Theiler's Virus Research Articles

Induction of Blood Brain Barrier Tight Junction Protein Alterations by CD8 T Cells

Disruption of the blood brain barrier (BBB) is a hallmark feature of immune-mediated neurological disorders as diverse as viral hemorrhagic fevers, cerebral malaria and acute hemorrhagic leukoencephalitis. Although current models hypothesize that immune cells promote vascular permeability in human disease, the role CD8 T cells play in BBB breakdown remains poorly defined. Our laboratory has developed a novel murine model of CD8 T cell mediated central nervous system (CNS) vascular permeability using a variation of the Theiler's virus model of multiple sclerosis. In previous studies, we observed that MHC class II−/− (CD4 T cell deficient), IFN-γR−/−, TNF-α−/−, TNFR1−/−, TNFR2−/−, and TNFR1/TNFR2 double knockout mice as well as those with inhibition of IL-1 and LTβ activity were susceptible to CNS vascular permeability. Therefore, the objective of this study was to determine the extent immune effector proteins utilized by CD8 T cells, perforin and FasL, contributed to CNS vascular permeability. Using techniques such as fluorescent activated cell sorting (FACS), T1 gadolinium-enhanced magnetic resonance imaging (MRI), FITC-albumin leakage assays, microvessel isolation, western blotting and immunofluorescent microscopy, we show that in vivo stimulation of CNS infiltrating antigen-specific CD8 T cells initiates astrocyte activation, alteration of BBB tight junction proteins and increased CNS vascular permeability in a non-apoptotic manner. Using the aforementioned techniques, we found that despite having similar expansion of CD8 T cells in the brain as wildtype and Fas Ligand deficient animals, perforin deficient mice were resistant to tight junction alterations and CNS vascular permeability. To our knowledge, this study is the first to demonstrate that CNS infiltrating antigen-specific CD8 T cells have the capacity to initiate BBB tight junction disruption through a non-apoptotic perforin dependent mechanism and our model is one of few that are useful for studies in this field. These novel findings are highly relevant to the development of therapies designed to control immune mediated CNS vascular permeability.

Acute hemorrhagic demyelination in a murine model of multiple sclerosis

Acute hemorrhagic leukoencephalomyelitis (AHLE) is a rare neurological condition characterized by the development of acute hemorrhagic demyelination and high mortality. The pathomechanism of AHLE, as well as potential therapeutic approaches, have remained elusive due to the lack of suitable animal models. We report the first murine model of AHLE using a variation of the Theiler's Murine Encephalitis Virus (TMEV) MS model. During acute TMEV infection, C57BL/6 mice do not normally undergo demyelination. However, when 7 day TMEV infected C57BL/6 mice are intravenously administered the immunodominant CD8 T cell peptide, VP2121–130, animals develop characteristics of human AHLE based on pathologic, MRI and clinical features including microhemorrhages, increased blood-brain barrier permeability, and demyelination. The animals also develop severe disability as assessed using the rotarod assay. This study demonstrates the development of hemorrhagic demyelination in TMEV infected C57BL/6 mice within 24 hours of inducing this condition through intravenous administration of CD8 T cell restricted peptide. This study is also the first demonstration of rapid demyelination in a TMEV resistant non-demyelinating strain without transgenic alterations or pharmacologically induced immunosuppression.

Bioinformatic analysis suggests that a conserved ORF in the waikaviruses encodes an overlapping gene

The genus Waikavirus belongs to the order Picornavirales, whose members all use a polyprotein expression strategy. With the exception of Theiler's virus, overlapping genes are essentially unknown in the order. Recently, we reported experimental verification for a new short overlapping coding sequence (CDS) in the Potyviridae-a family in which overlapping genes were previously unknown. Using the same bioinformatics software (MLOGD), we have identified an approximately 89-codon conserved open reading frame (ORF) with a strong coding signature in members of the genus Waikavirus. The ORF overlaps the polyprotein ORF but is in the +1 reading frame. Here, we describe the bioinformatic analysis.

