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MARÍA ÁNGELES GARCÍA Y COLS. AN. R. ACAD. NAC. FARM.

while in the presence of IPTG virus yields were inhibited. Induction
of PKR in TRAMP-C1 cells infected with VV-PKR could be due to leak-
iness of the promoter or as a result of the viral infection.

The above findings establish that VV-PKR can be used as a vector
to produce low levels of PKR in tumour TRAMP-C1 cells.

3.2. Attenuation of VV-PKR in infected mice

To evaluate whether low level expression of PKR could be biolog-
ically active leading to virus attenuation, we performed pathogenici-
ty studies in mice, by measuring survival, virus titration in tissues and
humoral immune responses to the virus. Thus C57/BL6 mice, 4 ani-
mals per group, were injected either by intraperitoneal (i.p.) or in-
tranasal (i.n.) routes with 5x107 plaque forming units (pfu) of either
VV-LUC, VV-PKR and VV-PKR(K296R) or with a solution of PBS.
Several signs of viral illness, like ruffled fur, rigidity, lack of activity
and mortality were evaluated with time of infection. As shown in
Figure 2A, i.n. inoculation of VV-LUC results in severe illness and the
animals had to be sacrificed by day 6 due to the loss of 30% of their
body weight, while animals that received VV-PKR recovered the
weight loss by day 7 after viral infection. Animals inoculated with VV-
PKR(K296R) behaved similarly as VV-LUC (not shown). The signs of
illness correlated well with virus abundance in lung tissues, with about
5 log reduction at day 3 in mice inoculated i.n. with VV-PKR in com-
parison with VV-Luc (Figure 2A, lower panel). When animals were in-
oculated with the different viruses by i.p. routes, VV-Luc or VV-
PKR(K296R) induced weight loss while infection with VV-PKR did
not (Figure 2B); this correlated with lower virus titers in the spleen
compared with animals administered with VV-LUC or VV-PKR
(K296R) (Figure 2B, lower panel). To provide further support for re-
duced replication of VV-PKR in mice, we evaluated by ELISA the an-
tibody response against the viral recombinants at 30 days after i.p. in-
oculation with the different viruses. As expected, VV-PKR induced
lower antibodies against VACV antigens than either VV-LUC or VV-
PKR(K296R) (Figure 3). The findings of Figures 2 and 3 establish that
VV-PKR is attenuated in mice when inoculated by i.n. or i.p. routes
and induces low levels of antibodies against the virus vector.

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