Reduction of the accumulation of viral mRNA and of cellular rRNA synthesis in MHV3-infected macrophages of resistant mice.

1. After MHV3 infection, only macrophages from resistant A/J mice partially restricted virus growth compared to those from susceptible BALB/c mice (2 logs of difference in virus titer). 2. Cellular ribosomal ribonucleic acid (rRNA) synthesis by MHV3-infected macrophages was decreased only in A/J mouse macrophages as indicated by accumulation of the 28S rRNA fraction. 3. The accumulation of viral messenger ribonucleic acids (mRNAs) in MHV3-infected macrophages was also reduced in A/J mouse macrophages compared to BALB/c mice. 4. In pulse-chase experiments of viral protein synthesis, the appearance, glycosylation and cleavage of glycoprotein S, as well as the metabolism of nucleoprotein N were delayed in A/J mouse macrophages. 5. These data show that MHV3 infection of A/J mouse macrophages induced an imbalanced accumulation of the 28S fraction of rRNA. Furthermore the synthesis of mRNAs correlated with viral protein synthesis in both A/J and BALB/c macrophages, but was delayed in A/J mice. 6. These results suggest that the partial restriction of MHV3 replication in macrophages of resistant A/J mice may take place during or before the mRNA synthesis, although it is correlated with the appearance, glycosylation, cleavage and metabolism of viral proteins.

Reduction of the accumulation of viral mRNA and of cellular rRNA synthesis in MHV3-infected macrophages of resistant mice

1. After MHV3 infection, only macrophages from resistant A/J mice partially restricted virus growth compared to those from susceptible BALB/c mice (2 logs of difference in virus titer). 2. Cellular ribosomal ribonucleic acid (rRNA) synthesis by MHV3-infected macrophages was decreased only in A/J mouse macrophages as indicated by accumulation of the 28S rRNA fraction. 3. The accumulation of viral messenger ribonucleic acids (mRNAs) in MHV3-infected macrophages was also reduced in A/J mouse macrophages compared to BALB/c mice. 4. In pulse-chase experiments of viral protein synthesis, the appearance, glycosylation and cleavage of glycoprotein S, as well as the metabolism of nucleoprotein N were delayed in A/J mouse macrophages. 5. These data show that MHV3 infection of A/J mouse macrophages induced an imbalanced accumulation of the 28S fraction of rRNA. Furthermore the synthesis of mRNAs correlated with viral protein synthesis in both A/J and BALB/c macrophages, but was delayed in A/J mice. 6. These results suggest that the partial restriction of MHV3 replication in macrophages of resistant A/J mice may take place during or before the mRNA synthesis, although it is correlated with the appearance, glycosylation, cleavage and metabolism of viral proteins

TNF alpha, IL-1 and O2- 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 (O2-), and A/J mouse macrophages were capable of releasing TNF alpha and IL-1 but not O2-. 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 O2- 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.

An attempt to identify gene products related to the induction of an antiviral state in macrophages resistant and sensitive to IFN-gamma.

The activation of bone-marrow-derived macrophages by IFN-gamma (IFN gamma) partially inhibits mouse hepatitis virus 3 (MHV3) replication only in cells from resistant A/J mice, and not in cells originating from susceptible BALB/c mice. The computer image analysis of gels obtained from 2D-SDS-PAGE of extracted proteins of IFN gamma-activated A/J or BALB/c macrophages enabled us to identify and tag several gene products that were synthesized at elevated or diminished levels. Comparisons of the patterns of non-activated and IFN gamma-activated A/J macrophages revealed 3 gene products which increased, 1 which newly appeared, 6 which decreased and 20 which disappeared upon IFN gamma activation. The protein pattern of BALB/c macrophages revealed 13 gene products which increased, 8 which decreased and 8 which disappeared in IFN gamma-activated BALB/c macrophages. Whether these proteins are involved in the induction of an antiviral state against MHV3 growth remains to be investigated. Macrophages from mice with different genetic background (A/J and BALB/c), upon IFN gamma activation, behave differently at a molecular level, and this observation is consistent with their distinct expression of antiviral state against MHV3.

Mouse hepatitis virus 3 and interferon gamma binding to extracted macrophage proteins correlate with virus growth in A/J and BALB/c mice.

1. After immunization, adult A/J mice are resistant and BALB/c mice are susceptible to MHV3 infection. After IFN gamma activation, only macrophages originating from A/J mice were able to partially restrict MHV3 growth. 2. When the binding of MHV3 and interferon (IFN) gamma to solubilized cytoplasmic and membrane macrophage proteins of mice was determined by ELISA, there was more binding of MHV3 to proteins extracted from BALB/c macrophages than to proteins extracted from A/J macrophages. When the proteins were obtained from IFN gamma-activated macrophages, decreased MHV3 binding was observed only in proteins originating from A/J macrophages. 3. ELISA showed a comparable binding of IFN gamma to A/J or BALB/c macrophage proteins. When the proteins were obtained from IFN gamma-activated macrophages, only IFN gamma-binding to A/J macrophage proteins was increased. 4. The results indicate a different expression and IFN gamma modulation of MHV3 receptors in macrophages from A/J and BALB/c mice, which directly correlated with their acquired resistance or susceptibility to MHV3 infection.

