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Sendai virus mutants, KDe-21 and KDe-62, which had undergone multiple cycles of replication in Madin Darby canine kidney (MDCK) cells in the absence of exogenous proteases were isolated. The fusion (F) protein of the mutants regained proteolytic cleavability in MDCK cells and chick embryos, but the F protein remained non-cleavable in other cell lines. Unlike the F protein of wild-type (wt) virus, the mutant F was resistant to trypsin but was sensitive to elastase and, to a lesser extent, to chymotrypsin. Sequence analyses of the F gene and the F protein revealed an amino acid substitution at the cleavage site, Arg(116) to Ile, which conferred trypsin resistance and enhanced cleavability at Ile(116) by elastase and host proteases present in MDCK cells and in chicken embryos. In contrast to the pneumopathogenicity in mice of wt Sendai virus, the KDe mutants were non-pathogenic; cleavage activation of the F protein did not occur in the lungs and thereby infection was terminated after an initial cycle of replication.