JHM Virus Research Articles

IL-10 expression in pyramidal neurons after neuropathogenic coronaviral infection.

The apoptosis of pyramidal neurons in CA2 and CA3 subregions of the hippocampus is induced after infection with Mu‐3 virus (Mu‐3), a neuropathogenic strain of the JHM virus (JHMV), at 4–5 days post‐inoculation (dpi). The viral antigens in the hippocampus are mainly found in the CD11b‐positive cells distributed in the stratum oriens located outside the pyramidal layer, and only a few pyramidal neurons are infected. Furthermore, the apoptotic cells, indicated as showing caspase 3 (Cas3) activation, consist of a high number of uninfected cells. Therefore, it is considered that the apoptotic lesions occur through the indirect effects of infection, and not as a result of direct infection with Mu‐3, similar to the reported neuronal apoptosis in the hippocampus after other types of infection. The apoptosis in the pyramidal neurons is accompanied by various types of proinflammatory cytokines depending on the causative agents. Thus, the local expression of proinflammatory cytokines was studied, revealing no correlation in the distribution of cytokine expression with the subregions showing apoptosis. However, the anti‐inflammatory cytokine IL‐10 was produced by pyramidal neurons of CA2 and CA3 at 3 dpi when there is no destructive change or viral invasion in the hippocampus.

Severe Acute Respiratory Syndrome Coronavirus Protein 6 Accelerates Murine Coronavirus Infections

One or more of the unique 3'-proximal open reading frames (ORFs) of the severe acute respiratory syndrome (SARS) coronavirus may encode determinants of virus virulence. A prime candidate is ORF6, which encodes a 63-amino-acid membrane-associated peptide that can dramatically increase the lethality of an otherwise attenuated JHM strain of murine coronavirus (L. Pewe, H. Zhou, J. Netland, C. Tangudu, H. Olivares, L. Shi, D. Look, T. Gallagher, and S. Perlman, J. Virol. 79:11335-11342, 2005). To discern virulence mechanisms, we compared the in vitro growth properties of rJ.6, a recombinant JHM expressing the SARS peptide, with isogenic rJ.6-KO, which has an inactive ORF containing a mutated initiation codon and a termination codon at internal position 27. The rJ.6 infections proceeded rapidly, secreting progeny about 1.5 h earlier than rJ.6-KO infections did. The rJ.6 infections were also set apart by early viral protein accumulation and by robust expansion via syncytia, a characteristic feature of JHM virus dissemination. We found no evidence for protein 6 operating at the virus entry or assembly stage, as virions from either infection were indistinguishable. Rather, protein 6 appeared to operate by fostering viral RNA and protein synthesis, as RNA quantifications by reverse transcription-quantitative PCR revealed viral RNA levels in the rJ.6 cultures that were five to eight times higher than those lacking protein 6. Furthermore, protein 6 coimmunoprecipitated with viral RNAs and colocalized on cytoplasmic vesicles with replicating viral RNAs. The SARS coronavirus encodes a novel membrane protein 6 that can accelerate replication of a related mouse virus, a property that may explain its ability to increase in vivo virus virulence.

Pathogenic Role for Virus-Specific CD4 T Cells in Mice with Coronavirus-Induced Acute Encephalitis

Acute viral encephalitis is believed to result from direct virus destruction of infected cells and from virus-induced host immune response, but the relative contribution of each remains largely unknown. For example, C57BL/6 (B6) mice infected with mouse hepatitis virus (JHM strain, JHMV) develop severe encephalitis, with death occurring within 7 days. Here, we show that the host response to a single JHMV-specific immunodominant CD4 T-cell epitope is critical for severe disease. We engineered a recombinant JHMV with mutations in the immunodominant CD4 T-cell epitope (rJ.MY135Q). Infection of naïve B6 mice with this virus resulted in mild disease with no mortality. However, introduction of a CD4 T-cell epitope from Listeria monocytogenes into rJ.MY135Q generated a highly virulent virus. The decrease in disease severity was not due to a switch from Th1 to Th2 predominance in rJ.MY135Q-infected mice, an effect on CD8 T-cell function, or differential expression of tumor necrosis factor-α by JHMV-specific CD4 T cells. These results show that the response to a single virus-specific CD4 T-cell epitope may contribute to a pathogenic host response in the setting of acute viral disease and that abrogation of this response ameliorates clinical disease without diminishing virus clearance.

