Infection of BALB/cByJ mice with the JHM strain of mouse hepatitis virus alters in vitro splenic T cell proliferation and cytokine production.

Earlier studies from this laboratory showed that infection of BALB/cByJ mice by a natural route with mouse hepatitis virus, strain JHM (MHV-JHM), results in functional splenic T cell suppression in vitro. This was evidenced by reduced concanavalin A-driven spleen cell proliferation and interleukin (IL)2 production measured after conventional intervals of cell culture (72 and 24 h, respectively). The purpose of the present work was to determine whether MHV-induced T cell dysfunction is kinetic or absolute and whether production of other T-cell derived cytokines is defective. Bioassays revealed that production of IL2, gamma interferon, and IL4 by spleen cells from acutely infected mice is suppressed and that some of the defects are kinetic as well as absolute. Proliferative responses of both CD4+ and CD8+ T cells were depressed, but neither cell type contained infectious virus. Cells that proliferated poorly in response to concanavalin A were fully capable of responding to specific virus stimulation. These results further emphasize the potential complications that MHV infection may pose to immunologic research using mice.     

Characterization of accessory cell function during acute infection of BALB/cByJ mice with mouse hepatitis virus (MHV), strain JHM.

Earlier studies revealed defective concanavalin A-stimulated proliferation and cytokine production by spleen cells derived from BALB/cByJ mice acutely infected with mouse hepatitis virus (MHV), strain JHM. Based on those observations, assays of in vitro antigen-presenting cell (APC) function were undertaken. APC function of unfractionated spleen cells from individual MHV-infected mice was highly variable. Experiments using pooled spleen cells derived from MHV-infected mice revealed that adherent spleen cell APC function was impaired to a much greater degree than B cell APC function. Adherent cells derived from peritoneal exudates of infected mice exhibited an APC defect that was similar in magnitude to that observed for splenic adherent cells. Splenic B cells derived from acutely infected BALB/cByJ mice harbored infectious MHV. In contrast, lysates of adherent spleen cells from acutely infected mice did not kill intracerebrally inoculated neonatal mice, but did induce seroconversion among all survivors. Despite impairment of APC function of cells derived from MHV-infected donors, neither indomethacin nor accessory cells from uninfected control mice restored concanavalin A-induced proliferative responses of spleen cells collected from acutely infected mice. These results and those of earlier studies suggest that, although APC function is impaired, in vitro T cell dysfunction exhibited by spleen cells from MHV-JHM-infected donors is probably related to an inherent proliferative defect subsequent to T cell activation. Defective concanavalin A-stimulated proliferation does not appear to be secondary to accessory cell function suppression or to inhibitory factors secreted by accessory cells.     

In vitro splenic T cell responses of diverse mouse genotypes after oronasal exposure to mouse hepatitis virus, strain JHM.

Mortality rates among BALB/cByJ, A/JCr, C3H/HeSnJ, and C57BL/6NCr mice inoculated oronasally with mouse hepatitis virus (MHV) strain JHM, ranged from 25 to 67%. Spleen cells harvested from the first three genotypes at 5 days postinoculation proliferated poorly in response to concanavalin A stimulation and produced significantly less interleukin (IL) 2 than cells from uninfected control mice. The function of spleen cells harvested at 14 days postinoculation varied and was host genotype-dependent. Despite clinical signs among some infected C57BL/6NCr mice, spleen cell function was relatively unaffected. C57BL/10ScNCr, B10.A, and SJL/JCr mice remained clinically normal after MHV inoculation. Proliferation and IL2 production by cells from inoculated C57BL/10ScNCr and B10.A mice were similar to responses of their respective controls. In contrast, cells from inoculated SJL/JCr mice were hyper-responsive and produced peak levels of IL2 earlier than control cells. Among the seven genotypes tested, only BALB/cByJ and C3H/HeSnJ spleen cells produced detectable IL4 after primary stimulation with concanavalin A or after priming and restimulation. Primary IL4 production by cells from these two genotypes was significantly reduced if donors were inoculated with MHV 5 days prior to spleen harvest. IL4 production by cells from acutely infected BALB/cByJ mice was considerably enhanced by priming and restimulation.     

