The overall project goal is to develop a contemporary live vaccine capable of inducing broad protection against multiple Bovine Parainfluenza 3 Virus (BPI3V) as well as Bovine Viral Diarrhea Virus (BVDV) strains. These pathogens play significant roles in causing Bovine Respiratory Disease Complex (BRDC), which leads to severe pneumonia and death of calves. The current BPI3Va vaccine is effective against some homologous, but not heterologous, strains. Notably, BPI3Va-specific serum is weak at neutralizing BPI3Vb and BPI3Vc strains that were previously reported in other countries, but they are now circulating in U.S. cattle herds. Similarly, BVDV vaccines are not very effective due, in part, to the immunosuppressive traits and failure of the vaccine virus to confer broad protection. Consequently, there is a need to develop better vaccines capable of conferring broad protection against diverse BPI3V and BVDV strains. The BPI3V structural surface Fusion (F) and Hemagglutinin-Neuraminidase (HN) glycoproteins contain protective epitopes. Immune responses directed at BVDV envelop (E2) and non-structural (NS2-5) antigens confer protection. Significant portions of these antigens are conserved among diverse strains and thus are ideal targets for the development of broadly protective vaccines. We have previously demonstrated that a prototype vaccine containing E2 and NS2-3 mosaic antigens that incorporate determinants that are conserved among BVDV-1a, b, and BVDV-2 genotypes, conferred protection against BVDV-1 & 2 strains. The mosaic antigens were designed from twenty BVDV genomes and to increase coverage, we designed novel E2 and NS2-5 mosaic antigens (E2-NS2-5) using data from >200 genome sequences. We also used this approach to design novel mosaic BPI3V antigens, designated F2 and HN2 (F2-HN2), respectively, using data from all sequenced genomes. More importantly, we designed a BPI3Vc backbone that contains defined mutations shown to attenuate BPI3Va and used it to generate attenuated BPI3Vc-F2-HN2 and BPI3Vc-E2-NS2-5 recombinant viruses. We hypothesize that immunization of calves with the recombinant BPI3Vc expressing the novel F2-HN2 or the E2-NS2-5 mosaic antigens, will confer broad protection against diverse BPI3V and BVDV strains. The hypothesis will be tested through completion of the following specific aims:Aim 1: Test whether immunization of calves with the recombinant BPI3Vc-F2-HN2 virus will safely confer protection against wildtype BPI3Va-c strains. Approach: In vivo attenuation, safety, immunogenicity, and protective efficacy of the recombinant BPI3Vc-F2-HN2 virus against representative BPI3Va-c, strains will be evaluated in calves following intranasal or subcutaneous immunization. Immune sera will be tested, in vitro, for cross-neutralization of disparate BPI3V strains.Aim 2: Determine whether immunization of calves with the recombinant BPI3Vc-E2-NS2-5 viruses, will confer protection against representative BVDV-1a, b, and BVDV-2 strains. Approach: Calves will be immunized as above and then challenged with representative BVDV-1a, b, or BVDV-2 strains to evaluate safety, immunogenicity, and protective efficacy. Immune sera will be tested, in vitro, for neutralization of diverse BVDV strains.