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.

Chemokine receptors CCR2 and CX3CR1 regulate viral encephalitis-induced hippocampal damage but not seizures.

Viral encephalitis is a major risk factor for the development of seizures, epilepsy, and hippocampal damage with associated cognitive impairment, markedly reducing quality of life in survivors. The mechanisms underlying seizures and hippocampal neurodegeneration developing during and after viral encephalitis are only incompletely understood, hampering the development of preventive treatments. Recent findings suggest that brain invasion of blood-born monocytes may be critically involved in both seizures and brain damage in response to encephalitis, whereas the relative role of microglia, the brain's resident immune cells, in these processes is not clear. CCR2 and CX3CR1 are two chemokine receptors that regulate the responses of myeloid cells, such as monocytes and microglia, during inflammation. We used Ccr2-KO and Cx3cr1-KO mice to understand the role of these receptors in viral encephalitis-associated seizures and neurodegeneration, using the Theiler's virus model of encephalitis in C57BL/6 mice. Our results show that CCR2 as well as CX3CR1 plays a key role in the accumulation of myeloid cells in the CNS and activation of hippocampal myeloid cells upon infection. Furthermore, by using Cx3cr1-creER+/-tdTomatoSt/Wt reporter mice, we show that, with regard to CD45 and CD11b expression, some microglia become indistinguishable from monocytes during CNS infection. Interestingly, the lack of CCR2 or CX3CR1 receptors was associated with almost complete prevention of hippocampal damage but did not prevent seizure development after viral CNS infection. These data are compatible with the hypothesis that CNS inflammatory mechanism(s) other than the infiltrating myeloid cells trigger the development of seizures during viral encephalitis.

Development of a reverse transcription recombinase polymerase amplification assay for rapid detection of Theiler's murine encephalomyelitis virus.

Theiler's murine encephalomyelitis virus (TMEV) is one of the most common viral pathogens that circulate widely in captive mouse colonies. A molecular biology detection method would be a useful tool to use in an integrated program to monitor and prevent TMEV infection and transmission. Thus, a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed to detect TMEV infection. The sensitivity of the RT-RPA assay approached 8 copies per reaction, which is equivalent to the sensitivity of RT-qPCR reactions. This assay did not detect RNA extracts from other murine pathogens included in this study or TMEV negative samples. Brain tissues and contaminated biological materials were used to assess the clinical performance of the RT-RPA. The detection results of RT-RPA and RT-qPCR were very similar, except that a contaminated biological material sample which was positive by RT-qPCR, with a CT value of 38, was negative by RT-RPA. In summary, the developed RT-RPA assay offers a rapid, sensitive and specific alternative method for monitoring of TMEV, especially in resource-limited conditions.

The influence of macrophage growth factors on Theiler's Murine Encephalomyelitis Virus (TMEV) infection and activation of macrophages.

Macrophages are common targets for infection and innate immune activation by many pathogenic viruses including the neurotropic Theiler's Murine Encephalomyelitis Virus (TMEV). As both infection and innate activation of macrophages are key determinants of viral pathogenesis especially in the central nervous system (CNS), an analysis of macrophage growth factors on these events was performed. C3H mouse bone-marrow cells were differentiated in culture using either recombinant macrophage colony stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), inoculated with TMEV (BeAn) and analyzed at various times thereafter. Cytokine RNA and protein analysis, virus titers, and flow cytometry were performed to characterize virological parameters under these culture conditions. GM-CSF-differentiated macrophages showed higher levels of TMEV viral RNA and proinflammatory molecules compared to infected M-CSF-differentiated cells. Thus, GM-CSF increases both TMEV infection and TMEV-induced activation of macrophages compared to that seen with M-CSF. Moreover, while infectious viral particles decreased from a peak at 12h to undetectable levels at 48h post infection, TMEV viral RNA remained higher in GM-CSF- compared to M-CSF-differentiated macrophages in concert with increased proinflammatory gene expression. Analysis of a possible basis for these differences determined that glycolytic rates contributed to heightened virus replication and proinflammatory cytokine secretion in GM-CSF compared to M-CSF-differentiated macrophages. In conclusion, we provide evidence implicating a role for GM-CSF in promoting virus replication and proinflammatory cytokine expression in macrophages, indicating that GM-CSF may be a key factor for TMEV infection and the induction of chronic TMEV-induced immunopathogenesis in the CNS.

