FIG. 3.

Multicycle replication of chimeric PIV3-PIV2 compared with that of the wild-type parents rPIV3/JS and PIV2/V94. (A) rPIV3-2TM, rPIV3-2TMcp45, rPIV3/JS, and PIV2/V94 were used to infect LLC-MK2 cells in six-well plates, each in triplicate, at a multiplicity of infection of 0.01. All cultures were incubated at 32°C. After a 1-h adsorption period, the inocula were removed, and the cells were washed three times with serum-free OptiMEM I. The cultures were overlaid with 2 ml of the same medium per well. For rPIV3-2TM- and rPIV3-2TMcp45-infected cells, p-trypsin (0.5 μg/ml) was included. Aliquots of 0.5 ml were taken from each well at 24-h intervals for 6 days, flash-frozen on dry ice, and stored at −80°C. Each aliquot was replaced with 0.5 ml of fresh medium with or without p-trypsin as appropriate. The virus present in the aliquots was titered on LLC-MK2 plates by terminal dilution at 32°C for 6 days, and the endpoints were identified with hemadsorption. Virus titers are expressed as means ± standard errors. (B) rPIV3-2CT and rPIV3-2CTcp45, along with the wild-type parents rPIV3/JS and PIV2/V94 were analyzed as described for panel A.