Induction of monocyte procoagulant activity by murine hepatitis virus type 3 parallels disease susceptibility in mice

Packaging, Cambridge, Mass.) in 1 ml DMEM fortified with 2% fetal calf serum and were infected at an MOI of 0.1 with MHV-3. Virus was adsorbed to the cells for 60 min at 37°C. Unadsorbed virus was removed by washing with DMEM, the media were replaced, and the cells were further incubated at 37°C. Cultures were frozen at appropriate intervals, treated as described above, and assayed for virus by plaque assay at serial 10-fold dilutions (Fig. 2) . In contrast to the growth of MHV-3 in a totally permissive cell line (17 CLI) , in BALB/c monocytes, a prolonged period of 16 h was observed postinfection, during which time no virus replication was evident. However, subsequent to the 16-h lag period, replication was progressive, with peak yields 24 h after infection. In contrast, no virus replication was detected in A/J or C3H/St monocytes within 48 h postinfection. These observations parallel the observations of acute fulminant hepatitis in BALB/c mice and resistance of A/J mice to overt hepatitis or death. Basal Cellular PCA. The total cellular content of PCA immediately after isolation of cells and before any form of in vitro culture was determined for A/J, BALB/c, and C3H/St mice and was remarkably consistent at 56-81 mU/106 PBM. This activity rises spontaneously to a maximum stable concentration of 515 mU/106 PBM within 6 h of culture in serum-free medium in the absence of added stimulus (Table II) . When the control cultured cells were fractionated by adherence into lymphocytes and MHV-3 Induction of PCA. PBM were isolated from each of the mice strains, and 1 X 108 PBM were incubated with 106 PFU of MHV-3 for 18 h at 37°C in 1 ml of RPMI 1640 supplemented with 2% heat-inactivated FCS at 37°C. The cells were washed three times with RPMI 1640, disrupted, and assayed for total PCA. As a positive control, 1 × 106 PBM from each strain was stimulated with Escherichia coli 011 l:B4 LPS at 10 #g/ml, as previously described (16) . Cells from all three strains responded to LPS with a six-to eightfold increase in PCA (Table III) , in accord with previous observations (16) . The PCA response of BALB/c PBM to 106 PFU MHV-3 was greater than previously observed for any stimulus, reaching levels of >70,000 mU/106 PBM (Table III) . In contrast to the profound PCA response of BALB/c PBM, no response to MHV-3 was observed in PBM from A/J mice, even though a normal PGA response was observed to LPS stimulation. A moderate PCA response was observed for PBM from C3H/St mice stimulated with MHV-3, but the response was only 70% of the response observed for LPS stimulation (Table III) . Thus, the PCA response of PBM to MHV-3 stimulation exhibited profound strain differences. The production of the PGA by PBM correlates with the susceptibility of the strain to hepatic disease. Furthermore, the PCA response of BALB/c mice was of a magnitude 15-20 times greater than that observed for the response to LPS or antigen-antibody complexes (14) (15) (16) . No increase in PCA above basal levels was observed when 1 X 106 PBM from BALB/c, A/J, or C3H/St mice were cultured with an aliquot from mockinfected DBT, L2, or 17 GL 1 cells (data not shown). Dose-response titrations were examined for MHV-3-induced PCA in which 1 x 106 PBM from BALB/c mice were incubated for 6 h with MHV-3. When 1 PFU of MHV-3 was used, a fourfold increase in PCA as compared with unstimulated control cultures was observed (Fig. 3) . A dose-dependent increase of PCA achieved a 168-fold maximum increase of