Fig. 5. Virulence assay using the intranasal model of infection. Groups of five BALB/c mice were mock-infected or infected with (a) 107 or (b) 4 106 p.f.u. of the indicated purified viruses. Mice were weighed daily, and results are the mean percentage weight change of each group ± SEM compared to the weight on the day of infection (upper panels). Animals were monitored daily for signs of illness, scored 1 to 4 (Alcami & Smith, 1992) (lower panels). Data are expressed as the mean ± SEM. P values were determined using the Student’s t-test and indicate the mean percentage weight changes or signs of illness that were significantly different between groups. To study v-slfn-mediated virus attenuation, recombinant VACV expressing an irrelevant protein (vH1) or non- attenuating v-slfn (v-176NHA) were used as controls. Mice were infected with 46106 p.f.u. vH1, v176-WT or v176- NHA (Fig. 5b) and virus titres were measured in lungs (Fig. 6a) and spleens (Fig. 6b) of infected animals. Initially, all viruses replicated similarly in the lungs (day 3), but from day 5 there was a reduction in virus titre after infection with v176-WT, compared with v176-NHA and vH1. This differ- ence was significant at day 7 p.i. for both control groups (Fig. 6a). In the spleen, the v176R-WT titre was below the level of detection at days 3 and 7 p.i., unlike vH1 and v176- NHA, indicating that v176R-WT spread to this organ later and was cleared earlier than control viruses (Fig. 6b). The early clearance of virus after infection with v176-WT compared with control groups suggested a more effective antiviral host response, and therefore the cellular inflam- matory response in lungs was analysed by flow cytometry (Fig. 7). At days 3, 5 and 7 p.i., cells recovered from BALs and lung cells from mice infected with v176-WT contained an increased number of lymphoid cells compared with mice infected with control viruses, and this difference was significant at day 7 p.i. in BALs (Fig. 7a) and lungs (Fig. 7b). To test whether this increase in lymphoid cells was due to an enhanced recruitment of a particular lymphoid subset, the percentage of CD4+ and CD8+ T lymphocytes, B220+ B lymphocytes and DX5+ natural killer (NK) cells present in lungs was analysed, but no significant difference between http://vir.sgmjournals.org Camelpox virus schlafen protein Fig. 6. Virus titres of vH1, v176-WT and v176-NHA in (a) lungs and (b) spleens. Groups of five mice were infected intranasally with 4 106 p.f.u. of the indicated virus, and the lungs and spleen were harvested at days 3, 5 and 7 p.i. Virus titres were determined by plaque assay on TK”143 cells. Virus titres are expressed as the mean log10 p.f.u. per organ ± SEM. P values were determined using Student’s t-test and indicate the mean virus titres that were significantly different between mice infected with v176-WT and mice infected with v176-NHA and vH1. Broken line indicates the minimum detection limit of the plaque assay. groups of mice was found (data not shown). Next, the activation of CD3+ T cells was investigated using the activation marker CD69+. This showed that although on day 7 p.i. there were more lymphocytes recruited to lungs infected with v-176WT compared with infection with vH1 or v176-NHA, CD3+ T cells were less activated (Fig. 7c, left panel), and this was reflected by a lower level of the activation marker CD69+ on both CD4+ and CD8+ lymphocytes (Fig. 7c, right panel). There was no difference between groups in CD3+ CD69+ lymphocytes at days 3 and 5 p.i. (data not shown). Another activation marker, 1673