Mouse Hepatitis Virus Infection Research Articles

Murine coronavirus replication-induced p38 mitogen-activated protein kinase activation promotes interleukin-6 production and virus replication in cultured cells.

Analyses of mitogen-activated protein kinases (MAPKs) in a mouse hepatitis virus (MHV)-infected macrophage-derived J774.1 cell line showed activation of two MAPKs, p38 MAPK and c-Jun N-terminal kinase (JNK), but not of extracellular signal-regulated kinase (ERK). Activation of MAPKs was evident by 6 h postinfection. However, UV-irradiated MHV failed to activate MAPKs, which demonstrated that MHV replication was necessary for their activation. Several other MHV-permissive cell lines also showed activation of both p38 MAPK and JNK, which indicated that the MHV-induced stress-kinase activation was not restricted to any particular cell type. The upstream kinase responsible for activating MHV-induced p38 MAPK was the MAPK kinase 3. Experiments with a specific inhibitor of p38 MAPK, SB 203580, demonstrated that MHV-induced p38 MAPK activation resulted in the accumulation of interleukin-6 (IL-6) mRNAs and an increase in the production of IL-6, regardless of MHV-induced general host protein synthesis inhibition. Furthermore, MHV production was suppressed in SB 203580-treated cells, demonstrating that activated p38 MAPK played a role in MHV replication. The reduced MHV production in SB 203580-treated cells was, at least in part, due to a decrease in virus-specific protein synthesis and virus-specific mRNA accumulation. Interestingly, there was a transient increase in the amount of phosphorylation of the translation initiation factor 4E (eIF4E) in infected cells, and this eIF4E phosphorylation was p38 MAPK dependent; it is known that phosphorylated eIF4E enhances translation rates of cap-containing mRNAs. Furthermore, the upstream kinase responsible for eIF4E phosphorylation, MAPK-interacting kinase 1, was also phosphorylated and activated in response to MHV infection. Our data suggested that host cells, in response to MHV replication, activated p38 MAPK, which subsequently phosphorylated eIF4E to efficiently translate certain host proteins, including IL-6, during virus-induced severe host protein synthesis inhibition. MHV utilized this p38 MAPK-dependent increase in eIF4E phosphorylation to promote virus-specific protein synthesis and subsequent progeny virus production. Enhancement of virus-specific protein synthesis through virus-induced eIF4E activation has not been reported in any other viruses.

Mouse hepatitis virus infection of the central nervous system: chemokine-mediated regulation of host defense and disease.

Infection of the central nervous system (CNS) of susceptible mice with mouse hepatitis virus (MHV), a positive-strand RNA virus that is a member of the Coronaviridae family, reproducibly results in an acute encephalomyelitis followed by a demyelinating disease similar to the human demyelinating disease multiple sclerosis (MS). MHV infection triggers a robust cell-mediated response in which both CD4+ and CD8+ T cells are essential in controlling viral replication and spread. However, viral clearance is incomplete and viral RNA and protein can persist within white matter tracts, areas of viral persistence are often associated with demyelinating lesions, and recent studies have indicated an important role for both T cells and macrophages in contributing to myelin destruction. The molecular mechanisms governing leukocyte trafficking and accumulation within the CNS of MHV-infected mice are just now being understood and recent studies indicate that chemokines and chemokine receptors have an important role in this process. This article will provide an overview on how these molecules regulate T cell and macrophage trafficking into the CNS of MHV-infected mice and illustrate the delicate balance that exists with regards to expression of chemokines and their receptors as it relates to both host defense and disease development.

Crystal structure of murine sCEACAM1a[1,4]: a coronavirus receptor in the CEA family.

