Prophylactic Administration of Gut Microbiome Metabolites Abrogated Microglial Activation and Subsequent Neuroinflammation in an Experimental Model of Japanese Encephalitis.

Short-chain fatty acids (SCFAs) are gut microbial metabolic derivatives produced during the fermentation of ingested complex carbohydrates. SCFAs have been widely regarded to have a potent anti-inflammatory and neuro-protective role and have implications in several disease conditions, such as, inflammatory bowel disease, type-2 diabetes, and neurodegenerative disorders. Japanese encephalitis virus (JEV), a neurotropic flavivirus, is associated with life threatening neuro-inflammation and neurological sequelae in infected hosts. In this study, we hypothesize that SCFAs have potential in mitigating JEV pathogenesis. Postnatal day 10 BALB/c mice were intraperitoneally injected with either a SCFA mixture (acetate, propionate, and butyrate) or PBS for a period of 7 days, followed by JEV infection. All mice were observed for onset and progression of symptoms. The brain tissue was collected upon reaching terminal illness for further analysis. SCFA-supplemented JEV-infected mice (SCFA + JEV) showed a delayed onset of symptoms, lower hindlimb clasping score, and decreased weight loss and increased survival by 3 days (p < 0.0001) upon infection as opposed to the PBS-treated JEV-infected animals (JEV). Significant downregulation of inflammatory cytokines TNF-α, MCP-1, IL-6, and IFN-Υ in the SCFA + JEV group relative to the JEV-infected control group was observed. Inflammatory mediators, phospho-NF-kB (P-NF-kB) and iba1, showed 2.08 ± 0.1 and 3.132 ± 0.43-fold upregulation in JEV versus 1.19 ± 0.11 and 1.31 ± 0.11-fold in the SCFA + JEV group, respectively. Tissue section analysis exhibited reduced glial activation (JEV group─42 ± 2.15 microglia/ROI; SCFA + JEV group─27.07 ± 1.8 microglia/ROI) in animals that received SCFA supplementation prior to infection as seen from the astrocytic and microglial morphometric analysis. Caspase-3 immunoblotting showed 4.08 ± 1.3-fold upregulation in JEV as compared to 1.03 ± 0.14-fold in the SCFA + JEV group and TUNEL assay showed a reduced cellular death post-JEV infection (JEV-6.4 ± 1.5 cells/ROI and SCFA + JEV-3.7 ± 0.73 cells/ROI). Our study critically contributes to the increasing evidence in support of SCFAs as an anti-inflammatory and neuro-protective agent, we further expand its scope as a potential supplementary intervention in JEV-mediated neuroinflammation.

C-C motif chemokine receptor 2 and 7 synergistically control inflammatory monocyte recruitment but the infecting virus dictates monocyte function in the brain.

Inflammatory monocytes (iMO) are recruited from the bone marrow to the brain during viral encephalitis. C-C motif chemokine receptor (CCR) 2 deficiency substantially reduces iMO recruitment for most, but not all encephalitic viruses. Here we show CCR7 acts synergistically with CCR2 to control this process. Following Herpes simplex virus type-1 (HSV-1), or La Crosse virus (LACV) infection, we find iMO proportions are reduced by approximately half in either Ccr2 or Ccr7 knockout mice compared to control mice. However, Ccr2/Ccr7 double knockouts eliminate iMO recruitment following infection with either virus, indicating these receptors together control iMO recruitment. We also find that LACV induces a more robust iMO recruitment than HSV-1. However, unlike iMOs in HSV-1 infection, LACV-recruited iMOs do not influence neurological disease development. LACV-induced iMOs have higher expression of proinflammatory and proapoptotic but reduced mitotic, phagocytic and phagolysosomal transcripts compared to HSV-1-induced iMOs. Thus, virus-specific activation of iMOs affects their recruitment, activation, and function.

Functional properties of measles virus proteins derived from a subacute sclerosing panencephalitis patient who received repeated remdesivir treatments.

