. Furthermore, using recombinant inbred strains of mice, we showed that genetic linkage between resistance/susceptibility to MHV-3 infection and induction of PCA was controlled by two non-H2-1inked recessive genes (5) . After infection with MHV-3, disturbances of the hepatic microcirculation associated with sinusoidal thrombosis occurred coincident with the rise in PCA (6, 7) . Intravenous infusion of macrophages, induced to express high amounts of PCA by MHV-3, resulted in rapid (2-5 min) death from disseminated intravascular coagulation in both susceptible and resistant mice (our unpublished observations). Together, these observations suggest that coagulative necrosis occurring as a result of induction of PCA may be a crucial feature of MHV-3-induced hepatic injury.
We have recently produced a panel of mAbs to MHVinduced macrophage PCA (8) . The antibodies did not react with purified viral proteins nor did they inhibit viral replication. One of these mAbs (3D4. 3) , an IgG2ak, strongly inhibited PCA expression in a one-stage dotting assay and inhibited conversion of prothrombin to thrombin (8) . This antibody had no reactivity with murine, rabbit, or human tissue factor. The monoclonal bound to a 70-kD protein that is distinct from murine and human tissue factor (42 kD) (9, 10) .
The purpose of this study was to determine whether treatment with this specific murine antiprothrombinase would modify the morbidity and/or mortality associated with murine hepatitis virus infection.
Virus. The origin and growth of MHV-3 have been previously described (2) . MHV-3, obtained from the American Type Culture Collection (Kockville, MD) (ATCC-VR262), was plaque purified on monolayers of DBT cells. Stock virus was grown to a titer of 2.8 x 107 PFU/ml in 17 CL1 cells. The virus was harvested by one cycle of freeze-thawing and clarified by centrifugation at 4,500 g for 1 h at 4~ Virus was then assayed on monolayers of L2 cells in a standard plaque assay as previously described (2) . To induce MHV-3 infection, each mouse received 103 PFU of MHV-3 by intraperitoneal injection.
Mice. BALB/cJ mice 8-10 wk of age, were obtained from Charles River Laboratories, (St. Constant, Quebec). Animals were housed in the D class facility at the University of Toronto fed a diet of standard chow and water ad libitum. Mice were killed on days 1, 3, 5, 7, 10, and 14 after infection. Blood was obtained by axillary bleeding. Splenic mononuclear cells were harvested and assayed for PCA both by immunofluorescence staining and in a onestage clotting assay. Livers were harvested for viral titers, histopathology, and PCA by immunofluorescence staining as described below. All mice used were screened by an ELISA for exposure to MHV, and were found to be negative (11) .
Anti-PCA mAt~ The preparation of mAb 3D4.3 has been previously described (8) . After injection of 106 hybridoma cells into Pristane-primed CAF1 mice, ascites was harvested. Animals were treated with ascitic fluid containing 25, 50, or 100 #g of mAb daily for 7 d preinfection and 7 d postinfection (p.i.) by intraperitoneal injection. As a standard control, 100 ~1 of ascites from animals that were infected with SP2 myeloma cells was injected into six separate mice. All ascitic fluid whether from SP2-or 3D4.3injected mice had no reactivity with MHV-3 either in the ELISA or plaque reduction assays (8) . To ensure that the protective effect of ascites containing mAb to PCA was specific, mAb to PCA (3D4.3) was isolated from hybridoma supematants by affinity chromatography using goat anti-mouse IgG immobilized on Sepharose 4B (Pharmacia, Montreal, Quebec). Immunoblot analysis of the column eluate confirmed that IgG was the only Ig present. Subclass analysis by ELISA, as previously described (8) , confirmed that IgG2ak was the only subclass present. 100 #g of the purified mAb was injected into 10 mice for 7 d preinfection and for 7 d p.i. as described above.
