The stable MDCK cell line expressing, in addition to GFP-CAT, the firefly luciferase gene under the control of the IFN- promoter was generated by the transfection of the above-described cell line with the plasmid pGL4.17 IFN firefly-luciferase. (25). It is still a mystery as to why influenza B viruses do not have as many natural hosts as the influenza A viruses. Influenza B viruses also continuously undergo antigenic drift (1,12,22), yet with a lower evolutionary rate than that of influenza A viruses (24), and reassortment among different influenza B virus strains occurs regularly Rabbit Polyclonal to FUK (18,36). Although influenza B viruses have only one recognized subtype, they have evolved into three major lineages, the earliest B/Lee/40 virus-like variants, B/Victoria/2/87-like variants, and B/Yamagata/16/88-like strains (22,28). The disease caused by influenza B viruses is generally milder than that caused by influenza GW6471 A viruses; however, in some cases B virus infections can lead to severe disease which requires hospitalization (23). The morbidity caused by infections with influenza B viruses may also have substantial social and economic impacts. Vaccination is by far the best means to protect people against disease from influenza virus infections (26). Live-attenuated cold-adapted virus and inactivated virus vaccines have been licensed by the FDA. Live-attenuated vaccines have some advantages over inactivated influenza virus vaccines; i.e., (i) they are easy to administer through intranasal delivery, and they mimic natural infection; (ii) they can provide a broader immunological response, including mucosal immunity and cellular immunity, than inactivated virus vaccines; and (iii) they can possibly provide longer-lasting immunity than inactivated virus vaccines. Although the current cold-adapted live-attenuated influenza A and B virus vaccine is effective (26), there is room for improvement (20). One strategy is to make a new live-attenuated influenza virus vaccine through modifying the influenza virus nonstructural protein 1 (NS1), which is an interferon (IFN) antagonist (34). This approach has proved to be successful in different animal models for influenza A virus infection (8,30,33). The NS1 of influenza B viruses has also been shown to be GW6471 an IFN antagonist (4,5,7), and IFN enhances the production of immunoglobulins and activates dendritic cells required for antigen presentation (13-15). The modification of the NS1 gene GW6471 of influenza B viruses by truncation and deletion would ensure that the mutant viruses are attenuated in their replication and enhanced in their induction of IFN compared to the wild-type (WT) virus (8,30,33). Hence, we hypothesize that immunization with NS1 mutant viruses would deliver an enhanced immune response compared with the responses induced by the current conventional live-attenuated or inactivated virus vaccines (34). Here, we report the novel construction of NS1 GW6471 mutant viruses in an GW6471 influenza B/Yamagata/16/88 virus background through reverse genetics. We also introduce a transgenic mouse model (C57BL/6 protein kinase R-deficient [PKR/] mice) in which non-mouse-adapted WT influenza B virus strains are able to cause morbidity and mortality. Using this mouse model, we shown the NS1 mutant viruses are attenuated and that they are safe in vivo. Finally, we shown their effectiveness against difficulties with homologous and heterologous viruses in the mouse model. == MATERIALS AND METHODS == == Cells and viruses. == 293T and MDCK cells were from the American Type Tradition Collection (ATCC, Manassas, VA) and were managed in Dulbecco’s revised Eagle’s medium (DMEM) and minimal essential medium (both from GIBCO, Carlsbad, CA), respectively, each supplemented with 10% fetal calf serum (HyClone, Logan, UT) and 1% penicillin-streptomycin (GIBCO). Influenza B/Yamagata/16/88 disease and rescued recombinant WT (rWT) viruses were propagated in 8-day-old embryonated chicken eggs for 3 days at 33C. Recombinant NS1 mutant viruses deltaNS1 (expressing amino acids 1 to 16 of NS1), NS1-80 (expressing NS1 amino acids 1 to 80), and NS1-110 (expressing NS1 amino acids 1 to 110) were propagated in IFN-deficient 7-day-old embryonated chicken.