Biomedical subjects

J O Fleming

Publications and source records attributed to J O Fleming.

At least 19 recordsLinked to original sources

PubMed1998

Quasispecies development by high frequency RNA recombination during MHV persistence.

Recent studies suggest that infectious viruses and particularly persisting viral RNAs often exist as diverse populations or "quasispecies". We have developed an approach to characterize populations of the murine coronavirus mouse hepatitis virus (MHV) generated during persistent infection which has allowed us to begin to address the role of the viral quasispecies in MHV pathogenesis. We analyzed the population of persisting viral RNAs using reverse-transcription polymerase chain reaction amplification (RT-PCR) of the S1 "hypervariable" region of the spike gene followed by differential colony hybridization to identify spike deletion variants (SDVs) from acute and persistently infected mice. Sequence analysis revealed that mice with the most severe chronic paralysis harbored the most complex quasispecies. Mapping of the SDVs to the predicted RNA secondary structure of the spike RNA revealed that an isolated stem loop structure is frequently deleted. Overall, these results are consistent with high frequency recombination at sites of RNA secondary structure contributing to expansion of the viral quasispecies and persisting viral pathogenesis.

Animals↗

PubMed1997

Evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection.

High-frequency RNA recombination has been proposed as an important mechanism for generating viral deletion variants of murine coronavirus. Indeed, a number of variants with deletions in the spike glycoprotein have been isolated from persistently infected animals. However, the significance of generating and potentially accumulating deletion variants in the persisting viral RNA population is unclear. To study this issue, we evaluated the evolution of spike variants by examining the population of spike RNA sequences detected in the brains and spinal cords of mice inoculated with coronavirus and sacrificed at 4, 42, or 100 days postinoculation. We focused on the S1 hypervariable region since previous investigators had shown that this region is subject to recombination and deletion. RNA isolated from the brains or spinal cords of infected mice was rescued by reverse transcription-PCR, and the amplified products were cloned and used in differential colony hybridizations to identify individual isolates with deletions. We found that 11 of 20 persistently infected mice harbored spike deletion variants (SDVs), indicating that deletions are common but not required for persistent infection. To determine if a specific type of SDV accumulated during persistence, we sequenced 106 of the deletion isolates. We identified 23 distinct patterns of SDVs, including 5 double-deletion variants. Furthermore, we found that each mouse harbored distinct variants in its central nervous system (CNS), suggesting that SDVs are generated during viral replication in the CNS. Interestingly, mice with the most severe and persisting neurological disease harbored the most prevalent and diverse quasispecies of SDVs. Overall, these findings illustrate the complexity of the population of persisting viral RNAs which may contribute to chronic disease.

Animals↗

PubMed1997

Generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted RNA secondary structure.

Coronavirus RNA evolves in the central nervous systems (CNS) of mice during persistent infection. This evolution can be monitored by detection of a viral quasispecies of spike deletion variants (SDVs) (C. L. Rowe, S. C. Baker, M. J. Nathan, and J. O. Fleming, J. Virol. 71:2959-2969, 1997). We and others have found that the deletions cluster in the region from 1,200 to 1,800 nucleotides from the 5' end of the spike gene sequence, termed the "hypervariable" region. To address how SDVs might arise, we generated the predicted folding structures of the positive- and negative-strand senses of the entire 4,139-nt spike RNA sequence. We found that a prominent, isolated stem-loop structure is coincident with the hypervariable region in each structure. To determine if this predicted stem-loop is a "hot spot" for RNA recombination, we assessed whether this region of the spike is more frequently deleted than three other selected regions of the spike sequence in a population of viral sequences isolated from the CNS of acutely and persistently infected mice. Using differential colony hybridization of cloned spike reverse transcription-PCR products, we detected SDVs in which the hot spot was deleted but did not detect SDVs in which other regions of the spike sequence were exclusively deleted. Furthermore, sequence analysis and mapping of the crossover sites of 25 distinct patterns of SDVs showed that the majority of crossover sites clustered to two regions at the base of the isolated stem-loop, which we designated as high-frequency recombination sites 1 and 2. Interestingly, the majority of the left and right crossover sites of the SDVs were directly across from or proximal to one another, suggesting that these SDVs are likely generated by intramolecular recombination. Overall, our results are consistent with there being an important role for the spike RNA secondary structure as a contributing factor in the generation of SDVs during persistent infection.