CEACAM1 is a member of the carcinoembryonic antigen (CEA) family. Isoforms of murine CEACAM1 serve as receptors for mouse hepatitis virus (MHV), a murine coronavirus. Here we report the crystal structure of soluble murine sCEACAM1a[1,4], which is composed of two Ig-like domains and has MHV neutralizing activity. Its N-terminal domain has a uniquely folded CC′ loop that encompasses key virus-binding residues. This is the first atomic structure of any member of the CEA family, and provides a prototypic architecture for functional exploration of CEA family members. We discuss the structural basis of virus receptor activities of murine CEACAM1 proteins, binding of Neisseria to human CEACAM1, and other homophilic and heterophilic interactions of CEA family members.

Acute hepatic failure in IFN-γ-deficient BALB/c mice after murine coronavirus infection

We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) persists in interferon-γ (IFN-γ)-deficient C57BL/6 (B6-GKO) mice and results in subacute fatal peritonitis, which bears a resemblance to feline infectious peritonitis. To examine the role of other host factors in MHV infection in mice, IFN-γ-deficient mice with a BALB/c background (BALB-GKO) were infected intraperitoneally with MHV and compared with B6-GKO mice. In contrast to B6-GKO mice, BALB-GKO mice died within 1 week due to acute hepatic failure. The viral titer of the liver in BALB-GKO mice was significantly higher than that in B6-GKO mice. All hepatocytes in BALB-GKO mice were necrotic at 5 days post-infection, which was clearly distinct from large but limited lesion in the liver from infected B6-GKO mice. The serum alanine aminotransferase activity of infected BALB-GKO mice were higher than that of B6-GKO mice and was paralleled with the severity of the pathological changes and viral titers in infected mice. Administration of exogenous IFN-γ to BALB-GKO partially inhibited the acute death. These results indicate that BALB-GKO and B6-GKO mice clearly show different diseases following MHV infection, although wild type counterparts of both mice apparently showed the same clinical course after MHV infection.

Soluble receptor potentiates receptor-independent infection by murine coronavirus.

Mouse hepatitis virus (MHV) infection spreads from MHV-infected DBT cells, which express the MHV receptor CEACAM1 (MHVR), to BHK cells, which are devoid of the receptor, by intercellular membrane fusion (MHVR-independent fusion). This mode of infection is a property of wild-type (wt) JHMV cl-2 virus but is not seen in cultures infected with the mutant virus JHMV srr7. In this study, we show that soluble MHVR (soMHVR) potentiates MHVR-independent fusion in JHMV srr7-infected cultures. Thus, in the presence of soMHVR, JHMV srr7-infected DBT cells overlaid onto BHK cells induce BHK cell syncytia and the spread of JHMV srr7 infection. This does not occur in the absence of soMHVR. soMHVR also enhanced wt virus MHVR-independent fusion. These effects were dependent on the concentration of soMHVR in the culture and were specifically blocked by the anti-MHVR monoclonal antibody CC1. Together with these observations, direct binding of soMHVR to the virus spike (S) glycoprotein as revealed by coimmunoprecipitation demonstrated that the effect is mediated by the binding of soMHVR to the S protein. Furthermore, fusion of BHK cells expressing the JHMV srr7 S protein was also induced by soMHVR. These results indicated that the binding of soMHVR to the S protein expressed on the DBT cell surface potentiates the fusion of MHV-infected DBT cells with nonpermissive BHK cells. We conclude that the binding of soMHVR to the S protein converts the S protein to a fusion-active form competent to mediate cell-cell fusion, in a fashion similar to the fusion of virus and cell membranes.

Lack of nitric oxide synthase type 2 (NOS2) results in reduced neuronal apoptosis and mortality following mouse hepatitis virus infection of the central nervous system.

The role of nitric oxide synthase type-2 (NOS2)-derived nitric oxide (NO) in the pathogenesis of mouse hepatitis virus (MHV)-induced central nervous system disease was examined. Infection of NOS2 knockout (−/−) and NOS2+/+ mice with MHV resulted in similar kinetics of viral clearance from the brain and comparable levels of demyelination. MHV-infected NOS2−/− mice displayed a marked decrease in mortality as compared to infected NOS2+/+ mice that correlated with a significant decrease (P ≤ 0.001) in the number of apoptotic cells (determined by TUNEL staining) present in the brain. Confocal microscopy revealed that the majority of cells (>70%) undergoing apoptosis were neurons. These studies indicate that NOS2-generated NO contributes to apoptosis of neurons but not demyelination following MHV infection.

