Tamoxifen Application Is Associated with Transiently Increased Loss of Hippocampal Neurons following Virus Infection.

Tamoxifen is frequently used in murine knockout systems with CreER/LoxP. Besides possible neuroprotective effects, tamoxifen is described as having a negative impact on adult neurogenesis. The present study investigated the effect of a high-dose tamoxifen application on Theiler's murine encephalomyelitis virus (TMEV)-induced hippocampal damage. Two weeks after TMEV infection, 42% of the untreated TMEV-infected mice were affected by marked inflammation with neuronal loss, whereas 58% exhibited minor inflammation without neuronal loss. Irrespective of the presence of neuronal loss, untreated mice lacked TMEV antigen expression within the hippocampus at 14 days post-infection (dpi). Interestingly, tamoxifen application 0, 2 and 4, or 5, 7 and 9 dpi decelerated virus elimination and markedly increased neuronal loss to 94%, associated with increased reactive astrogliosis at 14 dpi. T cell infiltration, microgliosis and expression of water channels were similar within the inflammatory lesions, regardless of tamoxifen application. Applied at 0, 2 and 4 dpi, tamoxifen had a negative impact on the number of doublecortin (DCX)-positive cells within the dentate gyrus (DG) at 14 dpi, without a long-lasting effect on neuronal loss at 147 dpi. Thus, tamoxifen application during a TMEV infection is associated with transiently increased neuronal loss in the hippocampus, increased reactive astrogliosis and decreased neurogenesis in the DG.

Bioinformatics Analysis of Gut Microbiota and CNS Transcriptome in Virus-Induced Acute Myelitis and Chronic Inflammatory Demyelination; Potential Association of Distinct Bacteria With CNS IgA Upregulation.

Virus infections have been associated with acute and chronic inflammatory central nervous system (CNS) diseases, e.g., acute flaccid myelitis (AFM) and multiple sclerosis (MS), where animal models support the pathogenic roles of viruses. In the spinal cord, Theiler's murine encephalomyelitis virus (TMEV) induces an AFM-like disease with gray matter inflammation during the acute phase, 1 week post infection (p.i.), and an MS-like disease with white matter inflammation during the chronic phase, 1 month p.i. Although gut microbiota has been proposed to affect immune responses contributing to pathological conditions in remote organs, including the brain pathophysiology, its precise role in neuroinflammatory diseases is unclear. We infected SJL/J mice with TMEV; harvested feces and spinal cords on days 4 (before onset), 7 (acute phase), and 35 (chronic phase) p.i.; and examined fecal microbiota by 16S rRNA sequencing and CNS transcriptome by RNA sequencing. Although TMEV infection neither decreased microbial diversity nor changed overall microbiome patterns, it increased abundance of individual bacterial genera Marvinbryantia on days 7 and 35 p.i. and Coprococcus on day 35 p.i., whose pattern-matching with CNS transcriptome showed strong correlations: Marvinbryantia with eight T-cell receptor (TCR) genes on day 7 and with seven immunoglobulin (Ig) genes on day 35 p.i.; and Coprococcus with gene expressions of not only TCRs and IgG/IgA, but also major histocompatibility complex (MHC) and complements. The high gene expression of IgA, a component of mucosal immunity, in the CNS was unexpected. However, we observed substantial IgA positive cells and deposition in the CNS, as well as a strong correlation between CNS IgA gene expression and serum anti-TMEV IgA titers. Here, changes in a small number of distinct gut bacteria, but not overall gut microbiota, could affect acute and chronic immune responses, causing AFM- and MS-like lesions in the CNS. Alternatively, activated immune responses would alter the composition of gut microbiota.

Impaired upregulation of Stat2 gene restrictive to pancreatic ß-cells is responsible for virus-induced diabetes in DBA/2 mice.

