Table 5 
Leakiness of Helper-2 Packaged Virus Stocks* 
Stock (titre b ) 
MOP 
LacZ + cells/IU d 
1 (1.2 x 10 9 /ml) 
95 
1/1.0 x 10 6 
2 (2.0 x 10 8 /ml) 
5 
1/5.6 x 10 5 
3 (2.5 x 10 8 /ml) 
6,5 
1/2.3 x 10 5 
3 (2.5 x lO'/ml) 
13 
1/2.3 x 10 s 
4 (0.7 x 10 8 /ml) 
3.6 
1/4.0 x 10 5 
4 (0.7 x 10 8 /ml) 
4.2 
1/3.7 x 10 5 
•Four different stocks were tested for infectivity without prior chymotrypsin treatment. 
•Titre of stock after activation; determined by I.F. 
'Refers to activated particles. 
d Number of infected cells relative to titer of activated stock. 
b. FREQUENCY OF REPLICATION-COMPETENT HELPER 2 VIRIONS 
In Vitro Studies with BHK Cells 
Critical to the biosafety of a recombinant SFV expression system is that few or 
no replication-competent virus be produced during the packaging of virions and 
subsequent infection of cells in culture. Based on the low frequency of 
recombinants formed between helper and vector RNAs and the low rate of 
reversion of the SQL phenotype, one would expect that the occurrence of both 
events in the same molecule would indeed be very rare. 
An initial test of the SFV3-lacZ/Helper 2 system was performed using 10 6 BHK 
cells infected at a MOI of 1-10 with activated and unactivated virus particles. In 
four individual experiments, culture supemates were collected and titered for 
plaque-forming units (PFU). None was found in any of the samples (Table 1). 
In contrast, low levels of PFU were evident with the Helper 1 construct, as noted 
earlier. Sampling culture supemates permits highly sensitive detection of even 
very low numbers of replication-competent virus, because with every replication 
cycle, the number of virus present is amplified a thousand-fold or more. 
Recombinant DNA Research, Volume 18 
[453] 