Recovery from Mouse Hepatitis Virus Infection Depends on Recruitment of CD8+ Cells Rather Than Activation of Intrahepatic CD4+αβ−TCRinter or NK-T Cells

Mouse hepatitis virus (MHV) provides an excellent animal model for the study of the immunopathological mechanisms involved in hepatic viral diseases. We previously generated an attenuated viral variant, YAC-MHV3, which induces a subclinical disease and recovery within 15 days. In contrast, the L2-MHV3 strain induces the development of a fulminant hepatitis, leading to death within 3 days. In this paper, we document intrahepatic and splenic T cell subpopulations involved in the hepatitis process and viral elimination identified in attenuated or pathogenic MHV3-infected C57BL/6 mice. Percentages of intrahepatic CD4+ cells decreased in attenuated YAC-MHV3-infected mice, while they increased in mice infected with pathogenic L2-MHV3, compared with uninfected animals. Moreover, in YAC-MHV3-infected mice, the percentages of intrahepatic CD8+ cells slightly decreased at 24 h pi, then increased until 15 days pi. In contrast, the CD4/CD8 ratios of splenic lymphoid subpopulations increased in the first days of infection and returned to normal values at 15 days pi. Intrahepatic NK1.1+αβ − TCRinter cells decreased in both virally infected groups of mice, while CD4+αβ − TCRinter LFA-1high cells increased in L2-MHV3-infected mice, in contrast with what was seen in YAC-MHV3-infected mice. However, these cells became anergic following Con A or PHA stimulation. Ex vivo studies showed that only the intrahepatic CD8+ cells that were increased in YAC-MHV3-infected mice could be stimulated by lectins. In addition, in vitro viral infections revealed that L2-MHV3 viral infection led to an increase of intrahepatic CD4+αβ − TCRinter cells in the absence of CD8+ cells only. These results indicate that the attenuated phenotype of the YAC-MHV3 virus is related to two different mechanisms: the first involves no increase of intrahepatic CD4+αβ − TCRinter or NK-T cells, while the second favors the recruitment and activation of CD8+ cells in liver. The results are discussed in relation to the integrity of intrahepatic immune tolerance mechanisms and immune-mediated viral elimination.

Neutralization of the chemokine CXCL10 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis.

Intracerebral infection of mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a chronic demyelinating disease with clinical and histological similarities with the human demyelinating disease multiple sclerosis (MS). Following MHV infection, chemokines including CXC chemokine ligand (CXCL)10 (IFN inducible protein 10 kDa), CXCL9 (monokine induced by IFN-gamma), and CC chemokine ligand 5 (RANTES) are expressed during both acute and chronic stages of disease suggesting a role for these molecules in disease exacerbation. Previous studies have shown that during the acute phase of infection, T lymphocytes are recruited into the CNS by the chemokines CXCL10 and CXCL9. In the present study, MHV-infected mice with established demyelination were treated with antisera against these two chemokines, and disease severity was assessed. Treatment with anti-CXCL10 reduced CD4+ T lymphocyte and macrophage invasion, diminished expression of IFN-gamma and CC chemokine ligand 5, inhibited progression of demyelination, and increased remyelination. Anti-CXCL10 treatment also resulted in an impediment of clinical disease progression that was characterized by a dramatic improvement in neurological function. Treatment with antisera against CXCL9 was without effect, demonstrating a critical role for CXCL10 in inflammatory demyelination in this model. These findings document a novel therapeutic strategy using Ab-mediated neutralization of a key chemokine as a possible treatment for chronic human inflammatory demyelinating diseases such as MS.

Reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor CCR5 following infection with a neurotropic coronavirus.

