The major goal of this application is to generate a novel class of modified live virus vaccines against influenza that elicit broadly protective immune responses.Avian influenza virus (AIV) is a devastating diseasewith demonstrated ability to cause the loss of hundreds of millions of birds.Control strategies for poultry against AIV is dependent on biosecurity to keep the virus out and vaccine application when the virus is present.Vaccine protection is principally mediated by an immune response to the subtype-specific hemagglutinin protein (HA) with inactivated virus vaccines the overwhelming choice in the field. However, this type of vaccine has a narrow range of activity that is not long lasting. In addition, not all HA subtypes of AIV are as immunogenic as others. It is widely known that live virus vaccines induce the greatest immune responses, but concerns of reversion, recombination and unintended transmission have slowed their development in poultry. Reverse genetics has allowed greater control of the viral genome which has helped minimize the perceived potential risks associated with live influenza virus vaccines; however, the mechanisms necessary to improve AIV vaccines with broader immunogenicity and increased suppression of virus transmission require further studies. The major goal of this application is to close such knowledge gap to ultimately improve the effectiveness of control programs for AIV and decrease its economic and public health burden. Since the H5 and H9 subtypes are two most widely spread AIVs in poultry around the world, we propose the following specific aims:Specific Aim 1:To generate and compare alternative modified live avian influenza virus vaccine platforms as vaccines against H5, and H9 subtypes of avian influenza viruses.Specific Aim 2:Test safety and protection of poultry against HPAI and LPAI challenge following MLV vaccination.Specific Aim 3:To characterize the impact of live virus vaccination on genomic mutation by deep sequencing analysis of virus shed from vaccinated birds.The application maximizes our current understanding of vaccines and immune responses against influenza in poultry and reverse genetics to produce MLV-AI vaccines with unique and novel set of features. These features are built-inin the context of high yield strains to potentiate protective mucosal responses while serving as molecular diagnostic markers that also reduce transmission and reassortment of the vaccine strains. If successful, our approach can be implemented in vaccine strategies against not only other AIV subtypes but also other viral respiratory pathogens of poultry and livestock.