Inhibition of Interferon-γ Signaling in Oligodendroglia Delays Coronavirus Clearance without Altering Demyelination

Infection of the central nervous system (CNS) by the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces an acute encephalomyelitis associated with demyelination. To examine the anti-viral and/or regulatory role of interferon-γ (IFN-γ) signaling in the cell that synthesizes and maintains the myelin sheath, we analyzed JHMV pathogenesis in transgenic mice expressing a dominant-negative IFN-γ receptor on oligodendroglia. Defective IFN-γ signaling was associated with enhanced oligodendroglial tropism and delayed virus clearance. However, the CNS inflammatory cell composition and CD8+ T-cell effector functions were similar between transgenic and wild-type mice, supporting unimpaired peripheral and CNS immune responses in transgenic mice. Surprisingly, increased viral load in oligodendroglia did not affect the extent of myelin loss, the frequency of oligodendroglial apoptosis, or CNS recruitment of macrophages. These data demonstrate that IFN-γ receptor signaling is critical for the control of JHMV replication in oligodendroglia. In addition, the absence of a correlation between increased oligodendroglial infection and the extent of demyelination suggests a complex pathobiology of myelin loss in which infection of oligodendroglia is required but not sufficient.

Very diverse CD8 T cell clonotypic responses after virus infections.

We measured CD8 T cell clonotypic diversity to three epitopes recognized in C57BL/6 mice infected with mouse hepatitis virus, strain JHM, or lymphocytic choriomeningitis virus. We isolated epitope-specific T cells with an IFN-gamma capture assay or MHC class I/peptide tetramers and identified different clonotypes by Vbeta chain sequence analysis. In agreement with our previous results, the number of different clonotypes responding to all three epitopes fit a log-series distribution. From these distributions, we estimated that >1000 different clonotypes responded to each immunodominant CD8 T cell epitope; the response to a subdominant CD8 T cell epitope was modestly less diverse. These results suggest that T cell response diversity is greater by 1-2 orders of magnitude than predicted previously.

Receptor-induced conformational changes of murine coronavirus spike protein.

Although murine coronavirus mouse hepatitis virus (MHV) enters cells by virus-cell membrane fusion triggered by its spike (S) protein, it is not well known how the S protein participates in fusion events. We reported that the soluble form of MHV receptor (soMHVR) transformed a nonfusogenic S protein into a fusogenic one (F. Taguchi and S. Matsuyama, J. Virol. 76:950-958, 2002). In the present study, we demonstrate that soMHVR induces the conformational changes of the S protein, as shown by the proteinase digestion test. A cl-2 mutant, srr7, of the MHV JHM virus (JHMV) was digested with proteinase K after treatment with soMHVR, and the resultant S protein was analyzed by Western blotting using monoclonal antibody (MAb) 10G, specific for the membrane-anchored S2 subunit. A 58-kDa fragment, encompassing the two heptad repeats in S2, was detected when srr7 was digested after soMHVR treatment, while no band was seen when the virus was untreated. The appearance of the proteinase-resistant fragment was dependent on the temperature and time of srr7 incubation with soMHVR and also on the concentration of soMHVR. Coimmunoprecipitation indicated that the direct binding of soMHVR to srr7 S protein induced these conformational changes; this was also suggested by the inhibition of the changes following pretreatment of soMHVR with anti-MHVR MAb CC1. soMHVR induced conformational changes of the S proteins of wild-type (wt) JHMV cl-2, as well as revertants from srr7, srr7A and srr7B; however, a major proportion of these S proteins were resistant to proteinase K even without soMHVR treatment. The implications of this proteinase-resistant fraction are discussed. This is the first report on receptor-induced conformational changes of the membrane-anchored fragment of the coronavirus S protein.