Viral infection is a putative causal factor for the development of type 1 diabetes, but the exact pathogenic mechanism of virus-induced diabetes (VID) remains unclear. Here, to identify the critical factors that regulate VID, we analyzed encephalomyocarditis D (EMC-D) VID-sensitive DBA/2 mice in comparison with resistant B6 mice. EMC-D virus-induced cell death occurred more frequently in DBA/2 ß-cells than in B6 ß-cells with 100U/ml IFN-ß priming in vitro. We therefore purified ß-cells using flow cytometry from mice two days after EMC-D virus infection and subjected them to microarray analysis. As a results, innate immune response pathway was found to be enriched in B6 ß-cells. The signal transducer and activator of transcription 2 (Stat2) gene interacted with genes in the pathway. Stat2 gene expression levels were lower in DBA/2 mice than in B6 mice, restrictive to ß-cells. Moreover, administration of IFN-ß failed to upregulate Stat2 gene in DBA/2 ß-cells than in those of B6 in vivo. The viral titer significantly increased only in the DBA/2 pancreas. Thus, these provided data suggest that impaired upregulation of Stat2 gene restrictive to ß-cells at the early stage of infection is responsible for VID development in DBA/2 mice.

Impact of Astrocyte Depletion upon Inflammation and Demyelination in a Murine Animal Model of Multiple Sclerosis.

Astrocytes play a key role in demyelinating diseases, like multiple sclerosis (MS), although many of their functions remain unknown. The aim of this study was to investigate the impact of astrocyte depletion upon de- and remyelination, inflammation, axonal damage, and virus distribution in Theiler`s murine encephalomyelitis (TME). Groups of two to six glial fibrillary acidic protein (GFAP)-thymidine-kinase transgenic SJL mice and SJL wildtype mice were infected with TME virus (TMEV) or mock (vehicle only). Astrocyte depletion was induced by the intraperitoneal administration of ganciclovir during the early and late phase of TME. The animals were clinically investigated while using a scoring system and a rotarod performance test. Necropsies were performed at 46 and 77 days post infection. Cervical and thoracic spinal cord segments were investigated using hematoxylin and eosin (H&E), luxol fast blue-cresyl violet (LFB), immunohistochemistry targeting Amigo2, aquaporin 4, CD3, CD34, GFAP, ionized calcium-binding adapter molecule 1 (Iba1), myelin basic protein (MBP), non-phosphorylated neurofilaments (np-NF), periaxin, S100A10, TMEV, and immunoelectron microscopy. The astrocyte depleted mice showed a deterioration of clinical signs, a downregulation and disorganization of aquaporin 4 in perivascular astrocytes accompanied by vascular leakage. Furthermore, astrocyte depleted mice showed reduced inflammation and lower numbers of TMEV positive cells in the spinal cord. The present study indicates that astrocyte depletion in virus triggered CNS diseases contributes to a deterioration of clinical signs that are mediated by a dysfunction of perivascular astrocytes.

Genetic and pharmacological manipulation of glial glutamate transporters does not alter infection-induced seizure activity.

The contribution of glial transporters to glutamate movement across the membrane has been identified as a potential target for anti-seizure therapies. Two such glutamate transporters, GLT-1 and system xc-, are expressed on glial cells, and modulation of their expression and function have been identified as a means by which seizures, neuronal injury, and gliosis can be reduced in models of brain injury. While GLT-1 is responsible for the majority of glutamate uptake in the brain, system xc- releases glutamate in the extracellular cleft in exchange for cystine and represents as such the major source of hippocampal extracellular glutamate. Using the Theiler's Murine Encephalomyelitis Virus (TMEV) model of viral-induced epilepsy, we have taken two well-studied approaches, one pharmacological, one genetic, to investigate the potential role(s) of GLT-1 and system xc- in TMEV-induced pathology. Our findings suggest that the methods we utilized to modulate these glial transporters, while effective in other models, are not sufficient to reduce the number or severity of behavioral seizures in TMEV-infected mice. However, genetic knockout of xCT, the specific subunit of system xc-, may have cellular effects, as we observed a slight decrease in neuronal injury caused by TMEV and an increase in astrogliosis in the CA1 region of the hippocampus. Furthermore, xCT knockout caused an increase in GLT-1 expression selectively in the cortex. These findings have significant implications for both the characterization of the TMEV model as well as for future efforts to discover novel and effective anti-seizure drugs.