Base Sequence↗

PubMed1996

Dissociation of demyelination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus JHM.

Infection of rodents with murine coronavirus JHM results in a subacute or chronic demyelinating disease which serves as a model for the human disease multiple sclerosis. Previous studies with JHMV have established a role for the immune system in both viral clearance and demyelination. To further clarify the role of the immune system in JHMV pathogenesis, several strains of congenitally immunodeficient mice were studied. Infection of immunocompetent C57BL/6 mice with JHMV resulted in severe paralysis and demyelination and complete clearance of infectious virus from the brain (C+D+ phenotype). In contrast, infected SCID mice showed little or no paralysis or demyelination and were unable to clear infectious virus (C-D- phenotype). Athymic nude mice and a proportion of mice lacking MHC Class I or II expression exhibited robust demyelination but did not completely clear infectious virus from the brain (C-D+ phenotype). These results are consistent with an immune-mediated mechanism for JHMV-induced demyelination, but indicate that the immune mechanisms which participate in demyelination and viral clearance are distinct. It may thus be possible to experimentally alter immunopathological responses without impairing antimicrobial immunity.

Animals↗

PubMed1996

Pathogenesis of mouse hepatitis virus-induced demyelination.

Infection of rodents with neurotropic mouse hepatitis virus (MHV) may result in lethal encephalitis or paralytic demyelinating disease resembling the human disease multiple sclerosis. The outcome of MHV infection is dependent on a number of variables, including the passage history of the viral isolate, dose and route of inoculation, and the age and immune status of the host. Alterations in surface glycoproteins, especially the spike protein, can profoundly influence pathogenesis. Innate resistance to MHV infection may be related to the expression of cellular receptors or to immunological factors. The immune system plays a major role in MHV pathogenesis, affecting encephalitis, viral clearance, and demyelination. Antiviral antibodies, CD4+ T lymphocytes, or CD8+ T lymphocytes may protect infected animals from lethal encephalitis, but both CD4+ and CD8+ T lymphocytes are required for effective viral clearance. Demyelination in MHV-infected animals has been attributed to the cytolytic effects of viral infection on myelin-producing oligodendrocytes, but more recent evidence supports an immunopathological mechanism for demyelination. Immunopathological models for demyelination include autoimmunity, direct immune cytotoxicity, and indirect 'bystander' damage. Although evidence exists supporting all of these models, the authors favor the bystander demyelination model. Much remains to be revealed about the processes leading to demyelination in MHV-infected mice, and information gained from these investigations may aid in the study of demyelinating disease in humans.

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PubMed1995

Immunotherapy in multiple sclerosis, Part 2.

The efficacies of corticosteroids and azathioprine (part 1) and of cyclophosphamide, immune globulin, cyclosporine, interferons, copolymer 1, and cladribine (part 2) in patients with multiple sclerosis (MS) are reviewed. MS is an inflammatory, demyelinating disease of the CNS that commonly affects young adults. The involvement of various immune mechanisms in MS suggests a role for immunomodulating therapy. The goals of immunotherapy vary with the clinical stage of the disease and include (1) improving recovery from exacerbations, (2) decreasing the number or severity of relapses, (3) preventing the development of chronic progressive disease from a relapsing-remitting course, and (4) decreasing further progression in patients with chronic progressive disease. In clinical trials, corticotropin and corticosteroids have been found to accelerate recovery from exacerbations. Tapering is often effective after high-dose induction therapy. Long-term maintenance regimens do not alter disease progression and are not recommended. Azathioprine produces modest benefits with respect to relapse rates and disease progression after two or more years of treatment; adverse effects are mild to moderate. Azathioprine should not be used in patients with aggressive disease who may approach severe disability in 6-18 months. Cyclophosphamide, because of its modest impact on disease progression and its potentially severe adverse effects, including cancer, should be reserved for patients with aggressive relapsing-remitting or chronic progressive disease in whom other treatments have failed to work; maintenance therapy is necessary after induction. Intravenous immune globulin may benefit patients with severe relapses; however, its efficacy remains unproven. Cyclosporine also cannot be recommended because of its modest efficacy, marked adverse effects, and high cost. Interferon beta-1b is a more specific immunotherapy that has been found to decrease the number and severity of relapses. This treatment should be considered in patients with relapsing-remitting disease who are having two or more exacerbations per year. Copolymer 1 and cladribine have shown some promising early results. Although various immunotherapeutic drugs can provide relief in patients with MS, none is capable of reversing disease progression, and some can cause serious adverse effects. Better understanding of the immunologic basis of MS may lead to more specific immunotherapies with more lasting benefits.

