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The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 ( cp 45) candidate vaccine was shown previously to be safe, immunogenic, and phenotypically stable in seronegative human infants. Previous findings indicated that each of the three amino acid substitutions in the L polymerase protein of cp 45 independently confers the temperature-sensitive ( ts ) and attenuation ( att ) phenotypes but not the cold-adaptation ( ca ) phenotype (29). cp 45 contains 12 additional potentially important point mutations in other proteins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in cis -acting sequences (the leader region and the transcription gene start [GS] signal of the N gene), and their contribution to these phenotypes was undefined. To further characterize the genetic basis for the ts , ca , and att phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant cp 45 virus that contained all 15 cp 45-specific mutations mentioned above, and found that it was essentially indistinguishable from the biologically derived cp 45 on the basis of plaque size, level of temperature sensitivity, cold adaptation, level of replication in the upper and lower respiratory tract of hamsters, and ability to protect hamsters from subsequent wild-type PIV3 challenge. We then constructed recombinant viruses containing the cp 45 mutations in individual proteins as well as several combinations of mutations. Analysis of these recombinant viruses revealed that multiple cp 45 mutations distributed throughout the genome contribute to the ts , ca , and att phenotypes. In addition to the mutations in the L gene, at least one other mutation in the 3′ N region (i.e., including the leader, N GS, and N coding changes) contributes to the ts phenotype. A recombinant virus containing all the cp 45 mutations except those in L was more ts than cp 45, illustrating the complex nature of this phenotype. The ca phenotype of cp 45 also is a complex composite phenotype, reflecting contributions of at least three separate genetic elements, namely, mutations within the 3′ N region, the L protein, and the C-M-F-HN region. The att phenotype is a composite of both ts and non- ts mutations. Attenuating ts mutations are located in the L protein, and non- ts attenuating mutations are located in the C and F proteins. The presence of multiple ts and non- ts attenuating mutations in cp 45 likely contributes to the high level of attenuation and phenotypic stability of this promising vaccine candidate.

Studies were initiated to define the genetic basis of the temperature-sensitive ( ts ), cold adaptation ( ca ), and attenuation ( att ) phenotypes of the human parainfluenza virus type 3 (PIV3) cp 45 live attenuated vaccine candidate. Genetic data had previously suggested that the L polymerase protein of cp 45, which contains three amino acid substitutions at positions 942, 992, and 1558, contributed to its temperature sensitivity (R. Ray, M. S. Galinski, B. R. Heminway, K. Meyer, F. K. Newman, and R. B. Belshe, J. Virol. 70:580–584, 1996; A. Stokes, E. L. Tierney, C. M. Sarris, B. R. Murphy, and S. L. Hall, Virus Res. 30:43–52, 1993). To study the individual and aggregate contributions that these amino acid substitutions make to the ts , att , and ca phenotypes of cp 45, seven PIV3 recombinant viruses (three single, three double, and one triple mutant) representing all possible combinations of the three amino acid substitutions were recovered from full-length antigenomic cDNA and analyzed for their ts , att , and ca phenotypes. None of the seven mutant recombinant PIVs was cold adapted. The substitutions at L protein amino acid positions 992 and 1558 each specified a 10 5 -fold reduction in plaque formation in cell culture at 40°C, whereas the substitution at position 942 specified a 300-fold reduction. Thus, each of the three mutations contributes individually to the ts phenotype. The triple recombinant which possesses an L protein with all three mutations was almost as temperature sensitive as cp 45, indicating that these mutations are the major contributors to the ts phenotype of cp 45. The three individual mutations in the L protein each contributed to restricted replication in the upper or lower respiratory tract of hamsters, and this likely contributes to the observed stability of the ts and att phenotypes of cp 45 during replication in vivo. Importantly, the recombinant virus possessing L protein with all three mutations was as restricted in replication as was the cp 45 mutant in both the upper and lower respiratory tracts of hamsters, indicating that the L gene of the cp 45 virus is a major attenuating component of this candidate vaccine.