NCM 2023 Houston: Enrichment strategies for recovery of Avian influenza virus from samples using MinION

Avian influenza virus (AIV) is a significant infectious agent worldwide. This became particularly evident during the ongoing Highly Pathogenic Avian Influenza (HPAI) outbreak. In this context of an outbreak response, rapid and accurate diagnosis is essential. The current diagnostic workflow relies on a combination of qPCR and genome sequencing, which can take days to weeks and is too slow for outbreak control. Nanopore sequencing offers many features which make it suitable for improving genome sequencing in veterinary diagnostics. However, working directly from clinical samples is challenging due to the overabundance of host nucleic acid, compromising the assay’s sensitivity. In this study, we aim to optimize the nanopore sequencing workflow for AIV identification and characterization from clinical samples by comparing different enrichment strategies. We tested host nucleic acid depletion, target-specific and target-independent enrichment approaches during the pre-processing steps. Host nucleic acid depletion treatment involved DNA and rRNA depletion. Treated and untreated RNA extracts were amplified via sequence-independent, single-primer amplification (SISPA) to increase viral reads. Enrichment and depletion strategies effectively reduced non-target DNA and RNA concentration from the samples, while SISPA increased viral detection. Additionally, target-specific enrichment, using Influenza A Universal Primers, will be used to specifically amplify our AIV target sequences. This amplicon-based approach will be compared to SISPA enrichment, in its sensitivity and genome coverage results. The final goal of this study is to generate a complete workflow for AIV diagnosis using nanopore sequencing, reducing the time for diagnostic, while keeping sensitivity and accuracy. Consequently, this will optimize the extraction of genetic information from clinical samples.