Mouse Hepatitis Virus 3 Infection Research Articles

Preliminary study on immunologic mechanism of chemokine receptor CXCR3 and its ligands involved in mouse fulminant hepatitis

Objective To investigate the role of the chemokine receptor CXCR3 and its ligands in the migration of lymphocytes and acute hepatic failure. Methods BALB/cJ mice (6-8 weeks, female) were intraperitoneally injected with 100 PFU mouse hepatitis virus-3(MHV-3). The proportions and numbers of T cells and NK cells in liver, spleen, and blood as well as the expression of CXCR3 in T cells, and NK cells post MHV-3 infection was analyzed by flow cytometry. The hepatic mRNA level of the CXCR3-associated chemokines(CXCL9 and CXCL10) was detected by real-time PCR. A transwell migration assay was used to assess the chemotactic effect of MHV-3-infected hepatocytes and CXCL10 on the splenic lymphocytes. Results Following MHV-3 infection, the number of hepatic NK cells and T cells and the frequencies of hepatic NK cells and T cells expressing CXCR3 increased markedly; however, in the spleen and peripheral blood, they both decreased significantly. Moreover, the hepatic mRNAs levels of CXCL9 and CXCL10 were significantly elevated post infection. The transwell migration assay demonstrated that MHV-3-infected hepatocytes have the capacity to attract and recruit the splenic NK cells and T cells, and CXCL10 plays a key role in lymphocyte mobilization from the spleen. Conclusion Interactions between CXCR3 and its ligands (CXCL9 and CXCL10),especially CXCL10 may play a key role in the recruitment of intrahepatic lymphocytes and subsequent necroinflammation and acute hepatic failure in MHV-3 infection. Key words: Chemokine; CXCR3; CXCL10; Fulminant hepatitis; Natural killer cell

Mouse Hepatitis Virus 3 Binding to Macrophages Correlates with Resistance to Experimental Infection

Mouse hepatitis virus 3 (MHV3) infection of A/J and BALB/c mice has been used as a model of resistance/susceptibility. A/J mice recover from a mild disease after 4–6 days of infection and the BALB/c mice develop an acute hepatitis and die after 3–4 days of infection. In view of studying the MHV3 binding to cells or cell extracts, we performed an enzyme‐linked immunosorbent assay‐like virus‐binding assay, preparing microplates with L929 cells, A/J or BALB/c mouse macrophages and also with proteins extracted from these cells. Higher MHV3 bindings were observed to proteins of BALB/c macrophages than to the A/J ones. The interferon‐γ (IFN‐γ) activation led to a reduction of MHV3 binding only to proteins of resistant A/J mouse macrophages. Our experiments contribute to the hypothesis that IFN‐γ activation of macrophages plays an important role against MHV3 infection by downregulating the expression of viral receptors.

Specific T-cell response correlates with resistance of genetic heterogeneous mouse populations to mouse hepatitis virus 3 infection.

In a recently published study [Vassão RC, Mello IGC, Pereira CA (1994) Arch Virol 137: 277-288] we have shown that the genetically selected high antibody responder mice (HIII) are susceptible and the low antibody responder counterparts (LIII) are resistant to death induced by experimental infection with mouse hepatitis virus 3 (MHV3). This report shows that the MHV3 titers in the peritoneal exudate (PE) of HIII mice, 3 days after infection, were more than 2 log greater than in the resistant LIII mice, the interferon gamma (IFN gamma) titers in the PE of both mouse populations being not significantly different. The treatment with monoclonal antibodies (mAb) against CD4+ or CD8+ T cells induced susceptibility among LIII mice. The depletion of CD4+ T-cell subset in LIII mice was evidenced by, and led to a significant reduction in, the IFN gamma synthesis in their PEs with a 100 fold increase in MHV3 titers. When lymph node cells (LNC) were harvested from MHV3-infected mice and stimulated "in vitro" with MHV3 inactivated by ultraviolet radiation (uv-MHV3), only LNC from LIII mice were capable of proliferating and synthesizing significant amounts of interleukin 2 (IL-2). The LNC proliferation and IL-2 synthesis were inhibited by treatment with mAbs against CD4 or CD8 molecules. The MHV3 infection induced in both lines of mice a profound depression of the mitogenic response of LNC to phytohemaglutinin (PHA). A correlation between the specific T-cell response and the resistance to MHV3 infection is discussed.