Cooperation between humoral factor(s) and Lyt-2+ T cells in effective clearance of Sendai virus from infected mouse lungs

The mechanism of cooperation between the L3T4+ and Lyt-2+ T cell subsets in effective clearance of Sendai virus from infected mouse lungs was studied by adoptive cell transfer using nude mice. Simultaneous transfer of a long-term-cultured Sendai virus-specific L3T4+ T cell line with L3T4+ cell-depleted immune spleen cell (L3T4-) fraction to infected nude mice could result in viral clearance, although single injection with either of these cells was not effective. Instead of the L3T4+ T cells, culture supernatants of the L3T4- T cell line or concanavalin A-stimulated mouse spleen cells and mouse serum immunized with the virus were also active in the cooperative viral clearance with L3T4- fraction. The role of the Sendai virus-sensitized L3T4- cell fraction in cooperative viral clearance with humoral factors could be replaced by neither T cell-deprived immune spleen cell fraction nor normal spleen cells. The 1,500 units of recombinant mouse interleukin 2 (IL-2), which was more than 12 times the IL-2 activity present in the supernatants of the T cell line or concanavalin A-stimulated spleen cells, failed to clear the virus in combination with the L3T4- fraction. Monoclonal antibodies to Sendai or mouse hepatitis viruses were also effective in the cooperative antiviral activity. IL-2 activity was not detected in these monoclonal antibodies and the mouse immune serum. Single injection of any humoral factors failed to clear the virus. These results indicate that Sendai virus-sensitized Lyt-2+ subset of T cells acts cooperatively with humoral factor(s) other than IL-2 or Sendai virus-specific antibody present in supernatants of the T cell line, of concanavalin A-stimulated spleen cells or hybridomas, and in mouse serum immunized with the virus.     

Responses of mice susceptible or resistant to lethal infection with mouse hepatitis virus, strain JHM, after exposure by a natural route

CF1 mice were resistant and BALB/cByJ mice were susceptible to the lethal effects of MHV-JHM infection after intranasal inoculation. Virus was recovered rarely from tissues of CF1 mice. In contrast, MHV-JHM was pantropic in BALB/cByJ mice. The peak systemic interferon response of Balb/cByJ mice occurred 30 hours later than that of CF1 mice. Exogenously administered interferon resulted in partial protection of Balb/cByJ mice challenged with MHV-JHM. Prior immunization of susceptible mice with MHV-JHM protected against challenge with homologous or heterologous (MHV-3) virus.     

Duration of mouse hepatitis virus infection: studies in immunocompetent and chemically immunosuppressed mice

The duration of mouse hepatitis virus (MHV) infection was examined in mice inoculated intranasally with selected strains of MHV. Following inoculation with virulent MHV-JHM, genetically susceptible BALB/c mice and resistant CD1 mice had detectable virus in the brain at 1 month, but not later intervals up to 12 months. BALB/c mice infected with avirulent MHV-S or MHV-1 had no detectable virus in brains at 1 month or thereafter. Immunosuppression of BALB/c mice with treatment regimens of hydrocortisone acetate or cyclophosphamide at 1 and 2 months after infection with MHV-JHM did not activate detectable virus in liver or increase the prevalence or degree of brain infection. Immunosuppression with these drugs during the acute phase of MHV-JHM infection influenced MHV infection, based on virus quantification in livers, but timing of drug treatment relative to MHV infection was critical. Mice infected with MHV developed IgG serum antibody titers that persisted without decline for up to 1 year after infection. Antibody titers varied with mouse genotype and infecting virus. These studies, using intranasal inoculation, support the conclusions of others, using other routes of inoculation, that MHV infection is not persistent in adult, immunocompetent mice.     

