Because only a limited number of cells (and virus particles) can be analyzed in this fashion, 
another strategy was employed to detect low levels of replication-competent virus. SFV3- 
lacZ/Helper 1 packaged virus stocks were used to infect BHK cells (lxlO 6 ) at MOI of 1-10, and 
infection allowed to proceed for up to 24 hours (Table 1). At 9 hours post-infection, the 
medium was collected and titrated for plaque-forming units (PFU), evidence of replication- 
competent virus. From a total of four individual experiments, 70 to 1000 PFU were detected, 
indicating that a low level of recombination ( < 10" 6 ) had occurred between the helper and SFV3- 
lacZ RNAs. 
Table 1 
Production of Replication-Competent Virus 
from Packaged Recombinant Virus 
Plaque-forming Units 1 
Helper 1 
Helper 2 
(SQL) 
Unactivated 
70-1000 
0,0 s 
Activated 
ND 4 
0,0 s 
lumbers of replication-proficient virions, detected as plaque-forming units (PFU), by 
infecting 10 6 BHK cells with packaged recombinant viruses, at MOI of 1-10, with or 
without protease activation. Helper 1 was assayed after 9 hours of culture; Helper 2 
after both 9 hours and 24 hours, corresponding to the two values given. 
2 Cytopathic effects were totally absent. 
3 All cells demonstrated active viral replication (CPE) and eventual cell death. 
4 ND: Not Determined. Helper 1 virus is infectious without activation. 
To obtain a more precise measure of the frequency of recombinants formed from 
SFV3-lacZ and Helper 1 RNAs under standard packaging conditions, the 
following experiments were performed. Confluent plates of BHK cells (2.5x10^ 
cells per plate) were infected with a stock of pSFV3-lacZ/Helper 1 packaged 
virus, using 10 4 , 10 5 or 10 6 particles per plate. In all, sixteen different virus 
stocks were evaluated. All tests were done in duplicate for each stock. After one 
hour, the cells were overlayered with agarose (dissolved in complete medium) and 
incubated for two days, after which the plates were stained for plaques. The 
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Recombinant DNA Research, Volume 18 