Role of macrophages, interferon gamma and procoagulant activity in the resistance of genetic heterogeneous mouse populations to mouse hepatitis virus infection.

SummaryGenetic heterogeneous mouse populations selected for high (HIII) and low (LIII) antibody response were used to study some aspects of mouse hepatitis virus 3 (MHV3) infection, such as the resistance pattern, virus replication in the liver and peritoneal exudate or in cultured peritoneal macrophages, the interferon (IFN) synthesis in the serum and peritoneal exudate and the procoagulant activity (PCA) of the peritoneal exudate (PEC) and spleen cells (SC). The HIII mice, when compared to their LIII mice counterparts, were susceptible to MHV3 infection showing higher virus titres in the liver and peritoneal exudate, comparable IFN alpha/beta or IFN gamma titres in the peritoneal exudate or in the serum, and higher levels of PCA of PEC and SC. A higher virus titre was detected in the supernatants of HIII mouse macrophages infected with MHV3. The activation of HIII mouse macrophages with LPS, IFN alpha/beta or IFN gamma, in contrast to that of LIII mouse macrophages, did not induce an antiviral effect with partial restriction of the MHV3 replication. The LPS antiviral activity was shown to be partially exerted by IFN alpha/beta synthesis. The IFN gamma was shown to be more effective in inducing an antiviral state in LIII macrophages, when compared to IFN alpha/beta. The data obtained are consistent with the notion that the resistance mechanisms to the MHV3 infection involve the PCA and the sensitivity of macrophages to IFN.

A genetic analysis of macrophage activation and specific antibodies in relation to the resistance of heterogeneous mouse populations to MHV3 infection.

SummaryThe genetically selected high antibody responder mice (HIII) are susceptible and the low antibody responder mice (LIII) are resistant to the experimental infection with Mouse Hepatitis Virus 3 (MHV3). The mortality rates of the F1 hybrids and of the F2 segregants showed the codominance of the susceptible and resistant characters. The direct individual intrapopulation correlation between the induction of antiviral state in macrophages activated by IFN gamma and the resistance to the virus infection, showed that an antiviral state could be induced in resistant mouse macrophages, whereas in susceptible mouse macrophages no restriction of virus replication could be observed. A direct inter- and intrapopulation correlation of pre-existing antibody titres against MHV3 with the mortality and a direct interpopulation correlation of those titres with the mean survival time of susceptible animals was shown. The data indicate, among the mechanisms of resistance against the virus infection, a role of IFN gamma macrophage-activation and of antibodies against MHV3 which may delay the mean survival time in susceptible animals.

TNF alpha, IL-1 and O -2 release by macrophages do not correlate with the anti-Mouse Hepatitis Virus 3 effect induced by interferon gamma

Macrophages have been described to be important in determining the resistance of A/J mice or the susceptibility of BALB/c mice to the experimental infection with Mouse Hepatitis Virus 3 (MHV3). The interferon gamma (IFN gamma) activation of A/J and BALB/c mouse macrophages was shown to partially restrict the MHV3 replication only in macrophages from the resistant A/J mice. The activation by IFN gamma and/or infection with MHV3 showed that BALB/c mouse macrophages were capable of releasing tumor necrosis factor alpha (TNF alpha), interleukin 1 (IL-1) and anion superoxide (O - 2), and A/J mouse macrophages were capable of releasing TNF alpha and IL-1 but not O - 2. Comparable amounts of TNF alpha or IL-1 were released by IFN gamma-activated A/J or BALB/c mouse macrophages. Following MHV3 infection or IFN gamma activation and MHV3 infection, BALB/c mouse macrophages were always capable of releasing higher amounts of TNF alpha, IL-1 or O - 2 than A/J mouse macrophages, which correlated with their susceptibility to the virus infection. The data indicate that the anti-MHV3 effect induced by IFN gamma in A/J mouse macrophages is not related to the studied extrinsic activities of these cells.

