Supporting molecular detection of A(H5N1) through development and characterization of RNA reference materials

In 2024, the US Centers for Disease Control and Prevention (CDC) and National Institute of Standards and Technology (NIST) initiated an interagency collaboration to bolster preparedness in the event of a pandemic scenario due to high pathogenicity avian influenza A(H5N1) clade 2.3.4.4b viruses that had been circulating in North America since 2021. Multiple spillover events into mammalian species, including an outbreak among dairy cattle and a surge in human cases, spurred the development of RNA reference materials to assist with new diagnostics development and validation. With input from CDC on sequences, NIST generated three synthetic RNA materials of A(H5N1) hemagglutinin, neuraminidase, and matrix proteins 1 and 2 gene segments. Here, we describe the development and characterization of these reference materials utilizing droplet digital reverse transcription polymerase chain reaction. Through this collaboration, we were able to rapidly provide quantified, homogeneous, stable, and epidemiologically relevant RNA reference materials to the public to aid new assay development and validation.