An amplicon-based tiled PCR scheme for the enrichment of avian metapneumovirus subtype B genomes prior to next generation sequencing
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Sonsiray Álvarez-Narváez1, David L Suarez1, Darrell R Kapczynski1
1U.S. National Poultry Research Center, United States Department of Agriculture, Agricultural Research Service, Athens, GA, United States.
Abstract:
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.
Insights
A new method, aMPVB-ATP, significantly improves avian metapneumovirus (aMPV) genome recovery from poultry samples. This advancement is crucial for tracking the spread of aMPV subtypes A and B in the U.S. and enhancing surveillance efforts.
Area of Science:
- Veterinary Virology
- Molecular Epidemiology
- Poultry Health
Background:
- Avian metapneumovirus (aMPV) is a significant global poultry pathogen.
- The U.S. confirmed outbreaks of aMPV subtypes A and B in 2023-2024, necessitating improved surveillance.
- Current epidemiological understanding of aMPV spread is limited, highlighting the need for advanced diagnostic tools.
Purpose of the Study:
- To evaluate and compare two avian metapneumovirus subtype B genome enrichment strategies.
- To assess the efficacy of a novel amplicon-based tiled PCR approach (aMPVB-ATP) against a standard method (HD-SISPA).
- To determine the suitability of aMPVB-ATP for both short-read and long-read next-generation sequencing.
Main Methods:
- Comparative analysis of two viral genome enrichment methods: HD-SISPA and aMPVB-ATP.
- Application of these methods to avian metapneumovirus subtype B samples.
- Next-generation sequencing (NGS) using both short-read and long-read platforms.
- Quantification of viral reads and genome recovery efficiency.
Main Results:
- The aMPVB-ATP method yielded a significantly higher proportion of aMPV reads (41%) compared to HD-SISPA (1%) in short-read NGS.
- aMPVB-ATP enabled the recovery of complete or near-complete aMPV genomes from samples with low viral loads (Ct < 22).
- aMPVB-ATP demonstrated compatibility with long-read NGS without compromising genome coverage or sequencing depth.
Conclusions:
- The aMPVB-ATP method represents a substantial improvement for avian metapneumovirus subtype B genome enrichment.
- This enhanced method facilitates more effective viral characterization and epidemiological surveillance.
- aMPVB-ATP is a versatile tool for aMPV genome recovery across different NGS platforms.
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