Developing a multiplex serology assay for the detection of highly pathogenic influenza

Since 2021, North America has experienced devastating outbreaks of Highly Pathogenic Avian Influenza virus (HPAIV) driven by HPAIV H5N1 clade 2.3.4.4b. Recently, it has spilled over into dairy cattle in the USA, raising concerns about its adaptation to mammals and potential human spread. Wild birds are the natural carriers of avian influenza viruses (AIV), but H5N1 can also infect wild mammals. Current HPAIV surveillance programs sample dead wildlife and use molecular methods that only detect active infection, underestimating the true number and distribution of infected animals. Serological tools measure antibodies from previous exposure and not active infection. However, current serology assays are not specific for HPAIV H5N1, can only be used in a limited number of species, and/or are too complex for large-scale implementation. Objectives: 1. Develop a species-independent multiplex assay for the detection of HPAIV H5N1 clade 2.3.4.4b, low pathogenicity (LPAIV) H5 and other HA subtypes 2. Validate the assay for use with filter paper strips with samples obtained from wildlife 3. Evaluate cross-reactivity between antibodies against H5N1 clade 2.3.4.4b, LPAIV H5 and other subtypes We will design a new assay on Meso Scale Discovery's platform to detect antibodies specific for AIV. The test will detect multiple subtypes in one sample and can be used on different animal species. Samples will be collected with filter paper strips from animals:1) positive for HPAIV H5N1, 2) positive for non-HPAIV H5N1, and 3) negative for all AIVs. A diversity of species will be included. Confirmatory testing will be done at the Canadian Food Inspection Agency and cross-validation at Public Health Ontario. This research will generate a standardized test crucial for nationwide comparative analyses. Its species independent nature allows for easy use in domestic animals and humans as needed, providing crucial insights into antibody reactivity, cross-reactivity, and infection reservoirs.