Characterization of a rare clinical isolate of A. spinulosporus following a central nervous system infection.

A rarely reported clinical specimen of Aspergillus spinulosporus was isolated from an immunocompetent 22-month-old boy who was suffering from central nervous system aspergillosis and meningitis. The patient had no comorbidity, organ transplantation, or other surgical operations that lead to invasive aspergillosis. A. spinulosporus is mostly a soil borne strain, and only three invasive aspergillosis cases involving this strain have been reported. We isolated this strain from cerebrospinal fluid, cultured it successfully on PDA medium, and named it BJCH M5. We performed a complete genomic and phenotypic analysis, evolutionary relationship, secondary metabolites analysis, identification of virulence factor, and pairwise synteny analysis. We sequenced the complete 31.6 MB genome of A. spinulosporus, including the eight chromosomes and mitochondria. 11,356 protein-coding genes were predicted. BJCHM 5 has a high sequence identity with ten virulent factors of Aspergillus fumigatus. It also encodes two unique BGCs (Biosynthetic gene clusters) which are involved in human infection. Pairwise synteny analysis demonstrated that this strain has chromosome arrangement differences from A. nidulans. In conclusion, we isolated a specimen of the rarely reported pathogen A. spinulosporus and performed a complete genome assembly and functional characteristic analysis.

Single-Cell RNA Sequencing Reveals the Diversity of the Immunological Landscape following Central Nervous System Infection by a Murine Coronavirus.

Intracranial (i.c.) infection of susceptible C57BL/6 mice with the neurotropic JHM strain of mouse hepatitis virus (JHMV) (a member of the Coronaviridae family) results in acute encephalomyelitis and viral persistence associated with an immune-mediated demyelinating disease. The present study was undertaken to better understand the molecular pathways evoked during innate and adaptive immune responses as well as the chronic demyelinating stage of disease in response to JHMV infection of the central nervous system (CNS). Using single-cell RNA sequencing analysis (scRNAseq) on flow-sorted CD45-positive (CD45+) cells enriched from brains and spinal cords of experimental mice, we demonstrate the heterogeneity of the immune response as determined by the presence of unique molecular signatures and pathways involved in effective antiviral host defense. Furthermore, we identify potential genes involved in contributing to demyelination as well as remyelination being expressed by both microglia and macrophages. Collectively, these findings emphasize the diversity of the immune responses and molecular networks at defined stages following viral infection of the CNS.IMPORTANCE Understanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the molecular signatures of immune cells within the CNS at defined times following infection with a neuroadapted murine coronavirus using scRNAseq. This approach has revealed that the immunological landscape is diverse, with numerous immune cell subsets expressing distinct mRNA expression profiles that are, in part, dictated by the stage of infection. In addition, these findings reveal new insight into cellular pathways contributing to control of viral replication as well as to neurologic disease.

Metagenomics for neurological infections - expanding our imagination.

Over the past two decades, the diagnosis rate for patients with encephalitis has remained poor despite advances in pathogen-specific testing such as PCR and antigen assays. Metagenomic next-generation sequencing (mNGS) of RNA and DNA extracted from cerebrospinal fluid and brain tissue now offers another strategy for diagnosing neurological infections. Given that mNGS simultaneously assays for a wide range of infectious agents in an unbiased manner, it can identify pathogens that were not part of a neurologist's initial differential diagnosis either because of the rarity of the infection, because the microorganism has not been previously associated with a clinical phenotype or because it is a newly discovered organism. This Review discusses the technical advantages and pitfalls of cerebrospinal fluid mNGS in the context of patients with neuroinflammatory syndromes, including encephalitis, meningitis and myelitis. We also speculate on how mNGS testing potentially fits into current diagnostic testing algorithms given data on mNGS test performance, cost and turnaround time. Finally, the Review highlights future directions for mNGS technology and other hypothesis-free testing methodologies that are in development.

Germ Line IgM Is Sufficient, but Not Required, for Antibody-Mediated Alphavirus Clearance from the Central Nervous System.

