An amplicon-based tiled PCR scheme for the enrichment of avian metapneumovirus subtype B genomes prior to next generation sequencing.

Avian metapneumovirus (aMPV) is an important respiratory pathogen of global concern in poultry. Until recently, the United States (U.S.) was considered free of aMPV following the eradication of subtype C in the early 2000s. However, in 2023-2024, both aMPV subtypes A and B were confirmed in the U.S., leading to widespread outbreaks in more than 30 states. The rapid spread of these viruses, despite enhanced biosecurity measures, highlights critical gaps in our epidemiological understanding and underscores the urgent need for active surveillance programs. Whole genome sequencing has become a valuable tool for the characterization and epidemiological investigation of viruses, and viral genome enrichment methods are essential to increase the proportion of viral genetic materials in a sample prior sequencing. In this study, we evaluated two aMPV subtype B genome enrichment strategies: the currently used combination of host and bacterial RNA depletion followed by sequence-independent single-primer amplification (HD-SISPA), and a newly developed amplicon-based tiled PCR approach (aMPVB-ATP). Samples treated with aMPVB-ATP yielded significantly higher proportion of aMPV reads (41%) following short-read next-generation sequencing (NGS) compared to those treated with HD-SISPA (1%). This improvement enabled the recovery of complete or nearly complete aMPV genomes from samples with RT-PCR cycle threshold (Ct) values below 22. The compatibility of aMPVB-ATP with long-read NGS was also assessed, showing no significant differences in genome coverage or sequencing depth compared to its performance combined with short-read platforms.