Histology. Histology was assessed by a blinded observer as previously described (8) . Briefly, li~r were cut into 1.0 x 0.2-cm blocks and fixed by immersion into 10% formalin in 0.1 molar phosphate buffer, pH 7.4. After fixation, the tissue was dehydrated in graded alcohols and xylene, then embedded in paraffin. 4-#m sections were cut, stained with Harris' hematoxylin for 4 min, and counterstained with eosin Y for 30 s. The sections were then washed with distilled water, dehydrated in graded alcohols and xylene, and mounted with Parmount. For each group five animals were used. To quantitate the effect of the mAb on liver histology, a digitalized image analysis system (HP-88; Hewlett Packard Co., Ltd., Mississauga, Ontario) with customized software was used. This constitutes a modification of a technique described previously (12) . The areas of necrosis were encircled as well as the entire section yielding a percentage figure representing the proportion of diseased liver present in that particular section. For each animal, three random sections were assayed in this fashion, and the mean +_ SD was calcuhted. Hbrin deposition was assessed by the Morris-Lendrum Hcro-Mallory stain as previously described (13) . Viral Titers. Livers that had been snap frozen at -70~ were homogenized in DMEM supplemented with 2% FCS and 4 mM glutamine as a 10% homogenate at 4~ as previously described (14) . Viral titers of liver homogenates were then determined on monolayers of L2 cells in a standard plaque assay (2, 13) .
Spleens were harvested aseptically and cells teased from splenic tissue and suspended in 8 ml of DMEM as previously described (15) . SMNC were isolated over Ficoll-Hypaque gradients (density, 1.0749) (Pharmcia) by centrifugation at 1,800g for 12 rain at 22~ Cells at the interface were collected. Viability was >98% as assessed by trypan blue exclusion. Cells were washed three times and resuspended in DMEM at a concentration of 4 x 106 SMNC/ml. Procoagutant Activity. Samples of frozen thawed SMNC were assayed for the capacity to shorten the spontaneous clotting time of human citrated platelet-poor plasma in a one-stage clotting assay as previously described (12) . Equal volumes (80 #1) of the cellular homogenate were admixed with citrated normal human plateletpoor plasma, and then 80 #1 of 25 mM CaClz was added at 37~ to start the reaction. The time in seconds for the appearance of a fibrin gel was then recorded. To establish arbitrary units, a rabbit brain thromboplastin standard at 36 mg dry mass/ml (Dade Division, American Hospital Supply, Miami, FL) was assigned a value of 100,000 mU. The assay was used over the range of 1-100,000 mU, and the results were linear with normal human plasma substrate. Data were converted to PCA per 10 6 splenic macrophages and expressed as the mean and standard deviation from six mice done in triplicate. Media and buffers were all without activity in this assay.
Inhibition of Viral Replication. mAb purified from hybridoma cell cultures containing 3D4.3 and ascites were assayed for their ability to inhibit replication of MHV-3 in a standard plaque reduction assay as previously described (8) . Briefly, 100 PFU of virus was admixed with dilutions of purified antibody or ascites, media as a negative control, or a high titered anti-MHV-3-neutralizing antibody as a positive control, for 30 min at 4~ The mixture was then added to a monolayer of L2 cells in culture medium, overlayed with 2% agarose, and incubated at 37~ in a 5% CO2 environment for an additional 48 h. The effect of antibody on viral replication was assessed by reduction of viral plaques (8) .
Immunofluorescence. Blocks of liver tissue were snap frozen in liquid nitrogen. Cryostat sections ('~4 #m thick) were fixed for 5 rain in acetone and air dried for 2 h as previously described (14) . Unoccupied sites were then blocked with 5% horse serum in PBS, pH 7.4, for 2 h. mAb 3D4.3 was conjugated with FITC (Sigma Chemical Co., St. Louis, MO) according to the method of Thi and Feltkamp (16) . The fluoresceinated antibody did not react with normal liver or uninduced normal peritoneal macrophages. Tissues were then stained with FITC-conjugated mAb 3D4.3 for 1 h at room temperature, washed three times, mounted in 90% glycerol in PBS, and viewed on a phase-epifluorecence microscope equipped with a x40 Fluotar objective (E. Leitz, Inc., Rockleigh, NJ).