Prevalence of natural virus infections in laboratory mice and rats used in Canada

Results of serological tests carried out over a period of 6 years to detect the presence of antibodies against 14 indigenous viruses in mice and rats used in 32 Canadian institutions are reported. Close to 20,000 individual sera were tested by the complement fixation or the hemagglutination inhibition technics. In order of mouse colony prevalence the six most common viruses present were pneumonia virus of mice, mouse hepatitis virus, rat virus, minute virus of mice, Sendai, and Theiler's mouse encephalomyelitis viruses. The most common viruses present in rat colonies were minute virus of mice, K virus, coronaviruses (rat coronavirus or sialodacryoadenitis virus), rat virus, H-1, pneumonia virus of mice, Theiler's mouse encephalomyelitis viruses, Sendai, and reovirus 3.     

CD4 and CD8 T cells have redundant but not identical roles in virus-induced demyelination

A chronic demyelinating disease results from murine infection with the neurotropic strain JHM of mouse hepatitis virus (MHV-JHM). Demyelination is largely immune mediated. In this study, the individual roles of CD4 and CD8 T cells in MHV-induced demyelination were investigated using recombination-activating gene 1-/- (RAG1-/-) mice infected with an attenuated strain of MHV-JHM. These animals develop demyelination only after adoptive transfer of splenocytes from mice previously immunized to MHV. In this study, we show that, following adoptive transfer, virus-specific CD4 and CD8 T cells rapidly infiltrate the CNS of MHV-JHM-infected RAG1-/- mice. Adoptive transfer of CD4 T cell-enriched donors resulted in more severe clinical disease accompanied by less demyelination than was detected in the recipients of undepleted cells. Macrophage infiltration into the gray matter of CD4 T cell-enriched recipients was greater than that observed in mice receiving undepleted splenocytes. In contrast, CD8 T cell-enriched recipients developed delayed disease with extensive demyelination of the spinal cord. MHV-JHM-infected RAG1-/- mice receiving donors depleted of both CD4 and CD8 T cells did not develop demyelination. These results demonstrate that the development of demyelination following MHV infection may be initiated by either CD4 or CD8 T cells. Furthermore, they show that CD4 T cells contribute more prominently than CD8 T cells to the severity of clinical disease, and that this correlates with increased macrophage infiltration into the gray matter.     

Host age and genotypic effects on enterotropic mouse hepatitis virus infection

Intestinal lesions due to infection with an enterotropic strain of mouse hepatitis virus (MHV-Y) were found to be more severe and wide-spread in BALB/cByJ and Cr1:CD-1(ICR) mice than in SJL mice inoculated at 1 week of age, using nonparametric ranking analysis. Lesions and viral antigen were limited largely to the bowel, but also occurred in the liver and brain of some mice. BALB/cByJ mice developed a particularly high prevalence of brain infection, resulting in mortality after the enteric phase of infection had ceased. MHV-Y antigen was present in neurons, glia and vascular endothelium in a vascular distribution. Cr1:CD-1(ICR) pups inoculated with MHV-Y at 4 or 7 days of age developed severe typhlocolitis, enteritis and encephalitis with moderate mortality. Pups infected at 2 or 3 weeks of age had mild intestinal lesions with minimal alteration of mucosal architecture, no encephalitis and no mortality. These results demonstrate that host age and genotype influence the course of enterotropic mouse hepatitis virus, as has been shown previously with non-enterotropic, respiratory-type strains of mouse hepatitis virus.     

Selective response of gamma delta T-cell hybridomas to orthomyxovirus-infected cells.

A gamma delta T-cell hybridoma established from influenza virus-infected mice responded to a reproducible way when cultured with influenza virus-infected stimulators. Subclones of this line responded to cells infected with influenza viruses A/PR/8/34 (H1N1), X-31 (H3N2), and B/HK/8/73 but not to cells infected with vaccinia virus or Sendai virus. This spectrum of response to both type A and type B orthomyxoviruses has never been recognized for the alpha beta T-cell receptor-positive subsets. There was no response to cells infected with a panel of recombinant vaccinia viruses expressing all individual influenza virus proteins, and so it is unlikely that the stimulating antigen is of viral origin. The alternative is that the antigen is a cellular molecule induced in influenza virus-infected cells. Infectious virus was required for stimulation, and immunofluorescence studies showed increased expression of heat shock protein 60 (Hsp60) in influenza virus- but not Sendai virus- or vaccinia virus-infected cells. Both the hybridoma generated from influenza virus-infected mice and an established hybridoma which uses the same gamma delta T-cell receptor combination responded to recombinant Hsp60. Furthermore, the Hsp60-reactive hybridoma, which was obtained from an uninfected mouse, also responded to influenza virus-infected cells, indicating that Hsp60 may indeed be the target antigen.     

