Development of an Avian Influenza H5N1, H7N9, H7N7, H5N6, and H9N2 Analytical Reference Material Set for Diagnostic Surveillance

Highly pathogenic avian influenza (HPAI) viruses, particularly H5N1 clade 2.3.4.4b, have significantly impacted the poultry and dairy industries, becoming a major public health issue. In the US, the H5N1 virus has affected over 130 million birds since 2022. The culling of infected poultry flocks and large-scale bird deaths have resulted in significant economic losses for farmers due to reduced income from egg production and meat sales. The discovery of H5N1 traces in milk and incidences of mild infections among dairy workers since the summer of 2024 has affected the entire US dairy industry, raising significant public concern. These circumstances have prompted the USDA and FDA to conduct comprehensive surveillance testing. 

Surveillance testing is vital for monitoring zoonotic HPAI strains and detecting early signs of pandemics. The reliable detection of new or emerging variant strains like H5N1, H5N6, H7N7, H7N9, H9N2, and H10N8 depends on using accurate analytical reference materials (ARMs) during diagnostics development. Without reliable ARMs, diagnostic tests may yield false results, undermining surveillance and public health efforts. As such, ARMs are crucial for calibrating diagnostic assays, ensuring they reliably detect these viruses in animal, human, and food samples and accurately measure viral load. 

In response to the rapid global emergence of H5N1 and other serotype outbreaks in Asia, ATCC® has developed a comprehensive suite of quantitative synthetic RNA for HPAI virus serotypes H5N1, H5N6, H7N7, H7N9, and H9N2. These ARMs represent the most concerning and relevant influenza strains; for example, the H5N1 synthetic RNA product is based on a recent virus strain sequence belonging to the emerging clade 2.3.4.4b. Each synthetic ARM contains the complete sequences from segments 4, 5, 6, 7, and 8, including the HA, NP, NA, M1, M2, NS1, and NEP/NS1 genes, covering 50% of the influenza genome. These segments are key diagnostic targets for molecular tests and provide sufficient genomic context for assessing assay specificity. These ARMs are manufactured using a highly reliable synthetic biology technology, verified through next-generation sequencing, and quantitated via Droplet Digital PCR™. Further, they do not contain any viable material and can be handled in BSL-1 settings. As such, they are intended to serve as safe and reliable positive controls for molecular tests for surveillance and diagnostics. 

The usability of the synthetic RNA ARM panel for HPAI was experimentally evaluated using several published quantitative PCR assays, including those from the Centers for Disease Control and Prevention, the World Health Organization, the World Organization for Animal Health, and other highly cited sources. Additionally, we conducted an in silico assessment of ARM compatibility with over 250 publicly available published assays. The data collectively demonstrate that all synthetic ARMs for HPAI are effectively designed and suitable for developing and validating molecular-based detection and quantification assays. Furthermore, our findings indicate that the synthetic RNA ARMs are equivalent to their corresponding native influenza RNA, making them a valuable BSL-1 alternative to BSL-3-derived material. Our results suggest that these synthetic ARMs can serve as reliable and safe controls for molecular assays used in diagnostics and surveillance.