Resistance of genetically selected mice to MHV3 infection is not dependent on the H2O2 release by macrophages

The genetically selected high antibody responder line of mice (HIII) for a natural immunogen were fully susceptible to mouse hepatitis virus 3 (MHV3) and the corresponding low antibody responder mice (LIII) were fully resistant, regardless of whether they were previously treated or not with the bacillus Calmette-Guérin (BCG). The resistance or susceptibility correlated with the virus growth in the peritoneum of mice. Peritoneal cells isolated from resistant mice released higher amounts of H2O2 after phorbol myristate acetate (PMA) stimulation than susceptible mice. No spontaneous in vitro H2O2 release by peritoneal exudate cells from HIII mice, shown to be mostly macrophages, were observed during the MHV3 infection, In contrast, the spontaneous in vitro H2O2 release by these cells from MHV3-infected LIII mice increased gradually, reaching a maximal value 3 days after infection, and decreased in parallel to the virus clearance from the peritoneum. The BCG treatment primed the macrophages of HIII mice for the production of H2O2 during the MHV3 infection, but did not confer resistance against the virus infection. The data obtained suggest that the acquired capability of macrophages to release H2O2 does not participate in the anti-MHV3 activity or resistance against the MHV3 infection.

The pattern of proteins synthesized in the liver is profoundly modified upon infection of susceptible mice with mouse hepatitis virus 3

Susceptible BALB/c mice, after experimental infection with mouse hepatitis virus 3 (MHV3), revealed virus titres in the liver that increased gradually to a peak of 8 × 105 PFU/g of tissue after 3 days' infection, when the mice died of acute hepatitis. BALB/c mice were infected with MHV3, subsequently labelled in vivo with 35S-methionine, and then the liver preparations from both infected and non-infected animals were subjected to two-dimensional gel electrophoresis. Comparisons of the patterns by computer image analysis revealed 17 gene products which increased, and 8 gene products which decreased, upon virus infection in their two-dimensional gel spot intensity. We conclude that during MHV3 infection of a susceptible strain of mice, a major modification in protein synthesis occurs. The pattern alterations were not related to the virus gene products but were mostly endogenous mouse proteins. Whether these proteins are a result of a defence attempt by the animal, or are dictated by the virus in order to prevent a protective response from happening, remains to be shown.

Effect of cyclosporin A on an experimental chronic viral infection of the central nervous system

The effects of cyclosporin A (CsA) on neuropathological lesions induced by a chronic viral infection have been tested in the experimental model of the mouse hepatitis virus 3 (MHV3) infection. Daily injections of CsA (50 mg/kg) inhibited the expression of the MHV3-induced ependymitis, meningitis, hydrocephalus and vasculitis. The effect was preserved even if CsA treatment was initiated 15 days after virus infection but was lost if CsA treatment was given later on or for a shorter period of time. Viral titers in brains of chronically infected mice were not affected by CsA treatment. During the first week following MHV3 infection, CsA treatment increased both the percentage of acute death (31 vs. 10%) and the viral titers in brain and liver of infected mice. In this model, the timing of CsA treatment appeared critical for the balance between its beneficial effect on CNS lesions and the risk of increased acute mortality.

Virus-pesticide interactions with murine cellular immunity after sublethal exposure to dieldrin and aminocarb.

