Streamlined CRISPR-based assays for detection and subtyping of avian influenza

Avian influenza viruses (AIVs) are zoonotic pathogens that pose an increasing global threat due to their potential for significant economic losses in agriculture, spillover into humans, and the risk of a pandemic should human-to-human transmission occur. These concerns underscore the need for rapid, sensitive and specific tools to detect and differentiate circulating AIV subtypes and clades. Current AIV diagnostic methods rely on specialized equipment and trained personnel, limiting their use in the field and in low-resource settings. Here, we extended SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a CRISPR-based platform, to detect and subtype AIVs. We designed, optimized, and validated SHINE assay for the H5 AIV detection using both fluorescence and lateral flow readout, achieving 100% specificity with PCR-based assays when tested on seasonal influenza-positive clinical samples, and a limit of detection of 121.7 copies/μL on vaccine-derived H5 viral seedstocks. To expand the scope of avian influenza detection, we also designed and validated a SHINE assay targeting the 2.3.4.4b A(H5N1) lineage, in response to the ongoing H5N1 outbreak in cattle in the United States, and a SHINE assay specific to Eurasian H7 lineage to discriminate against North American H7 lineage. Together, these SHINE assays offer a promising platform for AIV diagnosis and surveillance, particularly in settings with limited laboratory infrastructure.