Studies were performed to investigate the contributions of the CC chemokine receptor CCR5 in host defense and disease development following intracranial infection with mouse hepatitis virus (MHV). T cell recruitment was impaired in MHV-infected CCR5−/− mice at day 7 postinfection (pi), which correlated with increased (P ≤ 0.03) titers within the brain. However, by day 12 pi, T cell infiltration into the CNS of infected CCR5−/− and CCR5+/+ mice was similar and both strains exhibited comparable viral titers, indicating that CCR5 expression is not essential for host defense. Following MHV infection of CCR5+/+ mice, greater than 50% of cells expressing CCR5 antigen were activated macrophage/microglia (determined by F4/80 antigen expression). In addition, infected CCR5−/− mice exhibited reduced (P ≤ 0.02) macrophage (CD45highF4/80+) infiltration, which correlated with a significant reduction (P ≤ 0.001) in the severity of demyelination compared to CCR5+/+ mice. These data indicate that CCR5 contributes to MHV-induced demyelination by allowing macrophages to traffic into the CNS.

Targeted disruption of the Ceacam1 (MHVR) gene leads to reduced susceptibility of mice to mouse hepatitis virus infection.

The CEACAM1 glycoproteins (formerly called biliary glycoproteins; BGP, C-CAM, CD66a, or MHVR) are members of the carcinoembryonic antigen family of cell adhesion molecules. In the mouse, splice variants of CEACAM1 have either two or four immunoglobulin (Ig) domains linked through a transmembrane domain to either a short or a long cytoplasmic tail. CEACAM1 has cell adhesion activity and acts as a signaling molecule, and long-tail isoforms inhibit the growth of colon and prostate tumor cells in rodents. CEACAM1 isoforms serve as receptors for several viral and bacterial pathogens, including the murine coronavirus mouse hepatitis virus (MHV) and Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis in humans. To elucidate the mechanisms responsible for the many biological activities of CEACAM1, we modified the expression of the mouse Ceacam1 gene in vivo. Manipulation of the Ceacam1 gene in mouse embryonic stem cells that contained the Ceacam1a allele yielded a partial knockout. We obtained one line of mice in which the insert in the Ceacam1a gene had sustained a recombination event. This resulted in the markedly reduced expression of the two CEACAM1a isoforms with four Ig domains, whereas the expression of the two isoforms with two Ig domains was doubled relative to that in wild-type BALB/c (+/+) mice. Homozygous (p/p) Ceacam1a-targeted mice (Ceacam1aDelta4D) had no gross tissue abnormalities and were viable and fertile; however, they were more resistant to MHV A59 infection and death than normal (+/+) mice. Following intranasal inoculation with MHV A59, p/p mice developed markedly fewer and smaller lesions in the liver than +/+ or heterozygous (+/p) mice. The titers of virus produced in the livers were 50- to 100-fold lower in p/p mice than in +/p or +/+ mice. p/p mice survived a dose 100-fold higher than the lethal dose of virus for +/+ mice. +/p mice were intermediate between +/+ and p/p mice in susceptibility to liver damage, virus growth in liver, and susceptibility to killing by MHV. Ceacam1a-targeted mice provide a new model to study the effects of modulation of receptor expression on susceptibility to MHV infection in vivo.

MHV infection of the CNS: mechanisms of immune-mediated control.