Mouse hepatitis virus 3 and interferon gamma binding to extracted macrophage proteins correlate with virus growth in A/J and BALB/c mice

1. After immunization, adult A/J mice are resistant and BALB/c mice are susceptible to MHV3 infection. After IFN gamma activation, only macrophages originating from A/J mice were able to partially restrict MHV3 growth. 2. When the binding of MHV3 and interferon (IFN) gamma to solubilized cytoplasmic and membrane macrophage proteins of mice was determined by ELISA, there was more binding of MHV3 to proteins extracted from BALB/c macrophages than to proteins extracted from A/J macrophages. When the proteins were obtained from IFN gamma-activated macrophages, decreased MHV3 binding was observed only in proteins originating from A/J macrophages. 3. ELISA showed a comparable binding of IFN gamma to A/J or BALB/c macrophage proteins. When the proteins were obtained from IFN gamma-activated macrophages, only IFN gamma-binding to A/J macrophage proteins was increased. 4. The results indicate a different expression and IFN gamma modulation of MHV3 receptors in macrophages from A/J and BALB/c mice, which directly correlated with their acquired resistance or susceptibility to MHV3 infection

TNF alpha, IL-1 and O2- 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 (O2-), and A/J mouse macrophages were capable of releasing TNF alpha and IL-1 but not O2-. 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 O2- 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.

In vivo depletion of interferon-gamma leads to susceptibility of A/J mice to mouse hepatitis virus 3 infection.

The possible role of interferon-gamma (IFN-gamma) in the resistance of A/J mice to MHV3 infection was investigated. Monoclonal antibodies specific for IFN-gamma, CD4 and CD8 molecules were administered in vivo to deplete selectively the IFN-gamma synthesized or the appropriate subset of T cells. The animals were then infected with MHV3 and the course of infection was followed by studying different parameters, such as, the mortality, the virus growth in the tissues and the IFN-gamma synthesis in sera and peritoneal exudates. After MHV3 infection, a full resistance of control A/J mice was observed, in contrast to the high mortality rate observed among the depleted animals, where higher virus titers were found in different tissues. The IFN-gamma synthesis in sera and peritoneal exudates of depleted mice, after MHV3 infection, drastically decreased when compared to that detected in control mice. The data presented are consistent with the hypothesis that IFN-gamma plays an essential role in the resistance of A/J mice to 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 x 10(5) 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.

A comparative study of resistance to MHV3 infection in genetically homogeneous and heterogeneous mouse populations.

Resistance to MHV3 infection was investigated in genetically homogeneous inbred (A/J, BALB/c) and genetically selected (High, Low) mouse lines. The A/J and L lines are resistant and the BALB/c and H mice are susceptible. The genetic analysis was performed on the F1 hybrids, as well as on the genetically heterogeneous F2 populations and backcrosses bred from HxL and A/JxBALB/c lines. The mortality rates of the F1 hybrids showed codominance of susceptibility and resistance characters. The results indicate that the same MHV3 susceptibility genes are present in isogenic and selected lines and corroborate previous results showing that at least two major genes are involved in the control of this response.

A comparative study of resistance to MHV3 infection in genetically homogeneous and heterogeneous mouse populations

Resistance to MHV3 infection was investigated in genetically homogeneous inbred (A/J, BALB/c) and genetically selected (High, Low) mouse lines. The A/J and L lines are resistant and the BALB/c and H mice are susceptible. The genetic analysis was performed on the F1 hybrids, as well as on the genetically heterogeneous F2 populations and backcrosses bred from HxL and A/JxBALB/c lines. The mortality rates of the F1 hybrids showed codominance of susceptibility and resistance characters. The results indicate that the same MHV3 susceptibility genes are present in isogenic and selected lines and corroborate previous results showing that at least two major genes are involved in the control of this response

Acquired immunity of A/J mice to mouse hepatitis virus 3 infection: dependence on interferon-gamma synthesis and macrophage sensitivity to interferon-gamma.

Coronavirus-free A/J mice (A/J-), in contrast to those naturally infected with coronavirus (A/J+), were shown to be susceptible to experimental infection with our strain of mouse hepatitis virus 3 (MHV3). A/J- mice experimentally hyperimmunized with inactivated MHV3 (A/Ji) became resistant to challenge with this virus. BALB/c mice free of (BALB/c-) or naturally infected with (BALB/c+) coronavirus, or hyperimmunized with inactivated MHV3 (BALB/ci), were always fully susceptible. All susceptible mice developed an acute hepatitis with a high virus titre in the tissues. Resistance mice developed a mild disease in which the low virus titres detected in the tissues were cleared. After infection, interferon (IFN)-gamma synthesis in A/J- mice was lower than that in A/J+ and A/J mice; IFN-gamma synthesis was very high in BALB/c+ and BALB/ci mice, but low in BALB/c- mice. Studies of the anti-MHV3 effect induced in macrophages in vitro showed that only IFN-gamma-activated A/J mouse macrophages were able to restrict partially the growth of MHV3, regardless of whether the animals had been immunized. The effect occurred only when the cells were activated with IFN-gamma before virus infection. The results indicate that the resistance of A/J mice to our strain of MHV3 is not natural but is acquired after immunization, and that the mechanism involved is dependent on T cell activity, IFN-gamma production and the sensitivity of macrophages to IFN-gamma.