Analysis of Aichi virus and Saffold virus association with pediatric acute gastroenteritis.

BACKGROUND: Aichi virus (AiV) and Saffold virus (SAFV) have been reported in children with acute gastroenteritis and respiratory disease worldwide; however, their causative role in acute gastroenteritis remains ambiguous. OBJECTIVES: To assess the clinical association of AiV and SAFV with acute gastroenteritis in the pediatric population. STUDY DESIGN: A case-control study involving 461 paired stool samples from pediatric cases with diarrhea and healthy controls was conducted in China. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was used to screen AiV and SAFV. RESULTS: In the 461 paired samples, AiV and SAFV were more prevalent among asymptomatic children than children with acute gastroenteritis (0.87% vs. 0.43% and 2.8% vs. 1.5%, respectively), with no significant differences between groups (p=0.142 and p=0.478, respectively). Cox regression model analysis revealed no correlation between AiV (odds ratio, OR=2.24; 95% confidence interval, CI, 0.76-6.54) or SAFV infection (OR=1.36; 95% CI, 0.86-2.15) and diarrhea. High viral loads were found in both AiV- and SAFV-positive groups, with no significant difference in viral load between the groups (p=0.507 and p=0.677, respectively). No other known enteric pathogens were found in the AiV-positive samples but common in SAFV-positive cases. Phylogenetic analysis revealed that all 6 AiV subjects clustered with genotype B. All 7 SAFV-positive cases and 8 of 13 SAFV-positive controls were genotyped successfully; the genotypes identified included SAFV-1, SAFV-2 SAFV-3, and SAFV-6. CONCLUSION: Our study revealed no association of these viruses in acute gastroenteritis in children. These viruses may have the ability to replicate in humans; however, the infections are usually asymptomatic.

Saffold virus, an emerging human cardiovirus.

Saffold virus (SAFV) is an emerging human cardiovirus that has been shown to be ubiquitous. Initial studies of SAFV focused on respiratory and gastrointestinal infection; however, it has also recently been associated with diverse clinical symptoms including the endocrine, cardiovascular, and neurological systems. Given the systemic nature of SAFV, and its high prevalence, understanding its pathogenicity and clinical impact is of utmost importance. This comprehensive review highlights and discusses recent developments in epidemiology, human pathogenicity, animal, and molecular studies related to SAFV. It also provides detailed insights into the neuropathogenicity of SAFV. We argue that human studies have been confounded by coinfections and therefore require support from robust molecular and animal research. Thereby, we aim to provide foresight into further research to better understand this emerging virus.

Immunohistochemical insights into Saffold virus infection of the brain of juvenile AG129 mice.

BACKGROUND: Saffold Virus (SAFV) is a human cardiovirus that is suspected of causing infection of the central nervous system (CNS) in children. While recent animal studies have started to elucidate the pathogenesis of SAFV, very little is known about the mechanisms behind it. METHOD: In this study, we attempted to elucidate some of the mechanisms of the pathogenesis of SAFV in the brain of a juvenile mouse model by using immunohistochemical methods. RESULTS: We first showed that SAFV is able to infect both neuronal and glial cells in the brain of 2 week-old AG129 mice. We then showed that SAFV is able to induce apoptosis in both neuronal and glial cells in the brain. Lastly, we showed that SAFV infection does not show any signs of gross demyelination in the brain. CONCLUSION: Overall, our results provide important insights into the mechanisms of SAFV in the brain.

Development of a duplex real-time RT-PCR for the simultaneous detection and differentiation of Theiler's murine encephalomyelitis virus and rat theilovirus.

