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Proteolytic activation of Sendai virus in the lungs of mice is necessary to cause pneumopathogenicity. Using Sendai virus-infected lung block cultures, protease inhibitors were tested for their antiviral effect by examining inhibition of proteolytic activation. Among the inhibitors tested, a serine protease, aprotinin, was shown to be most effective. In vivo protection experiments demonstrated that aprotinin, when administered intranasally, could confer protection on mice against lethal Sendai virus pneumonia through the same mechanism as observed in the in vitro system. The present study provides an experimental basis for the use of protease inhibitors as antiviral drugs.