Mice infected with neurotropic strains of mouse hepatitis virus (MHV) clear infectious virus; nevertheless, viral persistence in the central nervous system (CNS) is associated with ongoing primary demyelination. Acute infection induces a potent regional CD8+ T-cell response. The high prevalence of virus specific T cells correlates with ex vivo cytolytic activity, interferon-gamma (IFN-gamma) secretion and efficient reduction in virus. Viral clearance from most cell types is controlled by a perforin dependent mechanism. However, IFN-gamma is essential for controlling virus replication in oligodendrocytes. Furthermore, CD4+ T cells enhance CD8+ T-cell survival and effectiveness. Clearance of infectious virus is associated with a gradual decline of CNS T cells; nevertheless, activated T cells are retained within the CNS. The loss of cytolytic activity, but retention of IFN-gamma secretion during viral clearance suggests stringent regulation of CD8+ T-cell effector function, possibly as a means to minimize CNS damage. However, similar CD8+ T-cell responses to demyelinating and non demyelinating JHMV variants support the notion that CD8+ T cells do not contribute to the demyelinating process. Although T-cell retention is tightly linked to the presence of persisting virus, contributions to regulating the latent state are unknown. Studies in B-cell-deficient mice suggest that antibodies are required to prevent virus recrudescence. Although acute JHMV infection is thus primarily controlled by CD8+ T cells, both CD4+ T cells and B cells make significant contributions in maintaining the balance between viral replication and immune control, thus allowing host and pathogen survival.

Expression of Mig (monokine induced by interferon-gamma) is important in T lymphocyte recruitment and host defense following viral infection of the central nervous system.

Induction of a Th1 immune response against viral infection of the CNS is important in contributing to viral clearance. The present studies demonstrate a role for the T cell chemoattractant chemokine Mig (monokine induced by IFN-gamma) in contributing to a Th1 response against mouse hepatitis virus infection of the CNS. Analysis of the kinetics of Mig expression revealed mRNA transcripts present at days 7 and 12 postinfection (p.i.) but not early (day 2) or late (day 35) in the infection. To determine functional significance, mouse hepatitis virus-infected mice were treated with anti-Mig antisera, and the severity of disease was evaluated. Such treatment resulted in a marked increase in mortality that correlated with a >3 log increase in viral burden within the brains as compared with control mice treated with normal rabbit serum. Anti-Mig-treated mice displayed a significant decrease (p < 0.005) in CD4(+) and CD8(+) T cell recruitment into the CNS as compared with normal rabbit serum-treated mice. In addition, anti-Mig treatment resulted in a significant decrease (p < 0.05) in levels of IFN-gamma and IFN-beta that coincided with increased (p < 0.02) expression of the anti-inflammatory Th2 cytokine IL-10 within the CNS. Collectively, these data indicate that Mig is important in contributing to host defense by promoting a protective Th1 response against viral infection of the CNS.

A study on mouse hepatitis virus receptor genotype in the wild mouse.

Whereas most of laboratory mouse strains are susceptible to mouse hepatitis virus (MHV) infection, only SJL mouse strain is resistant (Smith et al, 1984). MHV susceptible mouse strains have CEACAM1a (MHVR1) as a receptor for MHV, while SJL mice have CEACAM1b (MHVR2) (Ohtsuka and Taguchi, 1997). Although these molecules are functional receptors for MHV (Yokomori and Lai, 1992), there is a striking difference in receptor functionality between these two proteins. MHVR1 has 250∼500 times higher receptor-binding activity relative to MHVR2 as examined by a virus overlay protein blot assay and 10∼30 times higher receptor activity when expressed on cells without MHV receptor (Ohtsuka et al., 1996, Rao et al., 1997). These findings suggest that the difference in susceptibility to MHV infection between MHV-susceptible mouse strains and SJL could result from the difference in receptor functionality found between MHVR1 and MHVR2.

The role of B cells in mouse hepatitis virus infection and pathology.

Murine hepatitis virus (MHV) strain A59 infects the central nervous system (CNS) and liver of mice, causing encephalitis and hepatitis. Virus is cleared from both organs by 16 days post-infection (dpi) (Lavi et al, 1986; data not shown). Infection of the CNS by this virus is associated with demyelination. Interestingly, demyelination peaks at around 30 dpi, after infectious virus is cleared and only viral RNA and small amounts of protein remain (Lavi et al, 1984a, 1984b; Sutherland et al, 1997; data not shown).

CD8 T cell mediated immunity to neurotropic MHV infection.