Identification of a Noncanonical Signal for Transcription of a Novel Subgenomic mRNA of Mouse Hepatitis Virus: Implication for the Mechanism of Coronavirus RNA Transcription

Subgenomic RNA transcription of coronaviruses involves the interaction between the leader (or antileader) and the intergenic (IG) sequences. However, it is not clear how these two sequences interact with each other. In this report, a previously unrecognized minor species of subgenomic mRNA, termed mRNA5–1, was identified in cells infected with mouse hepatitis virus (MHV) strains JHM2c, JHM(2), JHM(3), A59, and MHV-1. Sequence analysis revealed that the leader-body fusion site of the mRNA is located at approximately 150 nucleotides (nt) downstream of the consensus IG sequence for mRNA 5 and did not have sequence homology with any known IG consensus sequences. To determine whether this sequence functions independently as a promoter, we cloned a 140-nt sequence (from ≈70 nt upstream to ≈70 nt downstream of the fusion site) from viral genomic RNA and placed it in front of a reporter gene in the defective-interfering (DI) RNA-chloramphenicol acetyltransferase (CAT) reporter vector. Transfection of the reporter RNA into MHV-infected cells resulted in synthesis of a CAT-specific subgenomic mRNA detected by reverse transcription-polymerase chain reaction (RT-PCR). The strength of this promoter was similar to that of the IG7 (for mRNA 7) as measured by the CAT activity. Deletion analysis showed that the sequence as few as 13 nt was sufficient to initiate mRNA transcription, while mutations within the 13-nt abolished mRNA transcription. In vitro translation study confirmed that the envelope (E) protein was translated from mRNA5–1, which encodes the open reading frame (ORF) 5b at its 5′-end, indicating that mRNA5–1 is a functional message. Furthermore, when the ORF5b was replaced with the CAT gene and placed in the DI in the context of viral mini-genome, CAT was expressed not only from the first ORF of mRNA5–1 but also from the second and third ORF of mRNA5 and genomic DI RNA, respectively, suggesting that more than one mechanism is involved in regulation of ORF5b expression. Our findings thus support the notion that base-pairing between the leader (or antileader) and the IG is not the sole mechanism in subgenomic RNA transcription.

No evidence for quasispecies populations during persistence of the coronavirus mouse hepatitis virus JHM: sequence conservation within the surface glycoprotein gene S in Lewis rats.

The surface glycoprotein S (spike) of coronaviruses is believed to be an important determinant of virulence and displays extensive genetic polymorphism in cell culture isolates. This led us to consider whether the observed heterogeneity is reflected by a quasispecies distribution of mutated RNA molecules within the infected organ. Coronavirus infection of rodents is a useful model system for investigating the pathogenesis of virus-induced central nervous system (CNS) disease. Here, we investigated whether genetic changes in the S gene occurred during virus persistence in vivo. We analysed the variability of S gene sequences directly from the brain tissue of Lewis rats infected with the coronavirus mouse hepatitis virus (MHV) variant JHM-Pi using RT-PCR amplification methods. The S gene sequence displayed a remarkable genetic stability in vivo. No evidence for a quasispecies distribution was found by sequence analysis of amplified S gene fragments derived from the CNS of Lewis rats. Furthermore, the S gene also remained conserved under the selection pressure of a neutralizing antibody. Only a few mutations predicted to result in amino acid changes were detected in single clones. The changes were not represented in the consensus sequence. These results indicate that to retain functional proteins under the constraints of a persistent infection in vivo, conservation of sequence can be more important than heterogeneity.

Mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis.