Interaction of two potential immunosuppressive factors, sublethal pesticide exposure and viral inhibition of lymphocyte mitogenesis, was examined in mixed lymphocyte reaction (MLR). Inbred (C57Bl/6 x A/J)F mice, semisusceptible to mouse hepatitis virus 3 (MHV3) infection were exposed to selected pesticides and subsequently infected with the MHV3 virus. The mortality of animals was examined as a function of pesticide exposure. Two pesticides were selected for further studies: the organochlorine pesticide dieldrin, which increased the cumulative mortality of animals, and the carbamate pesticide aminocarb, which did not affect the virus-induced cumulative mortality of animals. Spleen lymphocytes from dieldrin- and aminocarb-exposed C57Bl/6 mice (susceptible to MHV3 infection) were used as responder cells in one-way MLR. A marked immunosuppression of the MLR proliferative response was observed in the dieldrin group, whereas sublethal exposure to aminocarb did not affect the in vitro MLR response. The MLR cultures were subsequently infected in vitro with the MHV3 virus, which resulted in a time-dependent and virus dose-dependent inhibition of lymphocyte proliferation. However, no synergism was observed with the addition of either the MHV3 virus-induced inhibition of in vitro MLR lymphoproliferative response or dieldrin-related immunosuppression, since in vitro MHV3 infection of cells from dieldrin-exposed mice did not aggravate the dieldrin-related immunosuppression. In addition, no "hidden" aminocarb-related damage of the lymphoproliferative response was noted, as the kinetics of the virus-induced inhibition in the aminocarb group were analogous to the control. In conclusion, dieldrin-induced immunosuppression of the cellular immune response, rather than MHV3 virus-induced inhibition of lymphoproliferative activity itself, was the primary factor potentially responsible for the impaired cellular response. Furthermore, the data support the observation that cell-mediated immunity can be a potential target for the adverse effects of pesticide exposure.

Increased susceptibility to mouse hepatitis virus 3 of peritoneal macrophages exposed to dieldrin

Interaction of a single dose (36 mg/kg body wt) of the organochlorine pesticide dieldrin with mouse peritoneal macrophages was examined in C57Bl 6 , ( C57Bl 6 × A J )F 1 , and A J strains of different genetic resistance to mouse hepatitis virus 3 (MHV3) infection. In vivo studies showed increased susceptibility to MHV3 acute disease of C57Bl 6 and ( C57Bl 6 × A J )F 1 animals challenged with the pesticide. Significant decrease of mean time of death in dieldrin-exposed, MHV3-infected susceptible C57Bl 6 mice was observed similarly upon po or ip administration of a single, sublethal dose of dieldrin. In addition, decrease of humoral response to the virus was quantified by determination of anti-MHV3 IgG antibodies in spleen cell supernatant fractions and in blood sera of dieldrin-exposed C57Bl 6 mice. A single dose of dieldrin did not alter the in vivo resistance of A J animals to acute MHV3 disease. The resistant A J mice, however, showed increased mortality upon two subsequent exposures to dieldrin followed by infection with high lethal doses of MHV3. Phagocytic activity, cell adherence capacity, and attachment and uptake of 3H-radiolabeled MHV3 by C57Bl 6 peritoneal macrophages were determined by in vitro studies. These affector activities of peritoneal macrophages were slightly decreased or unchanged in cells originating from animals exposed to the pesticide. However, the intrinsic activity of MHV3 restriction appeared to be affected in macrophages derived from dieldrin-treated animals: (i) peritoneal C57Bl 6 macrophages collected from the early phase of acute MHV3 disease contained increased MHV3 antigen and (ii) increased cytolysis was observed after in vitro MHV3 infection of macrophages originating from dieldrin-exposed C57Bl 6 mice.

Natural resistance of mice to mouse hepatitis virus type 3 infection is expressed in embryonic fibroblast cells.

Mouse hepatitis virus 3 (MHV3) infection in mice varies according to the mouse strain used; they may show resistance, semi-susceptibility (paralysis) or full susceptibility (lethal acute hepatitis). In order to study the mechanism of inborn resistance, viral infection was carried out in primary cultures of embryonic fibroblasts originating from various mouse strains exhibiting different sensitivities to MHV3 infection. Virus-induced cytopathic effects and cell membrane antigens as well as virus replication and interferon synthesis were studied. Persistent infection was induced in four out of six primary embryonic fibroblast cultures and in six of six secondary cultures. Two primary embryonic fibroblast cultures from susceptible strains underwent total lysis. A high yield of virus was obtained, as tested by viral titres and cell membrane antigen detection. No significant difference in virus production or in interferon synthesis was observed in fibroblast cultures of the various mouse strains tested. Cytopathic effects characterized by cell lysis were related, however, to phenotypes in vivo. This effect was lost after a single trypsinization of the culture. In addition, resistance to virus-induced cell lysis was inhibited by actinomycin D treatment. These results indicate that the intrinsic resistance of fibroblasts is effective not against virus replication but against virus-induced cell lysis. Such a cellular control mechanism may be an important factor for resistance in vivo.