Glucocorticoid exposure alters the pathogenesis of Theiler's murine encephalomyelitis virus during acute infection

Previous research has shown that chronic restraint stress exacerbates Theiler's virus infection, a murine model for CNS inflammation and multiple sclerosis. The current set of experiments was designed to evaluate the potential role of glucocorticoids in the deleterious effects of restraint stress on acute CNS inflammatory disease. Exposure to chronic restraint stress resulted in elevated levels of corticosterone as well as increased clinical scores and weight loss (Experiment 1). In addition, corticosterone administration alone exacerbated behavioral signs of TMEV-induced sickness (i.e. decreased body weight, increased symptoms of encephalitis, and increased mortality) and reduced inflammation in the CNS (Experiment 2). Infected subjects receiving exogenous corticosterone showed exacerbation of acute phase measures of sickness and severe mortality as well as decreased viral clearance from CNS (Experiment 3). These findings indicate that corticosterone exposure alone is sufficient to exacerbate acute CNS inflammatory disease.

Replication of Theiler's virus requires NF‐κB‐activation: Higher viral replication and spreading in astrocytes from susceptible mice

To investigate viral replication and cell-cell spreading in astrocytes, recombinant Theiler's murine encephalomyelitis virus (TMEV) expressing green fluorescent protein (GFP) during the replication was generated. GFP and TMEV proteins were processed correctly in infected cells and production of viral proteins could be tracked by fluorescent microscopy. Viral replication of both wild-type TMEV and GFP-TMEV was dependent on the activation of NF-kappaB and partially MAP kinase, based on chemical inhibition studies. Viral replication was significantly reduced in primary astrocytes from NF-kappaB1 (p105)-deficient mice compared with that from wild-type control mice, whereas cytokine production was enhanced. These results suggest an association of canonical NF-kappaB subunits in viral replication, but not cytokine production. Viral replication was also suppressed in both IKKalpha and IKKbeta-deficient mouse embryonic fibroblasts (MEFs), compared with that in wild-type MEF. However, the inhibition was significantly greater in IKKbeta-deficient MEF, suggesting that IKKbeta plays a stronger role in supporting viral replication. Interestingly, viral replication and spreading in primary astrocytes from susceptible SJL/J mice were several-fold higher than those in astrocytes from resistant C57BL/6 mice, suggesting that higher viral replication levels in astrocytes may also contribute to the viral persistence in the central nervous system (CNS) of susceptible SJL/J mice. A relatively higher level of activated NF-kappaB was found in the nuclei of virus-infected SJL astrocytes compared with C57BL/6 astrocytes suggest that the NF-kappaB activation level affects on viral replication.

Roles of CD1d‐restricted Vα 14+ NKT cells in Theiler's virus infection, a viral model for multiple sclerosis

Most murine natural killer (NK) T cells express an invariant Vα 14 T cell receptor and are CD1d‐restricted. We investigated the contribution of NKT cells in an animal model for multiple sclerosis (MS), Theiler's murine encephalomyelitis virus (TMEV) infection. First, we treated susceptible SJL/J mice with an antibody against Vα 14 during TMEV infection. Treatment during the early stage of infection delayed the onset of clinical disease with relatively higher interleukin (IL)‐4 production. In contrast, treatment during the late stage or weekly injections resulted in more severe demyelination with higher virus persistence. Next, we compared TMEV infection in wild‐type versus CD1d KO mice in the background of TMEV‐resistant BALB/c mice. Although no wild‐type mice developed demyelination, 20 and 60% of CD1d KO mice developed demyelination at 3 and 5 weeks after infection, respectively. TMEV‐infected CD1d KO mice showed higher IL‐4 production and TMEV‐specific lymphoproliferative responses, compared with wild‐type mice. These results suggest that NKT cells could modulate inflammatory demyelinating disease by altering the cytokine profile and virus‐specific immune responses. This work was supported by the University of Utah (Funding Incentive Seed Grant to IT) and the National Institute of Health (NS34497 to RSF).