Perforin-deficient [perforin (-/-)] mice were infected with two strains of JHM virus (JHMV) to analyze the role of perforin-mediated cytotoxicity in acute lethal and subacute central nervous system (CNS) infections. During both acute and subacute infections, the overall mortality of the perforin (-/-) mice was not different from that of the controls. Perforin (-/-) mice survived longer than the controls, consistent with reduced morbidity. Both strains of virus were cleared from the perforin (-/-) mice as in the controls; however, the rate of clearance was delayed in the perforin (-/-) mice, indicating that perforin-mediated cytolysis is involved in viral clearance. The absence of perforin-mediated cytolysis did not prevent encephalomyelitis or extensive demyelination. Cells undergoing apoptosis were detected in the CNS of both the perforin (-/-) and control groups, indicating that perforin is not essential for programmed cell death. Neutralizing antibodies were not detected in either group of mice until day 9 postinfection, when the majority of the virus had been cleared. These data further confirm the importance of cell-mediated cytotoxicity and suggest that additional components of the immune response contribute to the clearance of JHMV from the CNS.

The JHM strain of mouse hepatitis virus induces a spike protein-specific Db-restricted cytotoxic T cell response.

Cytotoxic T lymphocyte (CTL) activity specific for mouse hepatitis virus (MHV) JHM strain (JHMV or MHV-4) was examined using in vitro stimulated spleen cells derived from immunized C57BL/6 (H-2b) mice. Target cells infected with JHMV were specifically recognized; however, analysis of target cells expressing the virus structural proteins via recombinant vaccinia viruses showed no recognition of the viral nucleocapsid (N), membrane (M), small membrane (sM) or haemagglutinin-esterase (HE) proteins. Only target cells expressing the virus spike (S) protein were recognized. Furthermore, the majority of CTL activity was restricted to target cells expressing the MHC class I Db molecules. Analysis of truncations and deletions of the S protein expressed by recombinant vaccinia viruses and peptide coated targets identified a single antigenic epitope, aa 510-518, conforming to the Db binding motif. These amino acids are contained within a domain deleted from a number of strains of mouse hepatitis virus, suggesting a role for immune pressure. To determine the potential for CTL specific for an epitope(s) within a non-structural protein, 24 CTL lines were established and characterized. No evidence for the induction of non-specific CTL activity or virus-specific CTL restricted to an epitope in a non-structural protein was obtained. These data indicate that the predominant CTL activity in JHMV-infected C57BL/6 mice is Db restricted and specific for a single epitope contained within aa 510-518 of the S protein.

Involvement of microtubules and the microtubule-associated protein tau in trafficking of JHM virus and components within neurons.

The neurotropic coronavirus JHM (JHMV) is capable of inducing various forms of CNS disease in rodents, ranging from an acute encephalomyelitis to a delayed onset demyelination1,2,3. In rats, during the early stages of the disease process, neurons become cellular targets3,4. When introduced by intranasal inoculation, JHMV can invade the CNS of mice and rats by spreading along the olfactory neurons5,6,7,8. Virus spread was shown to occur by the transneuronal route5,8. Subsequent spread within the CNS seems to involve specific neuronal populations and tracts6,8. In particular, in Wistar Furth rats, Purkinje and hippocampal neurons are extensively involved3,4. Moreover, neurons have been shown to provide a repository site where both RNA and virions can persist for prolonged periods9,10, 11. Recently, Pasick et al., (1994) demonstrated that trafficking of virus materials within neurons occurs asymmetrically along somatodendritic and axonal processes, and appears to be dependent on the integrity of the microtubular network, as evident from analyses by light and electron microscopy which revealed that JHMV nucleocapsids (N) are closely associated with microtubules12, 13. This report seeks to define, more directly, by immunoprecipitation and immunoblotting, interactions between N and the microtubular arrays.KeywordsDemyelinating DiseaseMouse Hepatitis VirusIntranasal InoculationRepository SiteSpecific Neuronal PopulationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Modulation of cellular macromolecular synthesis by coronavirus: implication for pathogenesis

Infection with the murine coronavirus strain JHM decreases cell surface expression of major histocompatibility complex class I antigens. Northern blots showed that JHM virus infection rapidly reduced the level of actin mRNA, whereas the levels of major histocompatibility complex class I and tubulin mRNAs were reduced only slightly. By contrast, the mRNA levels of interleukin 1 beta, colony-stimulating factor 1 receptor, and tumor necrosis factor alpha increased following infection.