ELISA for Anti-PCA Antibody. Titres of antibody to PCA were determined in a standard ELISA as previously described (8) . 96well enzyme immunoabsorbant assay (EIA) plates (Dynatech, McLean, VA) were coated with 50/~l/well of MHV-3-stimulated PCA-positive macrophage membranes or unstimuhted PCAnegative membranes (4 x 10 ~ macrophage/ml) at 4~ overnight. The plates were then washed three times with PBS, pH 7.5, conraining 0.05% Tween 20 (washing buffer), and the unoccupied sites were blocked with 200/zl of 5% Ig-free horse serum (Flow Laboratories, Mississauga, Ontario) that had been dissolved in washing buffer for 2 h at room temperature. 50-/~l/well dilutions of sera from treated animals were then added to the plates and incubated for 1 h at 37~ After washing three times, 50 #l/well of alkaline phosphatase-conjugated goat anti-mouse Ig in PBS containing 0.1% BSA and 0.05% Tween 20 was added for I h at 37~ After three washings, P-nitrophenyl phosphate in 0.1 M 2-amino-2-methyl-1, 3-proponediol buffer, pH 10.3 (Zymed Laboratories, San Francisco, CA), substrate was then added. The plates were then incubated at 22~ for 3 h and read at 405 nM with a plate reader (Titertek MCC/340; ICN/Flow, Mississauga, Ontario). Antibody levels were expressed (/~g/ml) by comparison to a standard curve.
StatisticalAnalysis. Statistical analysis was carried out using analysis of variance and the Wilcoxon ranked sum test. A p value of 0.05% or less was considered statistically significant.
Suwival. Mice infected with 1,000 PFU ofMHV-3 (n = 16) all succumbed to the infection within 5 d (Fig. 1) . This was consistent with previous data reported by our group as well as others (1, 2) . In contrast, there was survival in some animals that were treated with ascites containing antibody to PCA (Fig. 1 ). Animals treated with ascites containing 25 /~g/d had a 22% survival; 44% of those treated with ascites containing 50 #g/d and 100% of animals treated with 100 /~g/d survived. All animals survived when treated with 100 /~g of monodonal anti-PCA (IgG2ak), which had been purified from hybridoma superuatant, confirming that the beneficial effect of ascites was due to the monoclonal anti-PCA. Mice immunized with 100/~1 of ascites from SP2injected animals and infected with MHV-3 all died within 5 d (data not shown).
Liver Histology. MHV-3-infected mice that did not receive antibody developed histologic evidence of severe liver disease. By 24 h p.i., small, discrete loci of necrosis with a sparse PMN infiltrate could be seen. At 48 h, these lesions became both more pronounced and more numerous (Fig. 2 A) , and by 72-96 h, confluent liver necrosis was apparent (Fig. 2 B) .
In contrast, mice infected with MHV-3 but treated with antibody to PCA showed a marked reduction in liver disease in all groups (25, 50 , and 100/.r ( were a few small loci of inflammatory cells with no necrosis (Fig. 2 D) . Morphometric image analysis showed that the proportion of the liver that was necrotic was significantly different between the MHV-3-infected, anti-PCA-treated, and untreated groups at 48, 72 and 96 h (Fig. 3) . The histolog 7 of livers from all survivors at 10 and 14 d postinfection appeared normal. Fibrin deposits were seen in hepatic sinusoids as well as in areas of necrosis of untreated and MHV-3-infected mice (Fig. 4) . In animals treated with 25 and 50/~g of anti-PCA, a marked reduction in fibrin was noted. No fibrin was seen in the livers of infected mice treated with 100/~g of mAb. Treatment with anti-PCA alone resulted in no detectable histological evidence of liver disease in nonirLf~ed animals.
Viral Titers. By 24 h p.i., large amounts of infectious virus were recovered from liver homogenates of MHV-3-infected and untreated animals, and these persisted until the death of the animals (Fig. 5) . In animals treated with 25 and 50/~g of antibody to PCA, there was no significant difference in viral titers from those observed in untreated mice at days 1 and 3. Viral titers remained high at day 5, however, viral titers decreased by day 7 and no virus could be detected after day 10. In animals treated with the highest dose of antibody to PCA (100 #g/d), viral titers were markedly reduced (p <0.05) and approached those seen in resistant A/J mice (3, 5) .
Using the method of Reed and Meunch, the MHV-3 recovered from infected and antibody-treated animals was as pathogenic as stock MHV-3 or virus recovered from infected and untreated mice (data not shown) (17) .
PCA. In untreated, but MHV-3-infected animals, a sharp increase in splenic macrophage PCA was noted at 24 h p.i. (Fig. 6) . Maximal PCA was seen at 48 h and PCA remained elevated until the animals' death on day 4. Animals treated with low concentrations of antibody to PCA (25 and 50/zg/d) expressed high amounts of PCA at early time points, but by day 7, PCA levels fell and only basal levels of PCA were detected in SMNC by day 10. In contrast, no expression of PCA could be detected in animals treated with 100/zg of antibody to PCA during the course of infection (Fig. 6 ).
