The Building Resistance Against Viral Entities (BRAVE) Innovation Center

SUMMARY The introduction of highly pathogenic avian influenza (HPAI) virus of the Guangdong H5N1 lineage has resulted in severe disease outbreaks with widespread mortality in wild birds and poultry in the United States. Current control measures, including massive culling of infected flocks, vaccination efforts, and biosecurity procedures have failed to stop the spread of avian viruses including HPAI viruses resulting in significant economic losses and public health risks due to their zoonotic potential. There is a pressing need to explore alternative strategies to combat viral pathogens that inflict massive losses on the poultry industry. To tackle the susceptibility of chickens to avian viruses, we propose establishing the BRAVE (Building Resistance Against Viral Entities) Innovation Center. This initiative aims to generate chickens with intentional genome alterations that are resistant to viral pathogens of significant biological and economic importance. To identify the host factors that will be targeted for intentional genome alterations in chickens, we will perform a genome-wide CRISPR/Cas9 study with a sgRNA library in Aim 1. These experiments will focus on identifying pro-viral chicken genes that are essential for virus replication. We will begin with HPAI H5N1 virus, and then expand the study to HPAI H7N9 virus and other avian viruses including infectious bronchitis virus (IBV). Aim 1 will also identify host factor targets for intentional genome alterations to identify unique and common antiviral proteins that directly inhibit the replication of the viruses in this proposal. We will identify and rank the best host factors when knocked out (pro-viral factors) or overexpressed (antiviral factors) for the greatest inhibitory effect on virus replication. We will monitor cell viability and the lack of escape viruses that would mutate away from the resistance profile. Using the data collected in Aim 1, in Aim 2, we will generate an intentional genomically altered (IGA) chicken line resistant to HPAI virus infection and second line that has a multi-virus resistance phenotype. We will employ CRISPR/Cas9 or transposase methods to modify the genomes in chicken primordial germ cells (PGCs) and then inject the modified PGCs into recipient chicken embryos to establish germline-modified chickens and eventually flocks of birds for infection studies in Aim 3. Once an IGA chicken line is established, we will compare the susceptibility of the altered birds with that of wild-type birds to HPAI H5N1 and H7N9 virus infections. Virus replication, associated pathology, transmission, and emergence of mutant viruses associated with breakthrough infections will be assessed and compared to wild-type birds. We will also evaluate the susceptibility of the multi- virus-resistant chicken line to H5N1 and H7N9 viruses, along with other avian viruses including IBV. Non-infected IGA chicken lines will be characterized to ensure proper growth, longevity, and fertility along with any pathological changes compared to their wild-type counterparts.