Influenza A viruses (IAV) are significant human pathogens causing yearly epidemics and occasional pandemics. Past pandemics have resulted in significant morbidity and mortality. The 1918 influenza pandemic was thought to have resulted in the death of at least 675,000 people in the U.S., and 40 million people worldwide. Annual influenza A virus epidemics are also very significant, resulting in approximately 30,000 deaths in the U.S. per year. Pandemic strains of influenza emerge periodically and are thought to be derived ultimately from avian influenza A viruses. The current 2009 pandemic influenza virus emerged by reassortment of two pre-existing swine influenza virus lineages. The first lineage is the European avian-like swine lineage which emerged from an avian influenza virus in toto. The second lineage is the triple-reassortant H1N2 swine influenza virus containing gene segments derived from avian, human, and classical swine influenza viruses. The natural reservoir of influenza A viruses is thought to be wild waterfowl. Genetically and antigenically diverse influenza A viruses circulate in wild birds and viral strains from this pool can adapt to new hosts, including humans and domestic animals. Influenza A viruses are also significant pathogens for agriculturally important animals like poultry, swine, and horses. Understanding the mechanisms of host switching are very important for surveillance and pandemic preparedness. Understanding the molecular basis underlying the annual evolution of human influenza will aid in vaccine strain selection. Avian influenza A virus evolutionary biology: We evaluated the molecular epizootology of approximately 50 avian influenza viruses isolated from the same population of birds over a several month period in 2006. Low pathogenicity avian influenza viruses of both H5N1 and H7N3 subtypes were isolated. These subtypes have been associated with highly pathogenic avian influenza virus outbreaks. From August 2 to October 11, 2006, clusters of low pathogenicity (LP) North American lineage H5N1 and H7N3 avian influenza A viruses (AIV), and other subtypes, were recovered from free-flying, resident, wild mallards used as sentinels at one site. The antigenic subtypes, pathogenicity potential, and Sanger sequencing of the isolates determined the H5N1 and H7N3 isolates were only recovered from samples collected on 8/2/2006 and 9/8/2006, respectively. However, subsequent efforts using next-generation sequencing (NGS) and additional Sanger sequencing found partial H7 segments in other HA-NA virus combinations on 8/2/2006, 9/8/2006 and 10/11/2006. It is well established that over larger geographic areas and years AIVs form transient genomic constellations;this sequential sampling data revealed that over a short period of time the dynamics of AIVs can be active and newer sequencing platforms increase recognition of mixed infections. Both findings provide further insight into the natural history of AIVs in natural reservoirs. Despite the emergence of the pandemic H1N1 influenza A virus in 2009, seasonal H3N2 viruses continue to co-circulate in the population and may even predominate in coming influenza seasons. Because rapid subtype-specific diagnosis is often warranted to guide antiviral drug therapy, we developed a rapid molecular diagnostic test involving a specific minor groove binder TaqMan assay for H3N2 viruses with a detection limit of 16.5 standard DNA copies.