Characterization of Plaque-Sized Variants of Daniel's (DA) Strain in Theiler's Virus-Induced Epilepsy.

Epilepsy is a complex neurological disease characterized by recurrent seizures. Patients with viral encephalitis have a 16-fold increased risk of developing epilepsy, and this risk can persist for about 15 years after the occurrence of initial viral infection. Theiler's murine encephalomyelitis virus (TMEV) infection induces a well-characterized experimental model of epilepsy in C57BL/6 mice. In response to intracerebral (I.C.) injection of Daniel's (DA) strain of TMEV, there is vigorous immune response, which is detrimental to neurons and contributes to acute seizures, rendering mice susceptible to epilepsy. A comparative in vivo challenge study with either one of the two variants of the DA strain, small (DA-DS) or large (DA-CL) plaque forming variants, revealed differences in the diseases they induced in C57BL/6 mice. Compared to DA-CL-, DA-DS-infected mice exhibited significantly more seizures, higher clinical scores, neuroinflammation, and neuronal damage (mainly in the CA1-CA2 regions of hippocampus). Moreover, the brains of DA-DS infected mice contained approximately five-fold higher virus than those of DA-CL infected mice. A sequence comparison of the DA-CL and DA-DS genome sequences showed mutations in the leader (L) and L* proteins of DA-CL variant, which may be the cause of attenuating phenotype of DA-CL variant in the C57BL/6 mouse model of epilepsy.

Indoleamine 2,3-dioxygenase 1 deletion promotes Theiler's virus-induced seizures in C57BL/6J mice.

OBJECTIVE: Viral encephalitis increases the risk for developing seizures and epilepsy. Indoleamine 2,3-dioxygenase 1 (Ido1) is induced by inflammatory cytokines and functions to metabolize tryptophan to kynurenine. Kynurenine can be further metabolized to produce kynurenic acid and the N-methyl-d-aspartate receptor agonist quinolinic acid (QuinA). In the present study, we sought to determine the role of Ido1 in promoting seizures in an animal model of viral encephalitis. METHODS: C57BL/6J and Ido1 knockout mice (Ido1-KO) were infected with Theiler's murine encephalomyelitis virus (TMEV). Quantitative real-time polymerase chain reaction was used to evaluate hippocampal expression of proinflammatory cytokines, Ido1, and viral RNA. Body weights and seizure scores were recorded daily. Elevated zero maze was used to assess differences in behavior, and hippocampal pathology was determined by immunohistochemistry. RESULTS: Infected C57BL/6J mice up-regulated proinflammatory cytokines, Ido1, and genes encoding the enzymatic cascade responsible for QuinA production in the kynurenine pathway prior to the onset of seizures. Seizure incidence was elevated in Ido1-KO compared to C57BL/6J mice. Infection increased locomotor activity in Ido1-KO compared to C57BL/6J mice. Furthermore, the occurrence of seizures was associated with hyperexcitability. Neither expression of proinflammatory cytokines nor viral RNA was altered as a result of genotype. Immunohistochemical analysis revealed increased hippocampal pathology in Ido1-KO mice. SIGNIFICANCE: Our findings suggest that Ido1 deletion promotes seizures and neuropathogenesis during acute TMEV encephalitis.

Crucial role of Reg I from acinar-like cell cluster touching with islets (ATLANTIS) on mitogenesis of beta cells in EMC virus-induced diabetic mice.