On the role of different lymphocyte subpopulations in the course of coronavirus MHV IV (JHM)-induced encephalitis in Lewis rats.

The neurotropic strain of the mouse hepatitis virus (MHV), MHV-IV or JHM virus, causes neurological disorders in rats and mice when given intracerebrally 1,2. Depending on the rat strain and the age of the infected animal different courses of disease have been observed. Up to 2 weeks post partum Lewis (LEW) rats develop an acute fatal encephalitis, whereas animals older than 3 weeks usually do not succumb to the infection 3 but suffer from a subacute paralytic disease accompanied by multiple neurological disorders. However, the majority of them recovers completely roughly 3 weeks past infection 4. Since rats are expected to he immunologically competent at the age of 3 weeks, it is conceivable that maturity of the immune system probably plays a major role in the clinical course of the infection. We have recently shown, that T- and B-lymphocytes infiltrate into the central nervous system of these rats 4 and that the humoral immune response most likely contributes significantly to convalescence 5.KeywordsHumoral Immune ResponseLymphocyte SubsetInfectious VirusMouse Hepatitis VirusImmunize Spleen CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Persistence of viral RNA in the central nervous system of mice inoculated with MHV-4.

In order to study the role that viral persistence may play in chronic central nervous system (CNS) disease induced by murine coronaviruses, we have used the reverse transcriptase-polymerase chain reaction (RT-PCR) to study viral RNA in the brains of mice after intracerebral inoculation of JHM virus (JHMV or MHV-4). Quantitative RT-PCR showed that JHMV RNA decreased from approximately 2 ng/ug total brain RNA at day 6 post-inoculation (PI) to 0.1 pg/ug total brain RNA at 360 days PI. Double-stranded viral RNA could be detected up to day 20 PI. By the selective use of upstream or downstream primers during the RT step, it was possible to measure negative sense and positive sense JHMV RNA respectively, and we found that there was a marked rise in the ratio of positive to negative sense JHMV RNA after day 13 PI. Analysis of amplified products by dideoxy DNA sequencing showed that the characteristic mutation of our input virus (at position 3340 of gene 3) is maintained to at least day 42 PI. Taken together, these results favor a model of JHMV persistence in vivo in which viral RNA is present as double stranded forms initially and predominantly as single stranded, positive sense forms at late timepoints. Further analysis of this model in quantitative terms may contribute to our understanding of the biological significance of coronavirus persistence in the CNS.

Characterization of the Ld-restricted cytotoxic T-lymphocyte epitope in the mouse hepatitis virus nucleocapsid protein.

The mouse hepatitis virus (MHV) JHM strain (JHMV) produces primary demyelination in the central nervous system associated with acute encephalomyelitis. Humoral and cellular immune responses both participate in controlling the development of chronic MHV-induced demyelination. A subset of the CD8+ cytotoxic T lymphocytes (CTL) induced by immunization of BALB/c (H-2d) mice with JHMV is specific for the viral nucleocapsid protein. This CTL population recognizes an epitope located within the carboxy-terminal 149 amino acids in association with the Ld class I molecule (S. A. Stohlman, S. Kyuwa, M. Cohen, C. Bergmann, J. P. Polo, J. Yeh, R. Anthony, and J. G. Keck, Virology 189:217-224, 1992). Using a panel of vaccinia virus recombinants expressing truncated forms of the nucleocapsid protein and a series of overlapping synthetic peptides, we mapped the response to 15 amino acids. This sequence, encompassing the MHV epitope, contains the Ld-specific binding motif. The predicted 9-mer peptide (residues 318 to 326: APTAGAFFF) was sufficient and highly active in sensitizing target cells for CTL recognition when either added exogenously or synthesized intracellularly. Cross-reactivity of JHMV nucleocapsid protein-specific CTL with six other MHV strains indicated that natural sequence variations within the 9-mer epitope are tolerated in positions 4 and 5, whereas all other amino acids are conserved. These data define a novel 9-mer Ld-restricted CTL epitope which represents the first MHV CTL epitope. Characterization of this epitope provides a molecular basis to study the role of nucleocapsid protein-specific CTL in the clearance of JHMV from the central nervous system.