Being a mouse in a man's world: what TMEV has taught us about human disease

Choosing an appropriate animal model to study a disease is guided by a variety of factors including but not limited to the questions being asked, availability of reagents, knowledge of the animal species, personal biases of the researcher, and in some cases, cost and availability of facilities to effectively investigate the model. The validity of an animal model can be further complicated when the etiology of the disease is incompletely defined. Examples of these diseases include multiple sclerosis (MS) and type 1 diabetes (T1D). In addition to host genetics, epidemiological studies have implicated infectious agents, in particular viruses as triggers of these diseases. Thus many studies of these diseases have focused on modeling the interactions of viruses and the host immune response in vivo in small animals. Theiler's murine encephalomyelitis virus (TMEV) infection of mice has been used for over 30 years as a model of virus-induced demyelination. TMEV induces a MS-like disease in susceptible strains of mice but does not cause pathology in humans. While some researchers may question the rationale for using a non-human pathogen to model human disease, the TMEV model of central nervous system (CNS) demyelination has permitted study of some aspects of human MS which would have been difficult to address in other models of the disease. Despite being 'merely a disease of mice,' many of the findings in the Theiler's virus model are directly applicable to the human condition, and studies from the model are responsible for our current understanding of mechanisms of pathology and clinical disability in human MS. In this review we will present some of the key findings from the TMEV model in the context of human disease.

A survey of rodent-borne pathogens carried by wild-caught Norway rats: A potential threat to laboratory rodent colonies

Unintentional infection of laboratory rodents can compromise scientific research as well as the health of the animals and animal handlers. The source of contamination often is unknown, but may be introduced by wild rats from surrounding environments. To determine whether rats in Baltimore, Maryland, USA carry infectious agents commonly found in laboratory rodent colonies, we live-trapped 162 rats during 2005 to 2006 and screened them for a panel of viruses, bacteria and parasites. Antibodies against rat coronavirus/sialodacryoadenitis virus (91.7%), Mycoplasma pulmonis (72.9%), cilia-associated respiratory bacillus (52.1%), rat parvovirus/rat minute virus (29.2%), Kilham rat virus (10.4%), Toolan's H-1 virus (10.4%), Sendai virus (4.2%) and Theiler's mouse encephalomyelitis virus (4.2%), were detected in wild-caught Norway rats. Antibodies against reovirus and pneumonia virus of mice were not detected in wild Norway rats. Endoparasites, including Nippostrongylus braziliensis (71.6%), Rodentolepis nana or Hymenolepis diminuta (34.4%), Hetarakis spumosa (24.1%) and Trichuris muris (14.8%), as well as ectoparasites (14.8%), were identified in wild-caught rats. The risk of pathogen transmission from wild-caught rats to laboratory colonies needs to be mitigated by minimizing exposures rather than assuming wild animals represent a minimal hazard.

Axon Myelin Transfer of a Non-Enveloped Virus

We showed previously that Theiler's virus, a neurotropic non-enveloped picornavirus of mouse, traffics from the axon of infected neurons into the surrounding myelin. When this traffic is interrupted, as in the shiverer mouse which bears a mutation in the myelin basic protein gene, the virus is unable to persist in the central nervous system. In the present work, we used the Wlds mutant mouse, a strain in which axonal degeneration is considerably slowed down, to show that axon to myelin traffic takes place in the absence of axon degeneration. Our results suggest the existence of a mechanism of transfer of axonal cytoplasm into the myelin which Theiler's virus might exploit to ensure its persistence.

Targeting Inflammatory Demyelinating Lesions to Sites of Wallerian Degeneration

In Theiler’s murine encephalomyelitis virus (TMEV) infection, an animal model for multiple sclerosis (MS), axonal injury precedes inflammatory demyelinating lesions, and the distribution of axonal damage present during the early phase of infection corresponds to regions where subsequent demyelination occurs during the chronic phase. We hypothesized that axonal damage recruits inflammatory cells to sites of Wallerian degeneration, leading to demyelination. Three weeks after TMEV infection, axonal degeneration was induced in the posterior funiculus of mice by injecting the toxic lectin Ricinus communis agglutinin (RCA) I into the sciatic nerve. Neuropathology was examined 1 week after lectin injection. Control mice, infected with TMEV but receiving no RCA I, had inflammatory demyelinating lesions in the anterior/lateral funiculi. Other control mice that received RCA I alone did not develop inflammatory lesions. In contrast, RCA I injection into TMEV-infected mice induced lesions in the posterior funiculus in addition to the anterior/lateral funiculi. We found no differences in lymphoproliferative responses or antibody titers against TMEV among the groups. This suggests that axonal degeneration contributes to the recruitment of inflammatory cells into the central nervous system by altering the local microenvironment. In this scenario, lesions develop from the axon (inside) to the myelin (outside) (Inside-Out model).