Recently, we reported the presence of distinct cell clusters named acinar-like cell clusters touching Langerhans islets with thin interstitial surrounding (ATLANTIS) in human pancreas. A morphological study in humans demonstrated that ATLANTIS and islet cell clusters are found together in the microenvironment enclosed by a common basement membrane, and ATLANTIS releases vesicles containing Regenerating gene protein (REG Iα) to islet cell clusters. We examined 1) the presence or absence of ATLANTIS in homozygous Reg I (mouse homologue of human REG Iα) deficient (Reg I-/-) and wild-type mice, and 2) the possible role of ATLANTIS in the regeneration of beta cell clusters after encephalomyocarditis (EMC) virus (D-variant) infection in Reg I-/- and wild-type mice. ATLANTIS was found in both wild-type and Reg I-/- mice. In both groups, mean blood glucose increased transiently to greater than 14.0 mmol/L at 5 days after EMC virus infection and recovered to baseline at 12 days. At 12 days after EMC virus infection, lower BrdU labeling indices were observed in islet beta cells of Reg I-/- mice compared to wild-type mice. Beta cell volume 12 days after EMC virus infection in Reg I-/- mice did not differ from that of wild-type mice. These results suggest that Reg I, which is released from ATLANTIS to islet beta cell clusters, has a crucial role in beta cell regeneration in EMC virus-induced diabetes. The presence of mechanism(s) other than that mediated by Reg I in beta cell restoration after destruction by EMC virus was also suggested.

Kir 4.1 inward rectifier potassium channel is upregulated in astrocytes in a murine multiple sclerosis model.

Multiple sclerosis (MS) is a high prevalence degenerative disease characterized at the cellular level by glial and neuronal cell death. The causes of cell death during the disease course are not fully understood. In this work we demonstrate that in a MS model induced by Theiler's murine encephalomyelitis virus (TMEV) infection, the inward rectifier (Kir) 4.1 potassium channel subunit is overexpressed in astrocytes. In voltage clamp experiments the inward current density from TMEV-infected astrocytes was significantly larger than in mock-infected ones. The cRNA hybridization analysis from mock- and TMEV-infected cells showed an upregulation of a potassium transport channel coding sequence. We validated this mRNA increase by RT-PCR and quantitative PCR using Kir 4.1 specific primers. Western blotting experiments confirmed the upregulation of Kir 4.1, and alignment between sequences provided the demonstration that the over-expressed gene encodes for a Kir family member. Flow cytometry showed that the Kir 4.1 protein is located mainly in the cell membrane in mock and TMEV-infected astrocytes. Our results demonstrate an increase in K+ inward current in TMEV-infected glial cells, this increment may reduce the neuronal depolarization, contributing to cell resilience mechanisms.

Effects of Keratinocyte-Derived Cytokine (CXCL-1) on the Development of Theiler's Virus-Induced Demyelinating Disease.

CXCL-1, also called keratinocyte-derived cytokine (KC), is a predominant chemokine produced in glial cells upon infection with Theiler's murine encephalomyelitis virus (TMEV). In this study, we assessed the role of KC in the development of TMEV-induced demyelinating disease by utilizing polyclonal anti-KC antibodies as well as KC-expressing recombinant TMEV. Our results indicate that the level of KC produced after infection with TMEV or stimulation with various TLRs is significantly higher in various cells from susceptible SJL mice compared to those in cells from resistant B6 mice. SJL mice treated with rabbit anti-KC antibodies displayed accelerated development of TMEV-induced demyelinating disease, elevated viral loads in the CNS and decreased antiviral T cell responses. In addition, infection of susceptible SJL mice with recombinant KC-TMEV produced biologically active KC, which resulted in the accelerated pathogenesis of demyelinating disease and elevated T cell responses to viral antigens compared to mice infected with control recombinant HEL-TMEV. These results strongly suggest that both the lack of KC during TMEV infection and the excessive presence of the chemokine promote the pathogenesis of demyelinating disease. Therefore, a balance in the level of KC during TMEV infection appears to be critically important in controlling the pathogenesis of demyelinating disease.

microRNA-219 Reduces Viral Load and Pathologic Changes in Theiler's Virus-Induced Demyelinating Disease.