Sindbis virus (SINV) infection of neurons in the brain and spinal cord in mice provides a model system for investigating recovery from encephalomyelitis and antibody-mediated clearance of virus from the central nervous system (CNS). To determine the roles of IgM and IgG in recovery, we compared the responses of immunoglobulin-deficient activation-induced adenosine deaminase-deficient (AID-/-), secretory IgM-deficient (sIgM-/-), and AID-/- sIgM-/- double-knockout (DKO) mice with those of wild-type (WT) C57BL/6 mice for disease, clearance of infectious virus and viral RNA from brain and spinal cord, antibody responses, and B cell infiltration into the CNS. Because AID is essential for immunoglobulin class switch recombination and somatic hypermutation, AID-/- mice produce only germ line IgM, while sIgM-/- mice secrete IgG but no IgM and DKO mice produce no secreted immunoglobulin. After intracerebral infection with the TE strain of SINV, most mice recovered. Development of neurologic disease occurred slightly later in sIgM-/- mice, but disease severity, weight loss, and survival were similar between the groups. AID-/- mice produced high levels of SINV-specific IgM, while sIgM-/- mice produced no IgM and high levels of IgG2a compared to WT mice. All mice cleared infectious virus from the spinal cord, but DKO mice failed to clear infectious virus from brain and had higher levels of viral RNA in the CNS late after infection. The numbers of infected cells and the amount of cell death in brain were comparable. We conclude that antibody is required and that either germ line IgM or IgG is sufficient for clearance of virus from the CNS.IMPORTANCE Mosquito-borne alphaviruses that infect neurons can cause fatal encephalomyelitis. Recovery requires a mechanism for the immune system to clear virus from infected neurons without harming the infected cells. Antiviral antibody has previously been shown to be a noncytolytic means for alphavirus clearance. Antibody-secreting cells enter the nervous system after infection and produce antiviral IgM before IgG. Clinical studies of human viral encephalomyelitis suggest that prompt production of IgM is associated with recovery, but it was not known whether IgM is effective for clearance. Our studies used mice deficient in production of IgM, IgG, or both to characterize the antibody necessary for alphavirus clearance. All mice developed similar signs of neurologic disease and recovered from infection. Antibody was necessary for virus clearance from the brain, and either early germ line IgM or IgG was sufficient. These studies support the clinical observation that prompt production of antiviral antibody is a determinant of outcome.

Immune Surveillance of the CNS following Infection and Injury.

The central nervous system (CNS) contains a sophisticated neural network that must be constantly surveyed in order to detect and mitigate a diverse array of challenges. The innate and adaptive immune systems actively participate in this surveillance, which is critical for the maintenance of CNS homeostasis and can facilitate the resolution of infections, degeneration, and tissue damage. Infections and sterile injuries represent two common challenges imposed on the CNS that require a prompt immune response. While the inducers of these two challenges differ in origin, the resultant responses orchestrated by the CNS share some overlapping features. Here, we review how the CNS immunologically discriminates between pathogens and sterile injuries, mobilizes an immune reaction, and, ultimately, regulates local and peripherally-derived immune cells to provide a supportive milieu for tissue repair.

Genetic analysis of vibriosis and viral nervous necrosis resistance in Atlantic cod (Gadus morhua L.) using a cure model.

The aim of this study was to investigate whether observed time-until-death of Atlantic cod (Gadus morhua L.) juveniles in separate challenge tests with Vibrio anguillarum (causes vibriosis) and nodavirus [causes viral nervous necrosis (VNN)] are due to differences in susceptibility (whether at risk or not) or increased endurance (individual hazard, given that the animal is susceptible) using a cure mixture (CURE) model with Gibbs sampling. Observed time-until-death, prepared as sequential binary records, were analyzed with the CURE model and results were compared with cross-sectional threshold (SIMPLE) and an ordinary longitudinal survival score (NAÏVE) model (i.e., assuming that all animals are susceptible). Overall mortality at the end of the test was 86 and 71% for vibriosis and VNN, respectively. But the CURE model estimated 92 and 82% of the population to be susceptible to vibriosis and VNN, respectively. Hence, a substantial fraction among the survivors were considered to be susceptible but with high endurance. The underlying heritability of susceptibility was moderate for vibriosis (0.33) and extremely high for VNN (0.91), somewhat greater compared with classical SIMPLE model (0.19 and 0.76 for vibriosis and VNN, respectively), analyzing end survival as a cross-sectional binary trait. Estimates of the underlying heritability were low for single test-day scores of both endurance (0.02 and 0.15 for vibriosis and VNN, respectively) in the CURE model and for the NAÏVE model (0.02 and 0.18 for vibriosis and VNN, respectively). Based on the CURE model, the genetic correlation between susceptibility and endurance was low to moderately positive and significantly different from unity (P < 0.01) for both vibriosis (0.13) and VNN (0.47). Estimated breeding values from the SIMPLE and NAÏVE models showed moderate to high correlations (0.41 to 0.96) with EBV for susceptibility and endurance in the CURE model. The analyses indicate that susceptibility and endurance are apparently distinct genetic traits. Still, the genetic variation estimated in the SIMPLE and NAÏVE models seems to a large extent to be controlled by susceptibility and an efficient genetic selection for reduced susceptibility to vibriosis and VNN is therefore likely feasible even when using classical (noncure) models. Earlier termination of the challenge test or back truncation of survival data is not recommended as this likely shifts the focus of selection towards endurance rather than susceptibility.

