Rapid CRISPR Diagnostics for H5 and H7 Avian Influenza
Review written by: Megan Mauriello
Avian influenza viruses (AIVs) are zoonotic pathogens which threaten to cause human pandemics and massive losses in agriculture. AIVs are classified into subtypes based on their hemagglutinin (H) and neuraminidase (N) surface glycoproteins, and are additionally classified into low pathogenicity (LPAI) and high pathogenicity (HPAI) types. Particularly relevant AIVs at the moment are HPAI H5, which has devastated poultry farming, 2.3.4.4b A(H1N1) in cattle with some human cases, and Eurasian H7, which has caused outbreaks in humans. Existing AIV diagnostics require specialized laboratory equipment that is not readily available in the field or in low-resource settings, limiting their use in areas that potentially need them the most. CRISPR-based diagnostic assays (CRISPR-Dx) are specific, sensitive, and programmable, and have been previously used for AIV detection, but with complicated workflows with specific amplification and detection steps.
Streamlined Highlighting of Infections to Navigate Epidemics (SHINE) assays are streamlined CRISPR-Dx methods developed here for detection of AIVs. In SHINE assays, amplification and Cas13-based detection are combined in “one-pot” to increase accessibility of the assay. AIV genomes are reverse transcribed and amplified by a reverse transcriptase, recombinase polymerase amplification, and T7 RNA polymerase. When the target AIV genome is amplified, complementary base pairing with a Cas13-crRNA complex activates Cas13 trans cleavage of RNA-based reporters.
The authors designed, optimized, and validated a SHINE assay for H5 AIV detection (SHINE-H5), which achieved 100% specificity on seasonal influenza-positive clinical samples with a limit of detection of 121.7 copies per microliter. It was able to detect synthetic RNA targets spiked in milk. The SHINE-H5 assay detects the genome segment encoding the HA protein, which is unique to H5 viruses. SHINE assays were also developed to detect 2.3.4.4b A(H5N1) (SHINE-H5-CS) and Eurasian H7 (SHINE-H7-Eurasian) lineages.
SHINE-Avian assays are compatible with both fluorescence and lateral flow readouts. For the lateral flow readout version of the SHINE-H5 assay (SHINE-H5-LFA), a biotin-FAM reporter is used, with uncleaved reporters migrating to the lower band and cleaved ones migrating to the upper band. When a large reaction volume was used, SHINE-H5-FLA was able to detect H5N1 at 25 copies per microliter.
It is important to have quick, streamlined, accessible diagnosis and surveillance methods for AIV detection to the subtype and clade level for the sake of informing public health responses. This is especially true in resource-limited settings, which are often where public health responses are most stunted. The SHINE-H5 assay detected RNA targets in spiked milk, so it may be usable for dairy industry surveillance.
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READ MORE: Huang Y, Guo A, Adams G, Lemieux JE, Myhrvold C. Streamlined CRISPR-based assays for detection and subtyping of H5 and H7 avian influenza. iScience, 2026; 29. https://doi.org/10.1016/j.isci.2026.117556