Infection with Cytotoxic T-Lymphocyte Escape Mutants Results in Increased Mortality and Growth Retardation in Mice Infected with a Neurotropic Coronavirus

C57BL/6 mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis several weeks after inoculation. Previously, we showed that mutations in the immunodominant CD8 T-cell epitope (S-510-518) could be detected in nearly all samples of RNA and virus isolated from these mice. These mutations abrogated recognition by T cells harvested from the central nervous systems of infected mice in direct ex vivo cytotoxicity assays. These results suggested that cytotoxic T-lymphocyte (CTL) escape mutants contributed to virus amplification and the development of clinical disease in mice infected with wild-type virus. In the present study, the importance of these mutations was further evaluated by infecting naive mice with MHV-JHM variants isolated from infected mice and in which epitope S-510-518 was mutated. Compared to mice infected with wild-type virus, variant virus-infected animals showed higher mortality and morbidity manifested by decreased weight gain and neurological signs. Although a delay in the kinetics of virus clearance has been demonstrated in previous studies of CTL escape mutants, this is the first illustration of significant changes in clinical disease resulting from infection with viruses able to evade the CD8 T-cell immune response.     

Natural killer cell depletion enhances virus synthesis and virus-induced hepatitis in vivo

The role of natural killer (NK) cells in the natural resistance of mice to infections by several viruses was examined. Mice were specifically depleted of NK cells by i.v. injection of rabbit antiserum to asialo GM1, a neutral glycosphingolipid present at high concentrations on the surface of NK cells. Control mice were left untreated or were injected with normal rabbit serum. Four to 6 hr later, these mice were infected with lymphocytic choriomeningitis virus (LCMV), mouse hepatitis virus (MHV), murine cytomegalovirus (MCMV), or vaccinia virus. The mice were sacrificed 3 days post-infection and assayed for virus in liver and spleen, spleen NK cell activity, and plasma interferon (IFN). All mice treated with anti-asialo GM1 antibody had drastically reduced NK cell-mediated lysis. Correlating with NK cell depletion, these mice had significantly higher (up to 500-fold) titers of MCMV, MHV, or vaccinia virus in their livers and spleens as compared to control mice. NK cell-depleted MCMV and MHV-infected mice had higher levels of plasma IFN than controls, correlating with the higher virus titers. These NK cell-depleted, virus-infected mice had more extensive hepatitis, assayed by the number of inflammatory foci in their livers, as compared to control virus-infected mice; these foci were also larger and contained more degenerating liver cells than those in control mice. In contrast to the results obtained with MHV, MCMV, and vaccinia virus, NK cell depletion had no effect on virus titers in the early stages of acute LCMV infection or during persistent LCMV infection. Mice depleted of NK cells had similar amounts of LCMV in their spleens and similar plasma IFN levels. Because this antibody to asialo GM1 does not impair other detectable immunologic mechanisms, these data support the hypothesis that NK cells act as a natural resistance mechanism to a number of virus infections, but suggest that their relative importance may vary from virus to virus.     

Alteration of viral respiratory infections of mice by prior infection with mouse hepatitis virus

Pre-infection with mouse hepatitis virus (MHV) strains S, 3, or JHM reduced the ability of mice to seroconvert to PVM. Geometric mean antibody titers to PVM among MHV pre-infected mice were lower than those for control mice given only PVM, and dually infected mice seroconverted to PVM later than mice given PVM alone. PVM was not recovered from normally permissive respiratory tract tissues of MHV-S pre-infected mice. Pre-infection of DBA/2 mice with MHV-S compromised the susceptibility of these mice to lethal Sendai virus infection but did not substantially reduce the titers of infectious Sendai virus recovered from the lungs. Serologic responses to Sendai virus and lung Sendai virus titers were similar in Sendai virus-resistant C57BL/6 mice pre-infected or not with MHV-S.     