Antibody-secreting cells in the central nervous system in an animal model of MS: Phenotype, association with disability, and in vitro production of antibody

Little is known about antibody production within the central nervous system, called intrathecal antibody production (ITAbP). Mice infected with Theiler's murine encephalomyocarditis virus (TMEV) develop an immune-mediated demyelinating disease(TMEV-IDD), characterized by progressive weakness associated with robust ITAbP, CNS inflammation and demyelination. TMEV-IDD represents an excellent model of human multiple sclerosis (MS), especially its progressive disability. The mechanism of the weakness in this disease is unknown but there is considerable evidence that it is immune-mediated. Our hypothesis was that the disability in the model is induced by robust ITAbP by antibody-secreting cells(ASCs) in the CNS. We further hypothesized that these ASCs are predominantly CD138+ plasma cells, driven by the persistence of virus at high levels in the central nervous system (CNS) as well as high levels of B-cell activating factor (BAFF). In order to test these hypotheses we infected mice with TMEV, and correlated measures of ITAbP with disability. Measures of ITAbP were ELISpot and IgG gene expression, while disability was tested by Rotarod. We found that disability was highly correlated with ITAbP, assayed by number of CNS ASCs as well as amount of IgG mRNA. CNS ASCs were primarily CD138+, and produced high levels of IgG enriched for reactivity to TMEV. There were high levels of BAFF production in the CNS, but these levels were only minimally higher in infected mice than in uninfected controls. TMEV and IgG RNA were distributed throughout the spinal cord. These data are the first demonstrating that ITAbP is highly correlated with disability in TMEV infection, an excellent model of human MS. Our data raise the possibility that ITAbP contributes significantly to disability, both in this model and in MS.

TGF-β1 suppresses T cell infiltration and VP2 puff B mutation enhances apoptosis in acute polioencephalitis induced by Theiler's virus

GDVII and DA strains of Theiler's murine encephalomyelitis virus (TMEV) differ in VP2 puff B. One week after GDVII virus infection, SJL/J mice had large numbers of TUNEL + apoptotic cells with a relative lack of T cell infiltration in the brain. DA viruses with mutation in puff B induced higher levels of apoptosis than wild-type DA virus, but levels of inflammation in brains were similar between DA and DA virus mutants. The difference in inflammation among TMEVs could be due to TGF-β1 expression that was seen only in GDVII virus infection and negatively correlated with CD3 + T cell infiltration.

Differential Virus Replication, Cytokine Production, and Antigen-Presenting Function by Microglia from Susceptible and Resistant Mice Infected with Theiler's Virus

Infection with Theiler's murine encephalomyelitis virus (TMEV) in the central nervous system (CNS) causes an immune system-mediated demyelinating disease similar to human multiple sclerosis in susceptible but not resistant strains of mice. To understand the underlying mechanisms of differential susceptibility, we analyzed viral replication, cytokine production, and costimulatory molecule expression levels in microglia and macrophages in the CNS of virus-infected resistant C57BL/6 (B6) and susceptible SJL/J (SJL) mice. Our results indicated that message levels of TMEV, tumor necrosis factor alpha, beta interferon, and interleukin-6 were consistently higher in microglia from virus-infected SJL mice than in those from B6 mice. However, the levels of costimulatory molecule expression, as well as the ability to stimulate allogeneic T cells, were significantly lower in TMEV-infected SJL mice than in B6 mice. In addition, microglia from uninfected naïve mice displayed differential viral replication, T-cell stimulation, and cytokine production, similar to those of microglia from infected mice. These results strongly suggest that different levels of intrinsic susceptibility to TMEV infection, cytokine production, and T-cell activation ability by microglia contribute to the levels of viral persistence and antiviral T-cell responses in the CNS, which are critical for the differential susceptibility to TMEV-induced demyelinating disease between SJL and B6 mice.