Neurological disease induced by neurotropic coronaviruses has received considerable attention not only as a model for virus induced demyelination, but also of immune regulation and viral persistence within the central nervous system (CNS). The neurotropic JHM strain (JHMV) of mouse hepatitis virus (MHV) produces an acute CNS infection characterized by encephalomyelitis and demyelination. Several strains and variants have been derived from the lethal, parental JHMV to better study the demyelinating process as a subacute disease resembling the human CNS disease, Multiple Sclerosis. A concise overview of historical aspects, derivation of neurotropic Coronavirus strains and variants, together with types of neurological disease and immune responses associated with these viruses is provided in the previous volume of this series (Perlman, 1998) and in several other recent reviews (Houtman and Fleming 1996, Lane and Buchmeier 1997, Stohlman et al 1999). Advances in molecular immunology techniques, combined with the use of selective exclusion of immune components, either using antibody (Ab) mediated depletion or genetic disruption, has resulted in significant progress in dissecting the interactions between virus, CNS cells, and immune responses and their role in demyelination. This chapter therefore focuses on advances of the past 3-4 years in elucidating interactions between CNS infection and immune responses leading to persistence. As MHV variants differ vastly in tropism, spread, clinical symptoms and mortality, this review focuses largely on a murine model of infection using the neuroattenuated 2.2v-l variant of JHMV (Fleming et al 1986). This mAb selected variant establishes a persistent infection associated with extensive, subacute, ongoing CNS demyelination in the absence of infectious virus. During acute infection this virus replicates primarily in microglia, astrocytes and oligodendrocytes. Although the immune response of immunocompetent hosts generally eliminates infectious virus within 14 days post infection (p.i.), survivors remain persistently infected as evidenced by the detection of viral RNA (vRNA). Despite suffering from varying degrees of encephalitis and paralysis during acute infection, mice generally recover from clinical symptoms and remain asymptomatic during viral CNS persistence.

B cell mediated lysis of JHMV infected targets.

Humoral immunity has been implicated in various models of mouse hepatitis virus (MHV) central nervous system (CNS) infection (Stohlman, et al., 1999). However, the lack of neutralizing Ab until the virus has been cleared suggested that humoral immunity played little or no role in clearance of JHMV from the CNS. Infection of mice lacking the ability to produce Ab showed that following acute clearance, infectious virus reactivates within the CNS (Lin et al., 1999), demonstrating an essential role for humoral immunity in preventing CNS virus reactivation. The absence of both B cells and Ab in these mice leaves open the question of whether the B cells limit CNS virus reactivation, a role potentially masked by Ab mediated neutralization (Lin et al. 1999). B cells from naïve mice interact with MHV-A59 infected cells resulting in lysis (Holmes et al. 1986, Welsh et al. 1986). Lysis is blocked by neutralizing Ab (Wysocka et al. 1989), suggesting a role of the S protein. B cells, although resistant to MHV infection, express high levels of the MHV receptor (MHV-R) (Coutelier et al 1994). Interactions between the S protein and MHV-R appear to result in cytolysis via cell-cell fusion (Wysocka et al. 1989) and is independent of Fas/FasL interactions and TNF-α (Nishioka et al 1993). These studies were initiated to explore the potential role of B cell-MHV-R interactions in contributing to the suppression of infectious virus within the CNS.

Chemokine expression and viral infection of the central nervous system: regulation of host defense and neuropathology.

An effective host response against viral infection of the central nervous system (CNS) is the principal factor dictating the outcome of infection. It is the responsibility of the immune response to contain and control viral replication. Paradoxically, it is the immune response that may also contribute to the development of neuropathology. We have used mouse hepatitis virus (MHV), a positive-strand RNA virus, infection of the CNS to understand the dynamic interaction between viral replication, protection, and pathology with an emphasis on understanding how chemokines participate in these interrelated processes. Herein, we demonstrate the complexity of the chemokine response to MHV infection of the CNS and the delicate balance that exists between host defense and development of disease.