Adult↗

PubMed1995

Immunotherapy in multiple sclerosis, Part 1.

Adrenal Cortex Hormones↗

PubMed1995

Evolution of mouse hepatitis virus (MHV) during chronic infection: quasispecies nature of the persisting MHV RNA.

Coronavirus infection of mice has been used extensively as a model for the study of acute encephalitis and chronic demyelination. To examine the evolution of coronavirus RNA during chronic demyelinating infection, we isolated RNA from intracerebrally inoculated mice at 4, 6, 8, 13, 20, and 42 days postinfection and used reverse transcription-polymerase chain reaction amplification methods (RT-PCR) to detect viral sequences. RNA sequences from two viral structural genes, the spike gene and the nucleocapsid gene, were detected throughout the chronic infection. In contrast, infectious virus was not detectable from brain homongenates beyond 13 days postinfection. These results indicate that coronavirus RNA persists in the brain at times when infectious virus is not detected. To determine if genetic changes were occurring during viral replication in the host, we cloned and sequenced the RT-PCR products from the spike and nucleocapsid regions and analyzed the sequences for mutations. Sequencing of the cloned products revealed that a variety of mutant forms of viral RNA persisted in the CNS, including point mutants, deletion mutants, and termination mutants. The mutations accumulated during persistent infection in both the spike and the nucleocapsid sequences, with greater than 65% of the mutations encoding amino acid changes. These results show that a diverse population or quasispecies consisting of mutant and deletion variant viral RNAs (which may not be capable of producing infectious virus particles) persists in the central nervous system of mice during chronic demyelinating infection. The implications of these results for the role of persistent viral genetic information in the pathogenesis of chronic demyelination are discussed.

Amino Acid Sequence↗

PubMed1995

Demyelination induced by murine coronavirus JHM infection of congenitally immunodeficient mice.

Mouse hepatitis virus JHM (JHMV or MHV-4) induces demyelination in rodents and has been studied as a model for the human disease, multiple sclerosis (MS). As is proposed in MS, the mechanism of subacute demyelination induced by JHMV appears to be primarily immunopathological, since demyelination in JHMV-infected mice is abrogated by immunosuppressive doses of irradiation and restored by adoptive transfer of splenocytes. Thy-1+ cells play a critical role in transmitting disease to these recipient mice. To further characterize cells which may mediate JHMV-induced immunopathology, we inoculated congenitally immunodeficient mice with JHMV. By 12 days post-inoculation, both immunocompetent C57BL/6J controls and athymic nude C57BL/6 mice had severe paralysis and demyelination. In marked contrast, C57BL/6 mice with the severe combined immune deficiency (SCID) mutation had little or no paralysis or demyelination. Adoptive transfer of immune spleen cells from nude mice to infected SCID mice produced paralysis and demyelination. These findings suggest that a cell population present in immunocompetent C57BL/6J and nude mice but absent or non-functional in irradiated and SCID mice is essential for JHMV-induced demyelination. Identification of cells which mediate demyelination in this experimental system may have implications for our understanding of coronavirus pathogenesis and human demyelinating diseases.