Interleukin 22 Is a Candidate Gene forTmevp3, a Locus Controlling Theiler's Virus-Induced Neurological Diseases

After intracerebral inoculation, Theiler's virus induces in its natural host, the mouse, an acute encephalomyelitis followed, in susceptible animals, by chronic inflammation and primary demyelination. Susceptibility to demyelination among strains of laboratory mice is explained by the capacity of the immune system to control viral load during persistence. Also, differences of susceptibility to viral load between the susceptible SJL strain and the resistant B10.S strain are mainly due to two loci, Tmevp2 and Tmevp3, located close to the Ifng locus on chromosome 10. In this article, we show that the Tmevp3 locus controls both mortality during the acute encephalomyelitis and viral load during persistence. Most probably, two genes located in the Tmevp3 interval control these two different phenotypes with efficiencies that depend on the age of the mouse at inoculation. Il22, a member of the IL-10 cytokine family, is a candidate gene for the control of mortality during the acute encephalomyelitis.

Effectors of Demyelination and Remyelination in the CNS: Implications for Multiple Sclerosis

Most of the research on multiple sclerosis (MS) has focused on the early events that trigger demyelination and subsequent remyelination. Less attention has been given to the factors that directly mediate the demyelination that is the hallmark of the disease. Effector cells or molecules are those factors directly responsible for mediating the damage in the disease. Similarly, there are effector molecules that are critical for remyelination in the central nervous system (CNS). By understanding those effector molecules in demyelination and remyelination that directly influence the pathologic process, we should be able to generate specific therapies with the greatest potential for benefiting MS patients. This review focuses on effector cells and molecules that are critical for demyelination and remyelination in MS but also in experimental models of the disease including experimental autoimmune encephalomyelitis (EAE), virus-induced models of demyelination (Theiler's virus, murine hepatitis virus), and toxic models of demyelination (lysolecithin, ethidium bromide, and cuprizone). These are models in which the effector molecules for demyelination and remyelination have been most precisely evaluated.

The Role of Myelin in Theiler's Virus Persistence in the Central Nervous System

Theiler's virus, a picornavirus, persists for life in the central nervous system of mouse and causes a demyelinating disease that is a model for multiple sclerosis. The virus infects neurons first but persists in white matter glial cells, mainly oligodendrocytes and macrophages. The mechanism, by which the virus traffics from neurons to glial cells, and the respective roles of oligodendrocytes and macrophages in persistence are poorly understood. We took advantage of our previous finding that the shiverer mouse, a mutant with a deletion in the myelin basic protein gene (Mbp), is resistant to persistent infection to examine the role of myelin in persistence. Using immune chimeras, we show that resistance is not mediated by immune responses or by an efficient recruitment of inflammatory cells into the central nervous system. With both in vivo and in vitro experiments, we show that the mutation does not impair the permissiveness of neurons, oligodendrocytes, and macrophages to the virus. We demonstrate that viral antigens are present in cytoplasmic channels of myelin during persistent infection of wild-type mice. Using the optic nerve as a model, we show that the virus traffics from the axons of retinal ganglion cells to the cytoplasmic channels of myelin, and that this traffic is impaired by the shiverer mutation. These results uncover an unsuspected axon to myelin traffic of Theiler's virus and the essential role played by the infection of myelin/oligodendrocyte in persistence.

Exacerbated Pathology of Viral Encephalitis in Mice with Central Nervous System-Specific Autoantibodies

We examine here the outcome of viral encephalomyelitis [mouse hepatitis virus (MHV) A59, Theiler’s encephalomyelitis virus, and Coxsackievirus B3] in mice with autoantibodies to a central nervous system (CNS)-specific antigen, myelin oligodendrocyte glycoprotein, that usually develop no clinical disease. Morbidity and mortality of the acute viral CNS disease was augmented by the presence of the autoantibodies in all three viral infections. Transfer of serum containing the autoantibodies at the time of infection with MHV was sufficient to reproduce the exacerbated disease. The presence of the autoantibodies was found to result in increased infiltration of mononuclear cells into the brain. Early demyelination was severely augmented in brains and spinal cords of MHV-infected mice with CNS-specific autoantibodies. The antibody-mediated exacerbation was shown to be independent of the complement system but to require expression of Fc receptors, because it was observed in C′-3-deficient but not in Fc receptor-deficient mice. Our study illustrates the possibility that infections can lead to much more profound immunopathology in the presence of an otherwise latent autoimmune condition.