Analysis of microRNA (miR) expression in the central nervous system white matter of SJL mice infected with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) revealed a significant reduction of miR-219, a critical regulator of myelin assembly and repair. Restoration of miR-219 expression by intranasal administration of a synthetic miR-219 mimic before disease onset ameliorates clinical disease, reduces neurogliosis, and partially recovers motor and sensorimotor function by negatively regulating proinflammatory cytokines and virus RNA replication. Moreover, RNA sequencing of host lesions showed that miR-219 significantly downregulated two genes essential for the biosynthetic cholesterol pathway, Cyp51 (lanosterol 14-α-demethylase) and Srebf1 (sterol regulatory element-binding protein-1), and reduced cholesterol biosynthesis in infected mice and rat CG-4 glial precursor cells in culture. The change in cholesterol biosynthesis had both anti-inflammatory and anti-viral effects. Because RNA viruses hijack endoplasmic reticulum double-layered membranes to provide a platform for RNA virus replication and are dependent on endogenous pools of cholesterol, miR-219 interference with cholesterol biosynthesis interfered virus RNA replication. These findings demonstrate that miR-219 inhibits TMEV-induced demyelinating disease through its anti-inflammatory and anti-viral properties.

Macrophage depletion by liposome-encapsulated clodronate suppresses seizures but not hippocampal damage after acute viral encephalitis.

Viral encephalitis is a major risk factor for the development of seizures and epilepsy, but the underlying mechanisms are only poorly understood. Mouse models such as viral encephalitis induced by intracerebral infection with Theiler's virus in C57BL/6 (B6) mice allow advancing our understanding of the immunological and virological aspects of infection-induced seizures and their treatment. Previous studies using the Theiler's virus model in B6 mice have indicated that brain-infiltrating inflammatory macrophages and the cytokines released by these cells are key to the development of acute seizures and hippocampal damage in this model. However, approaches used to prevent or reduce macrophage infiltration were not specific, so contribution of other mechanisms could not be excluded. In the present study, we used a more selective and widely used approach for macrophage depletion, i.e., systemic administration of clodronate liposomes, to study the contribution of macrophage infiltration to development of seizures and hippocampal damage. By this approach, almost complete depletion of monocytic cells was achieved in spleen and blood of Theiler's virus infected B6 mice, which was associated with a 70% decrease in the number of brain infiltrating macrophages as assessed by flow cytometry. Significantly less clodronate liposome-treated mice exhibited seizures than liposome controls (P<0.01), but the development of hippocampal damage was not prevented or reduced. Clodronate liposome treatment did not reduce the increased Iba1 and Mac3 labeling in the hippocampus of infected mice, indicating that activated microglia may contribute to hippocampal damage. The unexpected mismatch between occurrence of seizures and hippocampal damage is thought-provoking and suggests that the mechanisms involved in degeneration of specific populations of hippocampal neurons in encephalitis-induced epilepsy are more complex than previously thought.

A CD103+ Conventional Dendritic Cell Surveillance System Prevents Development of Overt Heart Failure during Subclinical Viral Myocarditis.

Innate and adaptive immune cells modulate heart failure pathogenesis during viral myocarditis, yet their identities and functions remain poorly defined. We utilized a combination of genetic fate mapping, parabiotic, transcriptional, and functional analyses and demonstrated that the heart contained two major conventional dendritic cell (cDC) subsets, CD103+ and CD11b+, which differentially relied on local proliferation and precursor recruitment to maintain their tissue residency. Following viral infection of the myocardium, cDCs accumulated in the heart coincident with monocyte infiltration and loss of resident reparative embryonic-derived cardiac macrophages. cDC depletion abrogated antigen-specific CD8+ T cell proliferative expansion, transforming subclinical cardiac injury to overt heart failure. These effects were mediated by CD103+ cDCs, which are dependent on the transcription factor BATF3 for their development. Collectively, our findings identified resident cardiac cDC subsets, defined their origins, and revealed an essential role for CD103+ cDCs in antigen-specific T cell responses during subclinical viral myocarditis.

The role of peripheral interleukin-6 in the development of acute seizures following virus encephalitis.