Animals↗

PubMed1995

Mutations associated with viral sequences isolated from mice persistently infected with MHV-JHM.

Mouse hepatitis virus JHM (JHMV or MHV-4) induces subacute and chronic demyelination in rodents and has been studied as a model human demyelinating diseases, such a multiple sclerosis. However, despite intensive investigation, the state of JHMV during chronic disease is poorly understood. Using reverse transcription-polymerase chain reaction amplification (RT-PCR) to "rescue" viral RNA, we have found that JHMV-specific sequences persist for at least 787 days after intracerebral inoculation of experimental mice. Analysis of persisting viral RNA reveals that it is extensively mutated, and we hypothesize that the mutations observed reflect adaptation of the viral quasispecies to low-level intracellular replication during chronic disease.

Animals↗

PubMed1995

Coronaviruses.

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Animals↗

PubMed1993

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.

Animals↗

PubMed1993

Identification of an immunodominant linear neutralization domain on the S2 portion of the murine coronavirus spike glycoprotein and evidence that it forms part of complex tridimensional structure.

Numerous studies have demonstrated that the spike glycoprotein of coronaviruses bears major determinants of pathogenesis. To elucidate the antigenic structure of the protein, a panel of monoclonal antibodies was studied by competitive ELISA, and their reactivities were assayed against fragments of the murine coronavirus murine hepatitis virus strain A59 S gene expressed in prokaryotic vectors. An immunodominant linear domain was localized within the predicted stalk, S2, of the peplomer. It is recognized by several neutralizing antibodies. Other domains were also identified near the proteolytic cleavage site, in the predicted globular head, S1, and in another part of the stalk. Furthermore, competition results suggest that the immunodominant functional domain forms part of a complex three-dimensional structure. Surprisingly, some antibodies which have no antiviral biological activities were shown to bind the immunodominant neutralization domain.

Amino Acid Sequence↗

PubMed1992

Sequence analysis of the spike protein gene of murine coronavirus variants: study of genetic sites affecting neuropathogenicity.

Mouse hepatitis virus (MHV), a coronavirus, causes encephalitis and demyelination in susceptible rodents. Previous investigations have shown that the MHV spike (S) protein is a critical determinant of viral tropism and pathogenicity in mice and rats. To understand the molecular basis of MHV neuropathogenesis, we studied the spike protein gene sequences of several neutralization-resistant variants of the JHM strain of MHV, which were selected with monoclonal antibodies (MAbs) specific for the S protein. We found that variant 2.2-V-1, which was selected with MAb J.2.2 and primarily caused demyelination, had a single point mutation at nucleotide (NT) 3340, as compared to the parental JHM virus, which predominantly caused encephalitis. This site was in the S2 subunit of the S protein. In contrast, variant 7.2-V-1, which was selected with MAb J.7.2 and primarily caused encephalitis, had two point mutations at NT 1766 and 1950, which were in the S1 subunit. Finally, the double mutant 2.2/7.2-V-2, which was selected with both MAbs J.2.2 and J.7.2, and was attenuated with respect to both virulence and the ability to cause demyelination, had a deletion spanning from NT 1523 to 1624 in the S1 and a point mutation at NT 3340 in the S2. We conclude that at least two regions of the S protein contribute to neuropathogenicity of MHV. We have also isolated a partial revertant of 2.2-V-1, which was partially resistant to MAb J.2.2 but retained the same neuropathogenicity as the variant 2.2-V-1. This revertant retained the mutation at NT 3340, but had a second-site mutation at NT 1994, further confirming that NT 3340 contributed to the pathogenic phenotype of MHV. By comparing these results with MHV variants isolated in other laboratories, which had mutations in other sites on the S gene and yet retained the demyelinating ability, we suggest that the ability of JHM viruses to induce demyelination is determined by the interaction of multiple sites on the S gene, rather than the characteristics of a single, unique site. Our study also revealed the possible presence of microheterogeneity of S gene sequence, particularly in the S1 region, in these viruses. The sequence microheterogeneity may also contribute to the differences in their biological properties.

