Influenza A viruses (IAVs) cause occasional pandemics and seasonal epidemics, thus presenting continuous challenges to public health. Vaccination is the primary strategy for the prevention and control of influenza outbreaks. The antigenicity matched high-yield seed strain is critical for the success of influenza vaccine. Currently, there are several limitations for the influenza vaccine manufacture: 1) the conventional methods for generating such strains are time consuming; 2) egg-based vaccines, the predominant production platform, have several disadvantages including the emergence of viral antigenic variants that can be induced during egg passage; 3) vaccine seed viruses often do not grow efficiently in mammalian cell lines. Previous studies suggested that mutations in the receptor binding site (RBS) that locates at the globular head of the HA1 can change IAVs’ binding specificity, antigenicity, and yield and thus RBS would be an potential target for engineering vaccine seed strain. However, systematic analysis of the mutations on RBS affecting those viral phenotypes is lacking. Specifically, this dissertation has following aims: Firstly, we developed a novel method to rapidly generate high-yield candidate vaccine strains by integrating error-prone PCR, site-directed mutagenesis strategies, and reverse genetics. The error-prone PCR- based reverse genetic system could also be applied to gain-ofunction studies for influenza virus and other pathogens; Secondly, in this dissertation, we identified an Y161F mutation in the hemagglutinin (HA) that enhanced the infectivity and thermostability of virus without changing its original antigenic properties which would prompted the development of cell-based vaccines; Thirdly, the molecular mechanisms underlying host adaption of equine-origin influenza A(H3N8) virus from horses to dogs are unknown. This dissertation identified that a substitution of W222L in the HA of the equine-origin A(H3N8) virus facilitated its host adaption to dogs. This mutation increased binding avidity of the virus specifically to sialyl Lewis X motifs, which were found abundantly in the submucosal glands of dog trachea but not in equine trachea. To summary, this dissertation investigated the role of RBS in IAVs biology and expanded the current knowledge toward IAV vaccine strain engineering, IAV host adaption and evolution.