RNase L-independent specific 28S rRNA cleavage in murine coronavirus-infected cells.

We characterized a novel 28S rRNA cleavage in cells infected with the murine coronavirus mouse hepatitis virus (MHV). The 28S rRNA cleavage occurred as early as 4 h postinfection (p.i.) in MHV-infected DBT cells, with the appearance of subsequent cleavage products and a decrease in the amount of intact 28S rRNA with increasing times of infection; almost all of the intact 28S rRNA disappeared by 24 h p.i. In contrast, no specific 18S rRNA cleavage was detected in infected cells. MHV-induced 28S rRNA cleavage was detected in all MHV-susceptible cell lines and all MHV strains tested. MHV replication was required for the 28S rRNA cleavage, and mature cytoplasmic 28S rRNA underwent cleavage. In certain combination of cells and viruses, pretreatment of virus-infected cells with interferon activates a cellular endoribonuclease, RNase L, that causes rRNA degradation. No interferon was detected in the inoculum used for MHV infection. Addition of anti-interferon antibody to MHV-infected cells did not inhibit 28S rRNA cleavage. Furthermore, 28S rRNA cleavage occurred in an MHV-infected mouse embryonic fibroblast cell line derived from RNase L knockout mice. Thus, MHV-induced 28S rRNA cleavage was independent of the activation of RNase L. MHV-induced 28S rRNA cleavage was also different from apoptosis-related rRNA degradation, which usually occurs concomitantly with DNA fragmentation. In MHV-infected 17Cl-1 cells, 28S rRNA cleavage preceded DNA fragmentation by at least 18 h. Blockage of apoptosis in MHV-infected 17Cl-1 cells by treatment with a caspase inhibitor did not block 28S rRNA cleavage. Furthermore, MHV-induced 28S rRNA cleavage occurred in MHV-infected DBT cells that do not show apoptotic signs, including activation of caspase-3 and DNA fragmentation. Thus, MHV-induced 28S rRNA cleavage appeared to differ from any rRNA degradation mechanism described previously.

Demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus.

Demyelination is the pathologic hallmark of the human immune-mediated neurologic disease multiple sclerosis, which may be triggered or exacerbated by viral infections. Several experimental animal models have been developed to study the mechanism of virus-induced demyelination, including coronavirus mouse hepatitis virus (MHV) infection in mice. The envelope spike (S) glycoprotein of MHV contains determinants of properties essential for virus-host interactions. However, the molecular determinants of MHV-induced demyelination are still unknown. To investigate the mechanism of MHV-induced demyelination, we examined whether the S gene of MHV contains determinants of demyelination and whether demyelination is linked to viral persistence. Using targeted RNA recombination, we replaced the S gene of a demyelinating virus (MHV-A59) with the S gene of a closely related, nondemyelinating virus (MHV-2). Recombinant viruses containing an S gene derived from MHV-2 in an MHV-A59 background (Penn98-1 and Penn98-2) exhibited a persistence-positive, demyelination-negative phenotype. Thus, determinants of demyelination map to the S gene of MHV. Furthermore, viral persistence is insufficient to induce demyelination, although it may be a prerequisite for the development of demyelination.

Coronavirus-induced demyelination occurs in the absence of inducible nitric oxide synthase.

Demyelination induced by mouse hepatitis virus (MHV), strain JHM, is in large part immune mediated, but little is known about the mechanisms involved in this process. Previous results suggest that inducible nitric oxide synthase (NOS2) contributes transiently to MHV-induced demyelination. Herein, we show that equivalent amounts of demyelination were evident at day 12 after MHV infection in mice genetically deficient in NOS2 (NOS2(-/-)) and in C57BL/6 mice. Furthermore, using an established adoptive transfer model and pharmacological inhibitors of NOS2 function, we could demonstrate no effect on MHV-induced demyelination. These results indicate that NOS2 function is not required for demyelination in mice infected with MHV.