Cell Fusion Studies Identified Multiple Cellular Factors Involved in Mouse Hepatitis Virus Entry

We have previously reported that certain murine cell lines are susceptible to mouse hepatitis virus (MHV) A59 strain infection but resistant to JHM strain, despite the expression of the viral receptor for both strains. This restriction on viral infection has been shown to occur at the virus entry level (Yokomori et al., Virology 196, 45-56, 1993). To study whether JHM resistance of these cell lines is due to a defective cellular factor necessary for JHM virus entry, or due to the presence of an inhibitor, hybrid cells were obtained by polyethylene glycol (PEG)-mediated fusion between two resistant cell lines, i.e., a Balb/c mouse-derived cell line, BC10 cells, which are resistant to JHM infection but express a viral receptor, and the simian cell line COS cells, which are resistant to JHM because of the absence of the viral receptor. JHM could replicate in the hybrid BC10-COS cells, indicating that the restriction of viral infection in BC10 cells could be complemented by COS cells. This result indicates that the resistance of BC10 cells to JHM infection is due to the defectiveness of a cellular factor rather than the presence of an inhibitor. JHM virus-binding and penetration into BC10 cells appeared to be normal. However, JHM internalization into BC10 cells by PEG-induced virus-cell fusion did not lead to viral replication, suggesting that the restriction of JHM infection in BC10 cells is at the level of viral uncoating. This restriction could be complemented by PEG-mediated fusion with other murine cell lines which have the virion-uncoating activity, but not by cell lines which lack this activity. Furthermore, the viral resistance of most of other murine cell lines, which express MHV receptors, could not be overcome by fusion with COS cells, suggesting that different murine cell lines are defective in different viral entry functions. Therefore, we conclude that JHM viral entry process requires multiple cellular factors secondary to the viral receptor.

Retina and retinal pigment epithelial cell autoantibodies are produced during murine coronavirus retinopathy.

The murine coronavirus, mouse hepatitis virus (MHV), JHM strain, induces a biphasic retinal disease in adult BALB/c mice. In the early phase of the disease, day 1 to 7, a retinal vasculitis is noted and is associated with the presence of virus particles. In the late phase of the disease, day 10 to 140, a retinal degeneration is observed and is associated with the absence of both virus particles and inflammatory cells. We show that the retinal degenerative process is also associated with the presence of antiretinal autoantibodies. In total, 22 of 23 sera collected from 10 to 70 days after JHM virus inoculation contained antiretinal autoantibodies. These autoantibodies are not found in sera from normal or mock-injected mice. Antibodies to retinal tissue were identified as two distinct patterns of immunoperoxidase staining on frozen sections of normal rat eyes, retinal autoantibodies and retinal pigment epithelium (RPE) autoantibodies. The antiretinal autoantibodies first appeared as IgM class antibodies that shifted to IgG class autoantibodies. The anti-RPE cell autoantibodies were predominantly of the IgG class. Sera that were positive for these autoantibodies did not stain with liver or kidney sections but 2 of 3 did react with rat brain sections. A second mouse strain, CD-1, was also evaluated because these animals respond to JHM virus inoculation by developing only the early phase of this disease, i.e. vasculitis. On day 10 postinoculation, the retina architecture has a normal appearance. In these mice, which are free of a retinal degeneration, antiretinal autoantibodies are not produced. However, just as is noted in the BALB/c mice, antivirus neutralizing antibodies are produced in the infected CD-1 mice. These findings suggest a role for autoimmunity in the pathogenesis of murine coronavirus induced retinal degeneration. This study establishes an animal model for the study of humoral autoimmune responses in human retinal degenerations.