Theiler's murine encephalomyelitis virus (TMEV) and rat theilovirus (RTV), the member of the genus Cardiovirus, are widespread in laboratory mice and rats, and are potential contaminants of biological materials. Cardioviruses infection may cause serious complications in biomedical research. To improve the efficiency of routine screening for Cardioviruses infection, a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection and differentiation of TMEV and RTV. The duplex assay was specific for reference strains of TMEV and RTV, and no cross-reaction was found with seven other rodent viruses. The limits of detection of both TMEV and RTV were 4×10(1) copies RNA/reaction. Reproducibility was estimated using standard dilutions, with coefficients of variation <3.1%. 439 clinical samples were evaluated by both duplex real-time RT-PCR and conventional RT-PCR. For 439 clinical samples，95 samples were positive for TMEV and 72 samples were positive for RTV using duplex real-time RT-PCR approach, whereas only 77 samples were positive for TMEV and 66 samples were positive for RTV when conventional RT-PCR was applied. Mixed infections were found in 20 samples when analyzed by conventional RT-PCR whereas 30 samples were found to be mixed infection when duplex real-time RT-PCR was applied. This duplex assay provides a useful tool for routine health monitoring and screening of contaminated biological materials of these two viruses.

Detection of antibodies against Theiler's murine encephalomyelitis virus GDVII strain in experimental guinea pigs.

A disease affecting guinea pigs called 'guinea pig lameness' characterized by clinical signs of depression, lameness of limbs, flaccid paralysis, weight loss and death within a few weeks was first described by Römer in 1911. After a research group in our facility kept laboratory guinea pigs from two different origins together in one room, lameness was observed in two animals. Further investigations revealed a serological immune response against Theiler's murine encephalomyelitis virus (TMEV; GDVII strain) in these animals. Histopathology of the lumbar spinal cord of these animals showed mononuclear cell infiltration and necrotic neurons in the anterior horn. Therefore, all guinea pigs from this contaminated animal unit, from other units in our facility, as well as from different European institutions and breeding centres were screened for antibodies directed against GDVII. Our investigations showed that approximately 80% of all guinea pigs from the contaminated animal unit were seropositive for GDVII, whereas animals from other separate units were completely negative. In addition, 43% of tested sera from the different European institutions and breeding centres contained antibodies against GDVII. The present data confirm that an unknown viral infection causes an immune response in experimental guinea pigs leading to seroconversion against GDVII and that guinea pigs from a commercial breeder are the source of the infection.

Saffold virus infection associated with human myocarditis.

BACKGROUND: Saffold virus was described in 2007 as one of the first human viruses within the genus cardioviruses. Cardioviruses may cause severe infections of the myocardium in animals, and several studies have associated saffold virus with human disease. As a result, saffold virus has been isolated from different anatomical compartments, including the myocardium, but, until now, it has not been possible to demonstrate the accompanying histopathological signs of inflammation. OBJECTIVES: The aim of the study was to examine if saffold virus is capable of causing invasive infection in the human myocardium. STUDY DESIGN: Using real-time PCR, we retrospectively examined formalin-fixed paraffin embedded cardiac tissue specimens from 150 deceased individuals diagnosed with myocarditis at autopsy. The results were compared with histological findings. RESULTS AND CONCLUSIONS: Saffold virus was detected in the myocardium, lung tissue and blood of one child and was accompanied by histopathological inflammation in the heart and lungs, which was supportive of a viral infection. These findings suggest that cardioviruses may be associated with myocarditis in humans.

[Molecular genetic characteristics of the virus isolated from patients with human acute encephalomyelitis and multiple sclerosis].

The study of the antigenic and molecular genetic structure of human acute encephalomyelitis virus (HAEV) showed a high similarity of the HAEV N gene with the homologous gene of the fixed rabies virus strain. The results of the nucleotide sequence analysis indicate that HAEV belongs to the lyssavirus genotype 1. The N gene sequence is the closest to those of the ERA-CB20-M and RV-97 strains of the rabies virus. The need for further research into the role of the human acute encephalomyelitis virus in human pathology stems from past surveys that revealed the presence of the VNAs against this virus in 6 per cent of the blood received from donors in the USA and in each third among the patients with multiple sclerosis in the former USSR.

SAffold viruses in pediatric patients with diarrhea in Thailand.