Preferential infection of mature dendritic cells by mouse hepatitis virus strain JHM

Mouse hepatitis virus strain JHM (MHV-JHM) causes acute encephalitis and acute and chronic demyelinating diseases in mice. Dendritic cells (DCs) are key cells in the initiation of innate and adaptive immune responses, and infection of these cells could potentially contribute to a dysregulated immune response; consistent with this, recent results suggest that DCs are readily infected by another strain of mouse hepatitis virus, the A59 strain (MHV-A59). Herein, we show that the JHM strain also productively infected DCs. Moreover, mature DCs were at least 10 times more susceptible than immature DCs to infection with MHV-JHM. DC function was impaired after MHV-JHM infection, resulting in decreased stimulation of CD8 T cells in vitro. Preferential infection of mature DCs was not due to differential expression of the MHV-JHM receptor CEACAM-1a on mature or immature cells or to differences in apoptosis. Although we could not detect infected DCs in vivo, both CD8(+) and CD11b(+) splenic DCs were susceptible to infection with MHV-JHM directly ex vivo. This preferential infection of mature DCs may inhibit the development of an efficient immune response to the virus.     

Early stage of establishment of persistent Sendai virus infection: unstable dynamic phase and then selection of viruses which are tightly cell associated, temperature sensitive, and capable of establishing persistent infection

We obtained 157 cloned cell lines persistently infected with Sendai virus; these cell lines were generated independently of each other. Infectious viruses could be isolated from 123 of these cloned cell lines by inoculation of culture fluids or infected cells into embryonated eggs. The majority of the viruses carried by cells persistently infected with viruses showed high cytotoxicity and did not have the ability to establish persistent infection. The association of carried virus with cells became stronger and virus isolation correspondingly became more difficult as cells persistently infected with virus were subcultured. Viruses derived from virus-infected cells eventually acquired the ability to establish persistent infection, although the ways in which the viruses acquired this ability varied. The viruses also acquired temperature sensitivity as persistently infected cells were subcultured. First, the hemagglutinin-neuraminidase and M proteins acquired temperature sensitivity, and then the polymerase(s) did so. The M proteins were localized in the nuclei of cells infected with cloned viruses that had the ability to establish persistent infection. Cells infected with viruses capable of establishing persistent infection showed no or slight staining by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. Specific amino acid substitutions accumulated in the M protein and the L protein as virus-infected cells were subcultured. This study shows that there is an unstable dynamic phase at an early stage of the establishment of persistent Sendai virus infection (steady state), and then viruses capable of establishing persistent infection are selected.     

Ten-year long monitoring of laboratory mouse and rat colonies in French facilities: a retrospective study

From 1988 to 1997, a total of 69 mouse colonies and 36 rat colonies were examined for the presence of antibodies to 14 indigenous viruses of mice and rats. Among mouse viruses, high positivity rates were observed with mouse hepatitis virus (MHV), Theiler's encephalomyelitis virus (THEMV), minute virus of mice (MVM), Sendai virus and pneumonia virus of mice (PVM); the prevalence rates were high in rats with Khilam's rat virus (KRV), THEMV, Toolan's H-1 virus, Sendai virus, Parker's rat coronavirus (RCV/SDA) and PVM. During the last decade, the prevalence of some agents such as MHV, Sendai virus, THEMV, PVM and MVM has apparently decreased although they were still present in 1997 (except for PVM). Another point is the constant increase of colonies found free of viruses through this decade, demonstrating the efforts of the French research community to increase the quality of hygiene in laboratory animals.     

Inhibition of nitric oxide synthesis increases mortality in Sindbis virus encephalitis.