A Spike Protein-Dependent Cellular Factor Other Than the Viral Receptor Is Required for Mouse Hepatitis Virus Entry

Previous studies have shown that some mouse strains are resistant to mouse hepatitis virus (MHV) infection despite the presence of functional viral receptors (K. Yokomori and M. M. C. Lai, J. Virol. 66, 6931-6938, 1992). To determine the molecular requirement for MHV infection, several cell lines derived from both susceptible and resistant mouse strains were tested for their ability to support infection by two different MHV strains, JHM and A59. Most of the cell lines tested, including ones from susceptible mouse strains, exhibited selective resistance to JHM, but were susceptible to A59, suggesting that there is an additional cellular factor(s) discriminating JHM from A59 infection. Both RNA and protein syntheses of JHM were inhibited in the resistant cells; however, transfection of JHM genomic RNA into these cells led to the production of infectious virus, suggesting that the restriction step(s) is during an early stage of viral replication cycle. The mRNA for the MHV receptor (the murine homolog of the carcinoembryonic antigen) is expressed in all cell lines, and expression in COS cells of the receptor isolated from the resistant murine cell lines rendered the COS cells susceptible to both A59 and JHM infections. Furthermore, the transfection of additional MHV receptors into the resistant cells did not overcome the resistance to JHM virus infection. These results suggested that the viral receptor is functional; nevertheless, the JHM infection is restricted at an early step of infection in these cells. The study of the growth properties of the various recombinant viruses between A59 and JHM revealed that one of the viral genes determining viral replication in these cell lines is the S protein gene; thus, the second factor required for viral infection may interact directly or indirectly with the S protein at an early step of infection. Taken together, these studies suggest that expression of a functional viral receptor is not sufficient to establish MHV infection, and that an additional factor(s) is required for an early step of viral infection, possibly during virus entry.

Coronavirus (JHM) Replication within the Retina: Analysis of Cell Tropism in Mouse Retinal Cell Cultures

The murine coronavirus, mouse hepatitis virus, JHM strain, induces a retinal degenerative disease in adult BALB/c mice. Coronavirus infections are highly species specific with virus exhibiting a strong tissue and cell specificity. In this report we evaluated the cellular basis of JHM virus retinal tropism. Retinal cultures and retinal pigment epithelial (RPE)-retinal mixed cell cultures were prepared from eyes obtained from Balb/c mice. The ability of JHM virus to infect and replicate in these retinal cultures was evaluated by light microscopy, immunofluorescent staining, electron microscopy, and virus isolation. Cytopathology was not observed and virus could not be detected in supernatant fluid in retinal cultures. However, low levels of infectious virus could be detected within the cells for the first 4 days. This observation suggested that cell-to-cell interactions may be critical since virus particles and virus antigens can be seen in vivo within the neural retina and the RPE. In contrast to the retinal cultures, retinal-RPE mixed cultures were supportive to JHM virus replication. Syncytial cytopathology was observed for the first 4 days and virus was isolated from supernatant fluids. By electron microscopy, virus was found intracellularly within vacuoles and extracellularly at the plasma membrane. After Day 4, a persistent virus infection was established in which cells produced virus for 5 weeks without cytopathic effects or cell death. Double-labeling immunofluorescent studies of retinal-RPE mixed cultures showed that the virus antigen was co-expressed with a Muller cell marker, glutamine synthetase. This cell is the most prominent glial element in the retina. These studies demonstrate that JHM virus is capable of establishing a persistent virus infection in mixed retinal (Muller)-RPE cell cultures. Moreover, these data suggest that cell-to-cell interactions influence the establishment of coronavirus infections in the retina.

Coronavirus infects and causes demyelination in primate central nervous system

Two species of primates, Owl and African green monkeys, were inoculated intracerebrally with either the neurotropic mouse hepatitis virus JHM or the putative multiple sclerosis brain coronavirus isolate SD. These viruses caused an acute to subacute panencephalitis and/or demyelination in the infected animals. The course of pathogenesis and sites of detected viral RNA and antigen was dependent both on animal species and virus strain but the results clearly showed that these viruses replicated and disseminated in the central nervous system (CNS) of these primates. This study suggests that human CNS may be susceptible to coronavirus infection.