Saffold virus (SAFV) is a newly discovered human virus which is classified into the genus Cardiovirus of the family Picornaviridae. A total of 608 fecal specimens collected during January 2012 to December 2013 from children with diarrhea in Chiang Mai, Thailand were investigated for SAFV by RT-nested PCR and sequence analysis. Of these, nine out of 608 (1.5%) were positive for SAFVs and four genotypes were identified, SAFV1, SAFV2, SAFV3, and SAFV4. SAFV mono-infection was found in five cases (CMH-S038-12, CMH-S071-12, CMH-S102-12, CMH-N029-12, and CMH-S048-13), while co-infection with other viruses causing diarrhea was observed in four cases (CMH-S021-12, CMH-S115-12, CMH-N048-13 and CMH-N103-13). This study provides more information about the genetic background of SAFV circulating in pediatric patients with diarrhea in Thailand.

The level of viral infection of antigen-presenting cells correlates with the level of development of Theiler's murine encephalomyelitis virus-induced demyelinating disease.

UNLABELLED: Intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease in susceptible SJL/J mice but not in resistant C57BL/6 mice. Previous studies have indicated that the major histocompatibility complex (MHC) genes play the most prominent role in the development of TMEV-induced demyelinating disease. In this study, we used C57BL/6.S (B6.S) congenic mice, which carry H-2(s) MHC genes instead of H-2(b) MHC genes in conjunction with the C57BL/6 (B6) background genes. Our data show that virus-infected B6.S mice are free from disease and have significantly lower viral loads than susceptible SJL mice, particularly in the spinal cord. A strong protective Th1-type T helper response with virtually no pathogenic Th17 response was detected in B6.S mice, in contrast to the reduced Th1- and robust Th17-type responses in SJL mice. Notably, lower levels of viral infectivity in B6.S antigen-presenting cells (APCs) correlated with the disease resistance and T-cell-type response. In vitro studies using APCs from B6.S and SJL mice show that TLR2, -3, -4, and -7, but not TLR9, signaling can replace viral infection and augment the effect of viral infection in the differentiation of the pathogenic Th17 cell type. Taken together, these results strongly suggest that the viral replication levels in APCs critically affect the induction of protective versus pathogenic Th cell types via the signaling of pattern recognition receptors for innate immune responses. Our current findings further imply that the levels of viral infectivity/replication and TLR-mediated signaling play critical roles in the pathogenesis of chronic viral diseases. IMPORTANCE: This study indicates that innate immune cytokines produced in antigen-presenting cells stimulating the T cell immune responses during early viral infection play a critical role in determining the susceptibility of mice to the development of demyelinating disease. The level of innate immune cytokines reflects the level of initial viral infection in the antigen-presenting cells, and the level determines the development of T cell types, which are either protective or pathogenic. The level of initial viral infection to the cells is controlled by a gene or genes that are not associated with the major histocompatibility antigen complex genes. This finding has an important implication in controlling not only chronic viral infections but also infection-induced autoimmune-like diseases, which are closely associated with the pathogenic type of T cell responses.

Isolation of saffold virus type 2 in green monkey kidney cells.

Saffold viruses (SAFV) have been discovered recently and they are classified into Theilovirus species in genus Cardiovirus in the Picornaviridae family. SAFV, especially those belonging to the genotype 2, have been difficult to propagate in laboratory cell lines. This study describes the successful isolation of an efficiently growing SAFV-2 strain directly from a stool specimen by standard virological methods. The availability of SAFV isolates that can be propagated to high titers is crucial to the future studies on pathogenesis and epidemiology of these novel human viruses.

Melanoma differentiation-associated gene 5 is critical for protection against Theiler's virus-induced demyelinating disease.

