targeted enrichment methods: custom and off-the-shelf amplicon, and probe-based methods.
• Evaluation of a custom HA tiled-amplicon panel and a universal amplicon method.
• Comparison of probe-capture methods for resilience to RNA degradation.
• Assessment of ultrafiltration-based virus concentration versus large-volume direct extraction.

Main Results:

• The custom HA tiled-amplicon panel demonstrated sensitivity, speed, and cost-effectiveness for known subtypes.
• Universal amplicon methods performed poorly due to reliance on intact genome segments.
• Probe-capture methods showed resilience to RNA degradation and mismatches, but were costly and less sensitive.
• 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 may suit broader surveillance.
• Ultrafiltration is a superior method for concentrating viruses from wastewater for sequencing.
• This study provides crucial data for implementing routine IAV genomic sequencing in wastewater surveillance programs.