Seizure disorders are often associated with infectious etiologies. Infection, via the intracerebral (i.c.) route, of C57BL/6J mice with the Daniels (DA) strain of Theiler's murine encephalomyelitis virus (TMEV) results in approximately 50% of the mice developing acute behavioral seizures. TMEV-DA is the wild-type strain of the virus that replicates within the parenchyma of the brain. A variant of TMEV-DA, TMEV-H101, does not replicate within the parenchyma of the brain. However, infection with TMEV-H101 via the i.c. route still results in approximately 40% of the mice developing acute behavioral seizures. Infiltrating macrophages producing interleukin-6 (IL-6) have been implicated in the induction of acute seizures following TMEV-DA infection. We examined macrophage infiltration and microglial activation within the brain and cytokine levels in the periphery in mice infected with TMEV-DA or TMEV-H101 and assessed the effects of the addition of recombinant IL-6 to the periphery in wild-type and IL-6 knockout mice infected with TMEV-DA. We found that pathologic levels of IL-6 in the periphery may play a role in the development of seizures when viral replication within the brain is limited. Examination of the role played by the peripheral immune system in the development of seizures/epilepsy in the TMEV-induced seizure model, the first viral infection driven model for epilepsy, could lead to the elucidation of novel therapeutics.

Prostaglandin E2 produced following infection with Theiler's virus promotes the pathogenesis of demyelinating disease.

Infection of various cells with Theiler's murine encephalomyelitis virus (TMEV) activates the TLR- and melanoma differentiation-associated gene 5 (MDA5)-dependent pathways, resulting in the production of IL-1ß via the activation of caspase-1 upon assembly of the node-like receptor protein 3 (NLRP3) inflammasome. The role of IL-1ß in the pathogenesis of TMEV-induced demyelinating disease was previously investigated. However, the signaling effects of prostaglandin E2 (PGE2) downstream of the NLRP3 inflammasome on the immune responses to viral determinants and the pathogenesis of demyelinating disease are unknown. In this study, we investigated the levels of intermediate molecules leading to PGE2 signaling and the effects of blocking PGE2 signaling on the immune response to TMEV infection, viral persistence and the development of demyelinating disease. We demonstrate here that TMEV infection activates the NLRP3 inflammasome and PGE2 signaling much more vigorously in dendritic cells (DCs) and CD11b+ cells from susceptible SJL mice than in cells from resistant B6 mice. Inhibition of virus-induced PGE2 signaling using AH23848 resulted in decreased pathogenesis of demyelinating disease and viral loads in the central nervous system (CNS). In addition, AH23848 treatment caused the elevation of protective early IFN-γ-producing CD4+ and CD8+ T cell responses. Because the levels of IFN-ß were lower in AH23848-treated mice but the level of IL-6 was similar, over-production of pathogenic IFN-ß was modulated and the generation of IFN-γ-producing T cell responses was enhanced by the inhibition of PGE2 signaling. These results strongly suggest that excessive activation of the NLRP3 inflammasome and downstream PGE2 signaling contribute to the pathogenesis of TMEV-induced demyelinating disease.

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis.

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

Neuronal Injury, Gliosis, and Glial Proliferation in Two Models of Temporal Lobe Epilepsy.

It is estimated that 30%-40% of epilepsy patients are refractory to therapy and animal models are useful for the identification of more efficacious therapeutic agents. Various well-characterized syndrome-specific models are needed to assess their relevance to human seizure disorders and their validity for testing potential therapies. The corneal kindled mouse model of temporal lobe epilepsy (TLE) allows for the rapid screening of investigational compounds, but there is a lack of information as to the specific inflammatory pathology in this model. Similarly, the Theiler murine encephalomyelitis virus (TMEV) model of TLE may prove to be useful for screening, but quantitative assessment of hippocampal pathology is also lacking. We used immunohistochemistry to characterize and quantitate acute neuronal injury and inflammatory features in dorsal CA1 and dentate gyrus regions and in the directly overlying posterior parietal cortex at 2 time points in each of these TLE models. Corneal kindled mice were observed to have astrogliosis, but not microgliosis or neuron cell death. In contrast, TMEV-injected mice had astrogliosis, microgliosis, neuron death, and astrocyte and microglial proliferation. Our results suggest that these 2 animal models might be appropriate for evaluation of distinct therapies for TLE.