Immunofluorescence. By direct immunofluorescence staining, PCA could be detected at 24 h in livers from MHV-3- infected and untreated mice. PCA was seen in areas of inflammation and necrosis, and also in hepatic sinusoids, localized primarily in endothelial cells and Kupffer cells, but not expressed by hepatocytes (Fig. 7) . Livers from animals treated with antibody to PCA had significantly less PCA expression, although small amounts of PCA could be seen in macrophages and endothelial cells in hepatic sinusoids in animals treated with high-dose antibody to PCA (100 ~tg/d), even as late as day 14 (Fig. 7) .
Antibody to PCA. Sera were collected from animals at all time points and analyzed in an ELISA for the presence of antibody to PCA as previously described (8) . No antibody to PCA could be detected in sera from normal control animals or in animals infected with MHV-3 who had not received antibody to PCA (data not shown). Sera from animals treated with ascites containing 25-100/zg of antibody to PCA contained large amounts of anti-PCA before MHV-3 infection. After MHV-3 infection, the concentration of circulating antibody fell, but remained at >100 ng/ml in animals treated with 100/~g/d. In animals that were treated with 25 and 50/zg of antibody, by day 7, antibody was undetectable (<1 ng/ml) (Fig. 8) . Antibody to PCA either in ascites or purified from supernatants did not neutralize MHV-3 in a standard plaque reduction assay as previously described (8) To determine whether treated animals that had survived the acute infection developed long-term resistance to MHV-3 infection, mice previously infected with MHV-3 and treated with 100/~g of antibody (n = 15) were infected with 1,000 PFU of MHV-3, 21 d after their last exposure to virus. PCA antibody titers in this group were ~5 0 % of that present during antibody therapy, consistent with known rates of disappearance of IgG. The MHV-rechallenged mice experienced a 50% mortality rate, suggesting that protection was by passive immunization of antibody, not an acquired, active immune process (Fig. 9 ). In addition, no antibody to MHV-3 was detected in these mice either before or after rechallenge with virus.
MHV-3 infection produces fulminant hepatic failure and death in BALB/cJ mice (1, 3) . The availability of a mAb to PCA that neutralized acceleration of coagulation in vitro (8) provided us an opportunity directly to examine the role of PCA in vivo in murine hepatitis virus strain 3 infection. All BALB/cJ mice which were infected with 103 PFU of MHV-3 developed severe liver disease and died within 96-120 h. Administration of the mAb to PCA attenuated the hepatic necrosis, fibrin deposition and enhanced survival in a dosedependent manner. All of the animals receiving 100/~g, and 44% and 22% of the animals that received 50 and 25/~g/d, respectively, survived for 10 d and made a full recovery. Furthermore, the same protective effect was seen in mice treated with IgG2ak purified from hybridoma culture supernatants (3D4.3). The increased survival of the treated animals was specific for anti-PCA, since ascites from mice injected with SP2 cells alone, which contained no antibody to PCA, failed to protect mice from MHV-3 infection. Although antibodytreated, MHV-3-infected animals demonstrated clinical evidence of viral hepatitis early in the course of the infection, by 7-10 d their behavior appeared normal and liver sections showed little or no apparent disease. Examination of the livers of antibody-treated animals showed marked reduction in hepatic necrosis and inflammatory cells (neutrophils and mononuclear cells), each of which are prominent features of MHV-3 infection (3, 6, 7) . PCA expression is a feature of MHV-3 infection in this mouse strain (2) (3) (4) , and in untreated MHV-3-infected mice, PCA was detected by immunofluorescence staining in areas of necrosis as well as in hepatic sinusoids. Both macrophages and endothelial cells expressed detectable PCA, but hepatocytes did not. It is likely that the hepatic necrosis is secondary to ischemic changes resulting from induction of PCA, leading to the deposition of fibrin. Administration of mAb resulted in a dose-dependent reduction in the deposition of fibrin and in expression of PCA as detected by immunofluorescence staining in the liver and by a direct functional assay in SMNC.
Previously, we have shown that MHV-3 infection of peritoneal macrophages in vitro results in the production of PCA, TNF, leukotriene B4 (18) , and IL-1 (19) . Thus, expression of PCA by endothelial ceils might either be due to direct induction by MHV-3 or due to induction by IL.1 and/or TNF, which have previously been shown to induce PCA in endothelial cells in vitro (20, 21) .