Theiler's virus strain-dependent induction of innate immune responses in RAW264.7 macrophages and its influence on viral clearance versus viral persistence

Infection of susceptible mice with the DA strain of Theiler's murine encephalomyelitis virus (TMEV) induces a persistent central nervous system infection accompanied by demyelination that resembles multiple sclerosis. In contrast, Theiler's GDVII strain does not persist, because infected animals either clear the virus or die. Previously, the authors have shown that in vitro infection of RAW264.7 macrophages displays a similar strain-dependent outcome, resulting in the establishment of a persistent infection with the DA strain and clearance of the GDVII strain. Here, the authors show that when RAW264.7 cells were infected with both strains, the antiviral response triggered by the GDVII virus interfered with the DA virus' ability to induce a persistent infection. Treatment of cells with 2-aminopurine, a protein kinase R inhibitor, increased GDVII virus yields in contrast to DA virus yields. By comparing the antiviral activity of RAW264.7 macrophages against TMEV, it was found that GDVII-infected macrophages mounted a five times more potent antiviral response than the DA-infected ones, indicating that there are strain-dependent differences in the induction of host innate immune responses. Measurements of interferon (IFN) production confirmed this finding. In addition, it was found that the macrophages' antiviral response is dependent on the multiplicity of infection. The antiviral activity resulting from GDVII-infected macrophages could be partially neutralized with antibodies against IFN-alpha or IFN-gamma, but not with an anti-IFN-beta antibody. Because only a partial neutralization was reached, the authors speculate that apart from the investigated IFNs, other cellular factors contribute to the observed antiviral activity. Taken together, these results demonstrate the importance of host innate immune responses in determining the balance between viral clearance and viral persistence.

The immunodominant CD8+ T cell epitope region of Theiler's virus in resistant C57BL/6 mice is critical for anti-viral immune responses, viral persistence, and binding to the host cells

Theiler's virus infection induces an immune-mediated demyelinating disease, providing a relevant animal model of human multiple sclerosis. VP2121–130-specific CD8+ T cells in resistant H-2b mice account for the majority of CNS-infiltrating CD8+ T cells. To further study the role of the CD8+ T cells, we generated a panel of mutant viruses substituted with L, G, or T at the anchor residue (M130) of the VP2121–130 epitope. M130L virus (M130L-V) with a substitution of M with L displayed similar properties as wild-type virus (WT-V). However, M130G-V and M130T-V could not establish a persistent infection in the CNS. The level of both virus-specific CD8+ and CD4+ T cell responses is significantly reduced in mice infected with these variant viruses. While all mutant and wild-type viruses replicate comparably in BHK cells, replication of M130G-V and M130T-V in macrophages was significantly lower compared to those infected with WT-V and M130L-V. Interestingly, these mutant viruses deficient in replication in primary mouse cells showed drastically reduced binding ability to the cells. These results suggest that the anchor residue of the predominant CD8+ T cell epitope of TMEV in resistant mice is critical for the virus to infect target cells and this deficiency may result in poor viral persistence leading to correspondingly low T cell responses in the periphery and CNS. Thus, selection of the cellular binding region of the virus as the predominant epitope for CD8+ T cells in resistant mice may provide a distinct advantage in controlling viral persistence by preventing escape mutations.

Restraint stress facilitates systemic dissemination of Theiler's virus and alters its pathogenecity

Theiler's murine encephalomyelitis virus (TMEV), a Picornavirus used as a viral model for multiple sclerosis (MS), causes an acute encephalomyelitis and chronic demyelination. The failure to clear the virus, which can result from stress, is a prerequisite for development of the later disease. Similarly, stressful life events have been associated with the development of MS. In the present study, a restraint stress (RS) model was used to investigate the effect of stress on the systemic dissemination of TMEV during the early stage of disease. Experimental data demonstrated that repeated RS remarkably facilitated the spread of virus from the CNS to such systemic organs as the spleen, lymph nodes, thymus, lungs and heart and compromised the ability of viral clearance within those tissues. RS also altered the pathogenecity of TMEV, enabling it to become cardiotropic, resulting in higher myocardial infectivity. These results demonstrate the profound impact that RS has upon both the tissue and organ dissemination of the virus, and the organ tropism of TMEV. An additional finding associated with stress was hepatic necrosis in the restrained animals, regardless of whether or not they were infected.