Animals↗

PubMed1992

Sequential infection of glial cells by the murine hepatitis virus JHM strain (MHV-4) leads to a characteristic distribution of demyelination.

An antigenic variant of the neurotropic murine coronavirus JHMV, designated 2.2-V-1, causes marked demyelination in the relative absence of encephalitis. It is thus useful for the study of the pathogenesis of demyelinating lesions. To better understand the sequential events leading to demyelination, we have examined murine brain and spinal cord tissue at daily intervals after intracerebral inoculation, evaluating them for the distribution of viral antigen, leukocyte infiltration, and demyelination. Immunohistochemical staining indicated that virus established primary infection in the ependymal cells in both brain and spinal cord before spreading into nearby structures and along white matter tracts by cell-to-cell contact. Spread from brain to spinal cord appeared to occur via cerebrospinal fluid. Viral replication was focally cytocidal for ependymal cells, and essentially noncytocidal for other neural cells including glia. In brain, viral antigen and inflammation reached a peak at day 5 postinfection, and rapidly subsided by day 10 postinfection. In spinal cord, viral antigen was less abundant than in brain and was maximal between days 7 and 9 postinfection. The inflammatory response and demyelination, however, were more severe persisting from day 7 through day 19. In the spinal cord, demyelinating lesions developed initially in areas closer to the central canal and were detected most prominently in the anterior funiculi. This finding suggests that the permissiveness of the ependymal cell is crucial to viral entry and that sequential infection of glial cells leads to the characteristic distribution of demyelination.

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PubMed1990

Immunopathogenesis of demyelination induced by MHV-4.

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Animals↗

PubMed1990

Demyelination induced by murine hepatitis virus JHM strain (MHV-4) is immunologically mediated.

The neurotropic mouse hepatitis viruses (MHV), in particular strain JHM (JHMV or MHV-4), cause experimental central nervous system demyelination that pathologically resembles multiple sclerosis, an important human demyelinating disease. The mechanism of JHMV-induced demyelination remains unclear, though its tropism for oligodendrocytes had led to the belief that JHMV causes demyelination by direct lysis of these myelin-producing cells. However, several studies have also implicated the involvement of immune responses in the demyelinating process. In this communication, we present evidence that generalized immunosuppression with gamma irradiation prevents JHMV-induced demyelination, a finding that was not limited to a particular strain of JHMV or to one strain of mouse. In addition, significant paralytic-demyelinating disease was restored to infected, irradiated mice after the adoptive transfer of nylon wool nonadherent splenic cells and appeared to be restricted by the major histocompatibility complex (MHC). These observations indicate that the principal mechanisms of JHMV-induced demyelination are most likely immunopathological.

Animals↗

PubMed1990

Relapsing encephalomyelitis following transfer of partial immunity to JHM virus.

Mice infected with the JHM strain mouse hepatitis virus (JHMV) develop a fatal encephalomyelitis with evidence of demyelination. It has previously been shown that the adoptive transfer of 5 x 10(7) nylon wool adherent (NWA) spleen cells from immunized donors to lethally infected recipients clears virus from the central nervous system (CNS) and prevents demyelination. Adoptive transfer of a smaller number (1 x 10(7] of NWA spleen cells from immunized donors also protects from death but does not significantly alter virus replication in the CNS during the acute phase of the infection. Moreover, these mice develop a transient non-fatal encephalomyelitis which occurs approximately 3 weeks post-infection. This delayed encephalomyelitis is associated with a mononuclear cell infiltration into the CNS but little or no evidence of virus replication or increased viral antigen. A virus-specific delayed-type hypersensitivity (DTH) response precedes this delayed onset of disease by 24 to 48 h. Resolution of disease correlates with a selective and permanent suppression of the JHMV-specific DTH reactivity. In addition, no virus-specific DTH is detected following adoptive transfer of viral-specific DTH effectors derived from immunized donors. In contrast, these mice respond to a heterologous antigen, KLH, suggesting that the resolution of the encephalitis is accompanied by a profound suppression in viral-specific DTH response.

Animals↗