Treatment of mice with 100/~g of mAb to PCA not only increased survival and reduced hepatic necrosis, but also resulted One possible explanation for the decrease in viral replication observed in mice treated with antibody 3D4.3 is that this antibody reacts with the MHV receptor recently described by Holmes and coworkers (22, 23) . We regard this explanation as unlikely, since 3D4.3 does not show any neutraliza- tion of viral infectivity in a plaque reduction assay, whereas antibody to the MHV receptor does inhibit infectivity. Furthermore, the MHV receptor has a molecular mass of 110 kD, considerably different than that of the PCA molecule (74 kD). A second possible explanation for the effect of anti-PCA antibody on MHV-3 growth in mice is related to the normal cleavage of the MHV S protein. It has been shown that cleavage of S by proteases is necessary to activate the membrane-fusing properties of the S protein (24) . This fusion property facilitates the spread of virus to uninfected cells by cell-cell fusion and also increases the specific infectivity of MHV when compared with virus in which S has not been cleaved (24) . It is possible that in infected macrophages, PCA, a serine protease, mediates at least in part the proteolytic cleavage of S into $1 and $2, and thereby activates the fusion properties of this molecule. Consistent with this idea is the observation that infection of A/J macrophages, which do not produce PCA in response to MHV infection, does not result in the appearance of syncytial giant cells (our unpublished observations). Thus, antibody to PCA could inhibit the spread of virus to uninfected cells by decreasing the activation of the fusion properties of S. Although the mechanism by which anti-PCA treatment protects the susceptible animals is not clear, the use of antibody to PCA neutralizes PCA, thereby preventing activation of the coagulation system and inhibiting fibrin formation. The antibody could also result in complement-mediated destruction of MHV-3-infected cells that express membranebound PCA or promote macrophage activation with restriction of viral growth. In animals treated with 100 #g of antibody for 7 d before infection and 10 d p.i., concentrations of antibody to PCA in sera fell from 87/~g/ml before MHV-3 infection to 100 ng/ml during the active phase of disease (day 5), consistent with sequestration perhaps due to binding of the Ig to cells expressing PCA. Furthermore, in these mice, levels of PCA in SMNC remained at basal levels. In animals treated with lower amounts of antibody (50 or 25 #g/d) to PCA, hepatic necrosis and survival were only partially attenuated, and antibody was not detected (<1 ng/ml) after day 7 p.i. Together, the data strongly support the notion that anti-PCA antibody neutralizes PCA in vivo during the infection and that this may be the basis for its protective effect.
In a previous report we have demonstrated that dimethyl prostaglandin E2 inhibited procoagulant activity and prevented fulminant viral hepatitis, yet all animals still succumbed to the infection (13) . In PGE-treated mice, viral replication proceeded at a rate similar to that in untreated animals. Recently, we have demonstrated that although PGE2 inhibited functional PCA, antigenic expression of PCA was not altered as determined by Western immunoblotting (25) . We have proposed that PCA may exert its effect through activation of the coagulation system with microvascular and macrovascular thrombosis (6, 7), but the present results, in concert with the inhibition studies of PCA by PGE2, suggest that PCA has other sites of action as well. The protective effects of the mAb to PCA occurred even though the BALB/cJ mice failed to mount an antiviral humoral response. Our ob-servations are consistent with a recent report by Korner et al. (26) and support the notion that the murine antiviral antibody response may not be required for protection from acute viral infection.
Cytokines can play a potent role in the course of inflammatory injuries in vivo, and interference with their action can alter the course of certain inflammatory diseases (27, 28) . Treatment of rats with recombinant antibody to TNF has been shown to protect animals from the hypotension, hypothermia, and mortality of Gram-negative sepsis (29) , and treatment of rabbits with an IL-1R antagonist reduced the mortality associated with endotoxin shock (30, 31) . Our studies support the notion that induction of PCA during MHV-3 infection in mice is an integral and potentially central step in the disease. A previous report by Taylor et al. (32) demonstrated that lethal Escherichia coli septic shock can be prevented by blocking tissue factor (a distinct procoagulant) with mAb, demonstrating the importance of cellular coagulants in the pathophysiology of other infectious diseases. We conclude that PCA is a potent inflammatory mediator that plays a pivotal role in hepatic injury resulting from MHV-3 infection. 