A major role of macrophage activation by interferon-gamma during mouse hepatitis virus type 3 infection. II. Age-dependent resistance.

In contrast to adult mice, young A/J mice, developed an acute hepatitis following infection with Mouse Hepatitis virus type 3. 100% of the young animals died 4 to 5 days after the infection and high levels of virus were found in the liver and peritoneal exudate. Very low levels of IFN-gamma were found in the serum and peritoneal exudate of infected young mice. This was in contrast to the levels observed in adult mice. Spleen cells and macrophage cultures from young A/J mice, again in contrast to adult A/J mice, were shown to be unable to synthesize IFN-gamma and IFN-alpha/beta respectively. Macrophages from either young or adult A/J mice were able to be activated with exogenous recombinant IFN-gamma or IFN-alpha/beta, enabling both sets of cells to restrict MHV3 replication. The results indicate that the ability of the immune system to synthesize IFN-gamma and IFN-alpha/beta may play a major role in the age-dependent resistance of A/J mice to MHV3.

Macrophage activity in rabies virus infection of genetically selected high and low antibody responder lines of mice.

After infection with the Pasteur strain of fixed rabies virus, the onset of disease, mortality, interferon (IFN) synthesis and interaction of the virus with macrophages were investigated in high (HI) and low (LI) antibody responder lines of mice. The HI mice were shown to be more resistant than the LI mice, and resistance was age-dependent, since mice from both mouse lines were fully susceptible up to 2 weeks of age. IFN synthesis studies of the serum indicated that, after rabies infection, HI mice produced a slightly higher amount of IFN, which was determined to be predominantly IFN-gamma. In the brains of LI mice, only IFN-alpha/beta was found, in contrast to the mixture of IFN-alpha/beta and IFN-gamma observed in the brains of HI mice. Although macrophages from the two mouse lines expressed the same degree of extrinsic activity, their intrinsic activities were quite different; the LI mice showed a greater ability to uptake and process the virus or ingest C3 (IgM) sheep red blood cells. The present findings attribute the higher antibody response and IFN-gamma synthesis observed in HI mice during rabies infection to slower processing of the rabies antigen in their macrophages, thus conferring upon them a greater ability to present it to the immune system, leading to a higher degree of resistance to rabies infection.

A major role of macrophage activation by interferon-gamma during mouse hepatitis virus type 3 infection. I. Genetically dependent resistance.

Resistance of mice to mouse hepatitis virus type 3 (MHV3) infection is genetically determined. Normal adult A/J mice are resistant, and BALB/c mice are susceptible. Higher titers of virus and interferon (IFN) in vivo were found in MHV3-infected BALB/c mice compared with A/J mice. In vitro activation of macrophages (M phi) by lipopolysaccharide (LPS) delayed MHV3 replication only in cells that originated from A/J mice, although cell populations from both A/J and BALB/c mice were able to synthesize comparable amounts of IFN-alpha/beta. Using specific antibodies, we have shown that the delayed MHV3 replication in LPS-activated A/J M phi was due, in part, to IFN-alpha/beta. A/J M phi were found to be more sensitive to IFN-gamma than to IFN-alpha/beta, and BALB/c M phi did not develop an antiviral state to either IFN. Cultured spleen cells from A/J mice synthesized more IFN-gamma than BALB/c spleen cells after specific or non-specific stimulation. The results indicate that IFN-activated M phi may play a crucial role in the resistance to MHV3 infection. Since IFN-gamma is produced in large amounts by A/J spleen cells after specific stimulation with MHV3 and is efficient in activating the A/J M phi, a T cell-dependent mechanism is likely to be involved.

Nonfatal demyelinating encephalomyelitis induced by coronavirus (JHM strain) infection in mice.

C3H mice infected intravenously with the JHM strain of coronavirus showed high incidence of demyelination (44.8%) and low incidence of encephalitis-induced mortality (6.9%). High titers of virus were detectable in the brain and liver of mice only during the first 3 to 12 days of infection (10(3) and 10(4) PFU/g, respectively). Most of the animals recovered from the first phase of disease and some (11.1%) came down with paralysis 6 to 7 weeks after the infection, with no histological changes or virus detectable in their tissues.

Nonfatal demyelinating encephalomyelitis induced by coronavirus (JHM strain) infection in mice

C3H mice infected intravenously with the JHM strain of coronavirus showed high incidence of demyelination (44.8%) and low incidence of encephalitis-induced mortality (6.9%). High titers of virus were detectable in the brain and liver of mice only during the first 3 to 12 days of infection (10 and 10 PFU/g, respectively). Most of the animals recovered from the first phase of disease and some (1.1%) came down with paralysis 6 to 7 weeks after the infection, with no histological changes or virus detectable in their tissues