Finch, Courtney LaPagliaDue to the disease burden of influenza virus types A and B, vaccines, which are manufactured as formalin-inactivated killed virus (KV) and live-attenuated virus (LAIV), are produced to provide coverage against currently circulating influenza A (IAV) and B (IBV) viruses. Although the licensed LAIV vaccine provides enhanced coverage over the KV vaccine, it is only licensed for immunocompetent individuals ages 2-49 years without pre-existing conditions, so individuals who are most at risk cannot receive it. Previously, our lab showed that incorporation of an 8 amino acid HA tag in frame at the C-terminus of the PB1 open-reading frame (ORF) in addition to the mutations found in the PB2 and PB1 segments of the licensed LAIV vaccine yielded a stable, efficacious alternative LAIV vaccine for IAV; however, to develop a complete vaccine, a corresponding IBV candidate is required. Towards this goal, a contemporary IBV strain, B/Brisbane/60/2008, was cloned and recovered by reverse genetics (RG-B/Bris). Subsequently, it was demonstrated that the parental and RG-B/Bris show similar growth kinetics in vitro. An initial vaccine attempt, which combined PB2 cap-binding mutants with the HA tag in PB1, was made but led to the realization of the PB2 cap-binding mutations, PB2 W359F and F406Y, as virulence factors. In a subsequent vaccine attempt, mutations analogous to those found only in segment 2 of the A/Ann Arbor/6/60 cold-adapted LAIV backbone were introduced into the homologous segment of RG-B/Bris. The following mutations were introduced into the PB1 gene segment of RG-B/Bris, either in the presence or absence of a C-terminal HA tag: K391E, E580G, and S660A. Two viruses were rescued, referred to as RG-B/Bris <italic>ts</italic> and RG-B/Bris <italic>att</italic>, both containing the set of three amino acid mutations but differing in the absence or presence of the HA tag, respectively. Both viruses showed ideal attenuation, safety, and immunogenicity in DBA/2 mice and conferred protection against lethal IBV challenge. More importantly, RG-B/Bris <italic>att</italic>, but not RG-B/Bris <italic>ts</italic>, showed ideal stability with no reverting mutations over 8 passages in eggs. Taken together, a stable, immunogenic, and live attenuated virus alternative to the current live influenza B virus vaccine was produced.enReverse Genetics of Influenza B and the Development of a Novel LAIV VaccineDissertationVirologyMolecular biologyinfluenza Blive-attenuatedreverse geneticsvaccine