Sindbis virus (SV) is an alphavirus that causes acute encephalomyelitis in mice. The outcome is determined by the strain of virus and by the age and genetic background of the host. The mortality rates after infection with NSV, a neurovirulent strain of SV, were as follows v: 81% (17 of 21) in BALB/cJ mice; 20% (4 of 20) in BALB/cByJ mice (P < 0.001); 100% in A/J, C57BL/6J, SJL, and DBA mice; and 79% (11 of 14) in immunodeficient scid/CB17 mice. Treatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, increased mortality to 100% (P < 0.05) in NSV-infected BALB/cJ mice, to 95% (P < 0.001) in BALB/cByJ mice, and to 100% in scid/CB17 mice. BALB/cJ and BALB/cByJ mice had similar levels of inducible NOS mRNA in their brains, which were not affected by L-NAME or NSV infection. Brain NOS activity was similar in BALB/cJ and BALB/cByJ mice before and after infection and was markedly inhibited by L-NAME. NSV replication in the brains of BALB/cJ mice, BALB/cByJ mice, and mice treated with L-NAME was similar. Treatment of N18 neuroblastoma cells with NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside in vitro before infection increased cell viability at 42 to 48 h compared with untreated NSV-infected N18 cells with little effect on virus replication. These data suggest that NO protects mice from fatal encephalitis by a mechanism that does not directly involve the immune response or inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication.     

Characterization of a variant virus selected in rat brains after infection by coronavirus mouse hepatitis virus JHM.

The intracerebral inoculation of Lewis rats with the murine coronavirus MHV-JHM leads in the majority of animals to acute encephalitis and death within 14 days. Viral RNAs isolated from the brains of animals 5 to 7 days after infection were compared by Northern blot analysis with the RNAs produced during the lytic infection of Sac(-) or DBT cells with wild-type MHV-JHM (wt virus). Reproducibly, the subgenomic mRNAs 2 and 3 but no other viral RNAs were significantly larger in the brain-derived material. All viruses isolated from infected brain material displayed and maintained this altered mRNA profile when cultivated in Sac(-) or DBT cells. A virus isolated from the infected brain material, MHV-JHM clone 2 (cl-2 virus), has been further characterized. This isolate grew in tissue culture and induced cytopathic effects comparable to those induced by wt virus. However, the mRNAs 2 and 3 produced in cl-2 virus-infected cells had molecular weights ca. 150,000 larger than those produced in cells infected with wt virus. There was no detectable difference in genome-sized RNA (mRNA 1) or subgenomic mRNAs 4, 5, 6, and 7 as determined by electrophoresis in agarose gels. T1-resistant oligonucleotide analysis of genomic RNA revealed one additional and one missing oligonucleotide in the fingerprint of cl-2 virus compared with wt virus. The oligonucleotide fingerprints of intracellular mRNA 3 were identical for both viruses. Pulse-labeling with [35S]methionine in the presence of tunicamycin showed that the primary translation product of mRNA 3, the E2 apoprotein, was ca. 15,000 larger in molecular weight in cl-2 virus-infected cells. These data show that viruses with larger mRNAs 2 and 3 (the latter encoding an altered E2 glycoprotein) are selected for multiplication in rat brains. Mechanisms for the generation of such variants and the possible nature of their selective advantage are considered.     

T cells subsets responsible for clearance of Sendai virus from infected mouse lungs

T cell subsets responsible for clearance of Sendai virus from mouse lungs determined by adoptive transfer of immune spleen cell fractions to infected nude mice. T cells with antiviral activity developed in spleens by 7 days after intranasal infection. Spleen cell fractions depleted of Lyt-2+, Lyt-1+, or L3T4+ cells showed antiviral activity in vivo, although the degree of the activity was lower than that of control whole spleen cells. The antiviral activity of the Lyt-2+ cell-depleted fraction was consistently higher than that of L3T4+ (Lyt-1+)-depleted cells. In vitro cytotoxic activity against Sendai virus-associated, syngeneic lipopolysaccharide-blast cells was detected in stimulated cells from intraperitoneally immunized mice but was lost after depletion of Lyt-2+ cells. Multiple injection of anti-Sendai virus antibody into infected nude mice had no effect on lung virus titer. These results indicate that L3T4+ (Lyt-1+) and Lyt-2+ subsets are cooperatively responsible for efficient clearance of Sendai virus from the mouse lung.