Immortalized human brain endothelial cell line HCMEC/D3 as a model of the blood-brain barrier facilitates in vitro studies of central nervous system infection by Cryptococcus neoformans.

Cryptococcus neoformans cells must cross the blood-brain barrier prior to invading the central nervous system. Here we demonstrate that the immortalized human brain endothelial cell line HCMEC/D3 is a useful alternative to primary brain endothelial cells as a model of the blood-brain barrier for studies of central nervous system infection.

Natural host genetic resistance to lentiviral CNS disease: a neuroprotective MHC class I allele in SIV-infected macaques.

Human immunodeficiency virus (HIV) infection frequently causes neurologic disease even with anti-retroviral treatment. Although associations between MHC class I alleles and acquired immunodeficiency syndrome (AIDS) have been reported, the role MHC class I alleles play in restricting development of HIV-induced organ-specific diseases, including neurologic disease, has not been characterized. This study examined the relationship between expression of the MHC class I allele Mane-A*10 and development of lentiviral-induced central nervous system (CNS) disease using a well-characterized simian immunodeficiency (SIV)/pigtailed macaque model. The risk of developing CNS disease (SIV encephalitis) was 2.5 times higher for animals that did not express the MHC class I allele Mane-A*10 (P = 0.002; RR = 2.5). Animals expressing the Mane-A*10 allele had significantly lower amounts of activated macrophages, SIV RNA, and neuronal dysfunction in the CNS than Mane-A*10 negative animals (P<0.001). Mane-A*10 positive animals with the highest CNS viral burdens contained SIV gag escape mutants at the Mane-A*10-restricted KP9 epitope in the CNS whereas wild type KP9 sequences dominated in the brain of Mane-A*10 negative animals with comparable CNS viral burdens. These concordant findings demonstrate that particular MHC class I alleles play major neuroprotective roles in lentiviral-induced CNS disease.

The glial response to CNS HIV infection includes p53 activation and increased expression of p53 target genes.

HIV-associated dementia (HAD) is a chronic neuroinflammatory disease that remains an important clinical problem without available rational treatment. As HIV does not infect neurons, the pathogenesis of HAD is thought to be secondary to the impact of infected leukocytes, including parenchymal microglia, which can secrete inflammatory mediators and viral products that alter the function of surrounding uninfected cells. We previously reported that the transcription factor p53 accumulates in neurons, microglia, and astrocytes of HAD patients. We have also shown that microglia from p53-deficient mice fail to induce neurotoxicity in response to the HIV coat protein gp120 in a coculture system, supporting the hypothesis that p53 plays a pathogenic role in the chronic neuroinflammatory component of HIV-associated neurodegeneration. We analyzed the extent and cell type specificity of p53 accumulation in subcortical white matter of ten AIDS patients that had previously been shown to demonstrate white matter p53 accumulation. To determine if p53 activation functioned to alter gene expression in HAD, cortical tissue sections were also immunolabeled for the p53 target genes Bax and p21(WAF1). These studies reveal that microglia, astrocytes, and oligodendrocytes all demonstrate p53 activation in response to HIV infection. We observed immunoreactivity for both Bax and p21(WAF1) in neurons and glia from patients demonstrating elevated p53 immunoreactivity. Our findings demonstrate that widespread increased p53 expression is present in HAD. Activation of p53 mediated pathways in the glia of HAD patients may contribute to the neuroinflammatory processes that promote neurodegeneration by inhibiting glial proliferation and/or promoting glial cell dysfunction.

RNA interference for studying the molecular basis of neuropsychiatric disorders.

RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by sequence-specific mRNA degradation, which is triggered by short double-stranded RNA. This mechanism can be efficiently induced both in vitro and in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, or by the application of short interfering RNA (siRNA). The design of highly selective and efficacious siRNA and shRNA has become commonplace, owing to continuous progress in modeling, bioinformatics and chemistry. Thus, RNAi is attractive for use in functional genomics, target and pathway validation and, potentially, human therapeutics. This review highlights recent findings in the field of RNAi with emphasis on neuropsychiatric aspects.