Infection of dendritic and glial cells with Theiler's murine encephalomyelitis virus (TMEV) induces various cytokines via Toll-like receptor- and melanoma differentiation-associated gene 5 (MDA5)-dependent pathways. However, the involvement and role of MDA5 in cytokine gene activation and the pathogenesis of TMEV-induced demyelinating disease are largely unknown. In this study, we demonstrate that MDA5 plays a critical role in the production of TMEV-induced alpha interferon (IFN-α) during early viral infection and in protection against the development of virus-induced demyelinating disease. Our results indicate that MDA5-deficient 129SvJ mice display significantly higher viral loads and apparent demyelinating lesions in the central nerve system (CNS) accompanied by clinical symptoms compared with wild-type 129SvJ mice. During acute viral infection, MDA5-deficient mice produced elevated levels of chemokines, consistent with increased cellular infiltration, but reduced levels of IFN-α, known to control T cell responses and cellular infiltration. Additional studies with isolated CNS glial cells from these mice suggest that cells from MDA5-deficient mice are severely compromised in the production of IFN-α upon viral infection, which results in increased cellular infiltration and viral loads in the CNS. Despite inadequate stimulation, the overall T cell responses to the viral determinants were significantly elevated in MDA5-deficient mice, reflecting the increased cellular infiltration. Therefore, the lack of MDA5-mediated IFN-α production may facilitate a massive viral load and elevated cellular infiltration in the CNS during early viral infection, leading to the pathogenesis of demyelinating disease.

Interleukin-6, produced by resident cells of the central nervous system and infiltrating cells, contributes to the development of seizures following viral infection.

Cells that can participate in an innate immune response within the central nervous system (CNS) include infiltrating cells (polymorphonuclear leukocytes [PMNs], macrophages, and natural killer [NK] cells) and resident cells (microglia and sometimes astrocytes). The proinflammatory cytokine interleukin-6 (IL-6) is produced by all of these cells and has been implicated in the development of behavioral seizures in the Theiler's murine encephalomyelitis virus (TMEV)-induced seizure model. The assessment, via PCR arrays, of the mRNA expression levels of a large number of chemokines (ligands and receptors) in TMEV-infected and mock-infected C57BL/6 mice both with and without seizures did not clearly demonstrate the involvement of PMNs, monocytes/macrophages, or NK cells in the development of seizures, possibly due to overlapping function of the chemokines. Additionally, C57BL/6 mice unable to recruit or depleted of infiltrating PMNs and NK cells had seizure rates comparable to those of controls following TMEV infection, and therefore PMNs and NK cells do not significantly contribute to seizure development. In contrast, C57BL/6 mice treated with minocycline, which affects monocytes/macrophages, microglial cells, and PMNs, had significantly fewer seizures than controls following TMEV infection, indicating monocytes/macrophages and resident microglial cells are important in seizure development. Irradiated bone marrow chimeric mice that were either IL-6-deficient mice reconstituted with wild-type bone marrow cells or wild-type mice reconstituted with IL-6-deficient bone marrow cells developed significantly fewer behavioral seizures following TMEV infection. Therefore, both resident CNS cells and infiltrating cells are necessary for seizure development.

Identification of a novel neuropathogenic Theiler's murine encephalomyelitis virus.

Theiler's murine encephalitis viruses (TMEV) are divided into two subgroups based on their neurovirulence. Persistent strains resemble Theiler's original viruses (referred to as the TO subgroup), which largely induce a subclinical polioencephalomyelitis during the acute phase of the disease and can persist in the spinal cord of susceptible animals, inducing a chronic demyelinating disease. In contrast, members of the neurovirulent subgroup cause an acute encephalitis characterized by the rapid onset of paralysis and death within days following intracranial inoculation. We report herein the characterization of a novel neurovirulent strain of TMEV, identified using pyrosequencing technology and referred to as NIHE. Complete coverage of the NIHE viral genome was obtained, and it shares <90% nucleotide sequence identity to known TMEV strains irrespective of subgroup, with the greatest sequence variability being observed in genes encoding the leader and capsid proteins. The histopathological analysis of infected brain and spinal cord demonstrate inflammatory lesions and neuronal necrosis during acute infection with no evidence of viral persistence or chronic disease. Intriguingly, genetic analysis indicates the putative expression of the L protein, considered a hallmark of strains within the persistent subgroup. Thus, the identification and characterization of a novel neurovirulent TMEV strain sharing features previously associated with both subgroups will lead to a deeper understanding of the evolution of TMEV strains and new insights into the determinants of neurovirulence.