Social disruption alters pain and cognition in an animal model of multiple sclerosis.

Although pain and cognitive deficits are widespread and debilitating symptoms of multiple sclerosis (MS), they remain poorly understood. Theiler's murine encephalomyelitis virus (TMEV) infection is an animal model of MS where disease course is exacerbated by prior stressors. Here chronic infection coupled with prior social stress increased pain behavior and impaired hippocampal-dependent memory consolidation during the demyelinating phase of disease in SJL mice. These results suggest that the TMEV model may be useful in investigating pain and cognitive impairments in MS. However, in contrast to prior Balb/cJ studies, stress failed to consistently alter behavioral and physiological indicators of disease course.

Evaluating an etiologically relevant platform for therapy development for temporal lobe epilepsy: effects of carbamazepine and valproic acid on acute seizures and chronic behavioral comorbidities in the Theiler's murine encephalomyelitis virus mouse model.

Central nervous system infections can underlie the development of epilepsy, and Theiler's murine encephalomyelitis virus (TMEV) infection in C57BL/6J mice provides a novel model of infection-induced epilepsy. Approximately 50-65% of infected mice develop acute, handling-induced seizures during the infection. Brains display acute neuropathology, and a high number of mice develop spontaneous, recurrent seizures and behavioral comorbidities weeks later. This study characterized the utility of this model for drug testing by assessing whether antiseizure drug treatment during the acute infection period attenuates handling-induced seizures, and whether such treatment modifies associated comorbidities. Male C57BL/6J mice infected with TMEV received twice-daily valproic acid (VPA; 200 mg/kg), carbamazepine (CBZ; 20 mg/kg), or vehicle during the infection (days 0-7). Mice were assessed twice daily during the infection period for handling-induced seizures. Relative to vehicle-treated mice, more CBZ-treated mice presented with acute seizures; VPA conferred no change. In mice displaying seizures, VPA, but not CBZ, reduced seizure burden. Animals were then randomly assigned to acute and long-term follow-up. VPA was associated with significant elevations in acute (day 8) glial fibrillary acidic protein (astrocytes) immunoreactivity, but did not affect NeuN (neurons) immunoreactivity. Additionally, VPA-treated mice showed improved motor performance 15 days postinfection (DPI). At 36 DPI, CBZ-treated mice traveled significantly less distance through the center of an open field, indicative of anxiety-like behavior. CBZ-treated mice also presented with significant astrogliosis 36 DPI. Neither CBZ nor VPA prevented long-term reductions in NeuN immunoreactivity. The TMEV model thus provides an etiologically relevant platform to evaluate potential treatments for acute seizures and disease modification.

Prevalence and genetic characteristics of Saffold cardiovirus in China from 2009 to 2012.

The epidemiology and clinical features of the Saffold cardiovirus (SAFV) remain ambiguous. The present study was designed to systematically and intensively investigate the epidemiological features of SAFV in pediatric patients in China. Three cohorts of pediatric patients were recruited from 2009 to 2012. Cohort 1 comprised patients with acute respiratory tract infections. Cohort 2 comprised patients with diarrhea. Cohort 3 comprised hand, foot, and mouth disease (HFMD) patients. A total of 115 patients (1.6%) among 6052 (17/1647, 12/2013, and 86/2392 in cohorts 1, 2, and 3, respectively) were SAFV-positive. The samples from 82 SAFV-positive patients were successfully sequenced, and four genotypes were identified: 8 SAFV-1, 41 SAFV-2, 29 SAFV-3, and 4 SAFV-6. A significantly higher detection rate was found in the HFMD patients than in other two cohorts (both P <0.001). A higher frequency of severe clinical outcome and nervous system manifestation were also observed in the SAFV-positive HFMD patients. Additionally, 6 (3.5%) cerebrospinal fluid and 7 (2.2%) serum samples from the HFMD-associated encephalitis patients were SAFV-positive. Based on the VP1 sequences, all four genotypes displayed distinct geographical clustering. SAFV infection might be associated with a wide clinical spectrum and contribute to HFMD.