Caseous necrotic granuloma in the pituitary stalk due to nontuberculous Mycobacteria (Mycobacterium tokaiense) infection--case report.

A 36-year-old man with no history of immunosuppression presented with diabetes insipidus, but no other hormonal disturbances. Magnetic resonance imaging of the brain revealed an enhanced mass in the pituitary stalk appearing as a thickened pituitary stalk. The mass lesion was completely removed through the right optico-carotid space. Histological examination showed epithelioid cell granuloma with caseous necrosis, which strongly suggested mycobacterial infection. However, acid-fast staining detected no bacteria. Polymerase chain reaction (PCR) examinations of the gastric juice and cerebrospinal fluid for tuberculosis were negative. Nested PCR and deoxyribonucleic acid (DNA) sequencing of the DNA from the surgical specimen disclosed Mycobacterium tokaiense DNA sequences. This rare case of pituitary stalk granuloma caused by M. tokaiense shows that if the surgical specimen contains caseous necrosis, nested PCR and DNA sequencing are useful methods to identify mycobacterial infection.

Enfermedad neurologica por adenovirus / Neurologic disease due to adenovirus infection

El objetivo de este trabajo fue determinar la prevalencia de adenovirus (ADV) en las infecciones del sistema nervioso central (SNC). Se analizaron 108 muestras de líquido cefalorraquídeo (LCR) provenientes de 79 casos de encefalitis, 7 meningitis y 22 de otras patologías neurológicas, recibidas en el período 2000-2002. Cuarenta y nueve (47.35%) se obtuvieron de pacientes inmunocomprometidos. La presencia de ADV se investigó mediante reacción en cadena de la polimerasa en formato anidado (Nested-PCR). La identificación del genogrupo se realizó mediante análisis filogenético de la secuencia nucleotídica parcial de la región que codifica para la proteína del hexón. Se detectó la presencia de ADV en 6 de 108 (5.5%) muestras de LCR analizadas. Todos los casos positivos pertenecieron a pacientes con encefalitis que fueron 79, (6/79, 7.6%). No se observó diferencia estadísticamente significativa entre los casos de infección por ADV en pacientes inmunocomprometidos e inmunocompetentes (p>0.05). Las cepas de ADV detectadas se agruparon en los genogrupos B1 y C. En conclusión, nuestros resultados describen el rol de los ADV en las infecciones neurológicas en Argentina. La información presentada contribuye al conocimiento de su epidemiología, en particular en casos de encefalitis.

Infectious agents and age-related neurodegenerative disorders.

chlamdAs with other organ systems, the vulnerability of the nervous system to infectious agents increases with aging. Several different infectious agents can cause neurodegenerative conditions, with prominent examples being human immunodeficiency virus (HIV-1) dementia and prion disorders. Such infections of the central nervous system (CNS) typically have a relatively long incubation period and a chronic progressive course, and are therefore increasing in frequency as more people live longer. Infectious agents may enter the central nervous system in infected migratory macrophages, by transcytosis across blood-brain barrier cells or by intraneuronal transfer from peripheral nerves. Synapses and lipid rafts are important sites at which infectious agents may enter neurons and/or exert their cytotoxic effects. Recent findings suggest the possibility that infectious agents may increase the risk of common age-related neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and stroke. While scenarios can be envisioned whereby viruses such as Chlamydia pneumoniae, herpes simplex and influenza promote damage to neurons during aging, there is no conclusive evidence for a major role of these pathogens in neurodegenerative disorders. In the case of stroke, blood vessels may be adversely affected by bacteria or viruses resulting in atherosclerosis.

Differential regulation of Toll-like receptor mRNAs in experimental murine central nervous system infections.

Toll-like receptors (TLR) play a key role in the recognition of microbial components. We investigated the differential regulation of TLR mRNA expression in bacterial and viral mouse models of central nervous system infection. Streptococcus pneumoniae meningitis led to an enhanced expression of TLR2, TLR4 and TLR9 mRNA. In Escherichia coli meningitis, TLR2, TLR4 and TLR7 mRNA expression was increased and Herpes simplex encephalitis caused a rise of TLR4 mRNA. In organotypic hippocampal cultures treatment with S. pneumoniae R6 led to increased expression of TLR2 and TLR3 mRNA. Our data provide evidence that regulation of TLR mRNA is not fully specific for the molecular patterns of the infectious pathogen. The TLR mRNA regulation observed probably represents a combination of specific response to the causative pathogen and non-specific activation of the innate immune system.