Cold adaptation of parainfluenza virus type 3: induction of three phenotypic markers

In order to attenuate parainfluenza type 3 virus, a wild type strain that was isolated from a child with respiratory disease was adapted to replicate in African green monkey kidney cells at 20 degrees C. Replication at 20 degrees C was not a property of the wild type virus. The virus was serially passaged 45 times in the cold, and clones were selected following passage levels 7, 12, 18, and 45. The population of cold-adapted virus was found to be progressively enriched with temperature sensitive (ts) mutants. After 7 passages in the cold, 1 of 9, and after 12 passages in the cold, 3 of 12 clones were temperature sensitive. Following 18 passages in the cold, 80% of the clones were temperature sensitive and after 45 passages in the cold, all clones were temperature sensitive. In addition to being temperature sensitive each ts clone manifested the tiny plaque morphology. Each temperature-sensitive clone was also cold adapted. Some clones were cold adapted but were not temperature sensitive. The mutants were found to be genetically stable when serially passaged at 32 degrees, 35 degrees, or 39 degrees C. The mutants may possess the necessary degree of attenuation for use as live attenuated intranasal vaccines.