Using 1 x 10 8 packaged SFV3-lacZ/Helper 2 (activated), we infected 1 x 10 cells of the 
following types: H293 (human), COS (monkey), and CHO (hamster). Supemates were 
collected after 36 and 72 hours. Thirty percent of the supemates of each culture then 
was used (nonactivated) to infect BHK cells, and we assayed for plaques as evidence of 
replication-competent virus. None of the three cultures revealed any plaques. 
In vivo Studies 
Although the cDNA clone (SFV4) used to construct the present SFV expression system 
was derived from an attenuated variant of the prototype SFV strain L10, SFV4 virions 
are still relatively virulent when administered to neonatal mice. In vivo studies thus 
provide a highly sensitive indicator of the presence of replication-competent virus in a 
stock of packaged virus, as well as a test of the propensity of the Helper 2 constructs to 
revert the SQL mutation and/or to undergo recombination to form wild type virus in the 
complex cellular milieu of the whole animal. 
Therefore, each of the SFV constructs discussed earlier was tested by intracerebral (i.c.) 
injection into neonatal BALB/c mice (Table 9). The SFV-SQL virus, when injected 
(2.5xl0 7 IU) in either the nonactivated form or the activated form, killed all of the 
injected animals. 
SFV-lacZ/Helper 1 virus (lxlO 7 IU) also proved relatively virulent, rapidly killing four 
of the nine animals injected. But considering that 10 7 IU were innoculated, and that the 
frequency of recombinants (Table 2) is approximately 10"*, it is surprising that less than 
half of the animals were killed. A reasonable explanation is that the recombination event 
disrupts the viral genome, resulting in virus that is attenuated, compared to wildtype 
SFV4. 
In marked contrast to the above viruses, SFV-lacZ/Helper 2 virus injected i.c., activated 
or nonactivated, killed none of the 24 injected animals (1 x 10 7 IU per animal, 2.4xl0 8 
IU, total). 
When the same set of viruses was injected intranasally (i.n.) into 40-day-old mice, at a 
ten-fold lower dosage of virus, none of the mice was killed. That all of the mice injected 
with SFV-SQL acquired resistance to challenge by a lethal dose of SFV strain L10 and 
that two-thirds demonstrated brain lesions, indicates that some level of virus replication 
occurred in these animals. But based on the measured reversion frequency (10"*) of the 
SQL phenotype (Table 4), i.n. injection of 2.5 x 10 6 IU would be expected to result in 
revertants far exceeding in number the LD^ for SFV4, (10 IU). This apparent 
attenuation may reflect a lowered cleavage efficiency of the second-site revertants. 
In contrast, neither SFV-lacZ/Helper 1 virus nor SFV-lacZ/Helper 2 virus (injected i.n.) 
demonstrated evidence of replication (2.5 x 10 6 IU per animal, 3.3 xlO 7 IU total). 
Recombinant DNA Research, Volume 18 
[4571 
