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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Exotic & Emerging Avian Viral Diseases Research » Research » Publications at this Location » Publication #431438
Research Project: Control Strategies to Prevent and Respond to Diseases Outbreaks Caused by Avian Influenza Viruses

Location: Exotic & Emerging Avian Viral Diseases Research

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

Author
item Alvarez Narvaez, Sonsiray
item Suarez, David
item Kapczynski, Darrell

Submitted to: Frontiers in Cellular and Infection Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/20/2026
Publication Date: 3/17/2026
Citation: Alvarez Narvaez, S., Suarez, D.L., Kapczynski, D.R. 2026. An amplicon-based tiled PCR scheme for the enrichment of avian metapneumovirus subtype B genomes prior to next generation sequencing. Frontiers in Cellular and Infection Microbiology. Volume 16. https://doi.org/10.3389/fcimb.2026.1782197.
DOI: https://doi.org/10.3389/fcimb.2026.1782197

Interpretive Summary: Avian metapneumovirus (aMPV) is responsible for a highly infectious respiratory disease of chickens and turkeys. Recently, introduction of these viruses in the U.S. has resulted in significant economic impacts to the poultry industry. The mechanisms for the virus to transmit between geographic areas are unknown. In an effort to assist the poultry industry, we developed novel techniques to aid in molecular characterization of these viruses. The results of these studies allow us to provide near complete genome sequence of these viruses to increase our knowledge of their origins and movement. These results will aid the poultry industry in our understanding in transmission and control of aMPV.

Technical 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-SPA (1%). This improvement enabled the recovery of complete or nearly complete aMPV genomes from samples with RT-PCR cycle threshold 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.