Subacute sclerosing panencephalitis (SSPE) is a rare but fatal late neurological complication of measles, caused by persistent measles virus (MeV) infection of the central nervous system. There are no drugs approved for the treatment of SSPE. Here, we followed the clinical progression of a 5-year-old SSPE patient after treatment with the nucleoside analog remdesivir, conducted a post-mortem evaluation of the patient's brain, and characterized the MeV detected in the brain. The quality of life of the patient transiently improved after the first two courses of remdesivir, but a third course had no further clinical effect, and the patient eventually succumbed to his condition. Post-mortem evaluation of the brain displayed histopathological changes including loss of neurons and demyelination paired with abundant presence of MeV RNA-positive cells throughout the brain. Next-generation sequencing of RNA isolated from the brain revealed a complete MeV genome with mutations that are typically detected in SSPE, characterized by a hypermutated M gene. Additional mutations were detected in the polymerase (L) gene, which were not associated with resistance to remdesivir. Functional characterization showed that mutations in the F gene led to a hyperfusogenic phenotype predominantly mediated by N465I. Additionally, recombinant wild-type-based MeV with the SSPE-F gene or the F gene with the N465I mutation was no longer lymphotropic but instead efficiently disseminated in neural cultures. Altogether, this case encourages further investigation of remdesivir as a potential treatment of SSPE and highlights the necessity to functionally understand SSPE-causing MeV.IMPORTANCEMeasles virus (MeV) causes acute, systemic disease and remains an important cause of morbidity and mortality in humans. Despite the lack of known entry receptors in the brain, MeV can persistently infect the brain causing the rare but fatal neurological disorder subacute sclerosing panencephalitis (SSPE). SSPE-causing MeVs are characterized by a hypermutated genome and a hyperfusogenic F protein that facilitates the rapid spread of MeV throughout the brain. No treatment against SSPE is available, but the nucleoside analog remdesivir was recently demonstrated to be effective against MeV in vitro. We show that treatment of an SSPE patient with remdesivir led to transient clinical improvement and did not induce viral escape mutants, encouraging the future use of remdesivir in SSPE patients. Functional characterization of the viral proteins sheds light on the shared properties of SSPE-causing MeVs and further contributes to understanding how those viruses cause disease.

EBV infected cells in the multiple sclerosis brain express PD-L1: How the virus and its niche may escape immune surveillance.

The presence of EBV infected B cells in postmortem multiple sclerosis (MS) brain tissue suggests immune evasion strategies. Using immunohistochemical techniques we analysed the expression of the immune checkpoint molecule PD-L1 and its receptor PD-1 in MS brains containing B cell-enriched perivascular infiltrates and meningeal follicles, a major EBV reservoir. PD-1 and PD-L1 immunoreactivities were restricted to CNS-infiltrating immune cells. PD-L1 was expressed on B cells, including EBV infected B cells, while PD-1 was expressed on many CD8+ T cells, including EBV-specific CD8+ T-cells, and fewer CD4+ T cells. PD-L1+ cells and EBV infected cells were in close contact with PD-1+ T cells. PD-L1 expressed by EBV infected B cells could favour local immune evasion leading to EBV persistence and immunopathology in the MS brain.

Single-Nucleus Sequencing of Silkworm Larval Brain Reveals the Key Role of Lysozyme in the Antiviral Immune Response in Brain Hemocytes.

INTRODUCTION: The brain is considered as an immune-privileged organ, yet innate immune reactions can occur in the central nervous system of vertebrates and invertebrates. Silkworm (Bombyx mori) is an economically important insect and a lepidopteran model species. The diversity of cell types in the silkworm brain, and how these cell subsets produce an immune response to virus infection, remains largely unknown. METHODS: Single-nucleus RNA sequencing (snRNA-seq), bioinformatics analysis, RNAi, and other methods were mainly used to analyze the cell types and gene functions of the silkworm brain. RESULTS: We used snRNA-seq to identify 19 distinct clusters representing Kenyon cell, glial cell, olfactory projection neuron, optic lobes neuron, hemocyte-like cell, and muscle cell types in the B. mori nucleopolyhedrovirus (BmNPV)-infected and BmNPV-uninfected silkworm larvae brain at the late stage of infection. Further, we found that the cell subset that exerts an antiviral function in the silkworm larvae brain corresponds to hemocytes. Specifically, antimicrobial peptides were significantly induced by BmNPV infection in the hemocytes, especially lysozyme, exerting antiviral effects. CONCLUSION: Our single-cell dataset reveals the diversity of silkworm larvae brain cells, and the transcriptome analysis provides insights into the immune response following virus infection at the single-cell level.

Characterization of CD8+ T cells in immune-privileged organs of ZIKV-infected Ifnar1-/- mice.

Zika virus (ZIKV) infection caused neurological complications and male infertility, leading to the accumulation of antigen-specific immune cells in immune-privileged organs (IPOs). Thus, it is important to understand the immunological responses to ZIKV in IPOs. We extensively investigated the ZIKV-specific T cell immunity in IPOs in Ifnar1-/- mice, based on an immunodominant epitope E294-302 tetramer. The distinct kinetics and functions of virus-specific CD8+ T cells infiltrated into different IPOs were characterized, with late elevation in the brain and spinal cord. Single epitope E294-302-specific T cells can account for 20-60% of the total CD8+ T cells in the brain, spinal cord, and testicle and persist for at least 90 days in the brain and spinal cord. The E294-302-specific TCRαßs within the IPOs are featured with the majority of clonotypes utilizing TRAV9N-3 paired with diverse TRBV chains, but with distinct αß paired clonotypes in 7 and 30 days post-infection. Specific chemokine receptors, Ccr2 and Ccr5, were selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of virus-specific CD8+ T cells after infection. Overall, this study adds to the understanding of virus-specific CD8+ T cell responses for controlling and clearing ZIKV infection in IPOs.IMPORTANCEThe immune-privileged organs (IPOs), such as the central nervous system and testicles, presented pathogenicity and inflammation after Zika virus (ZIKV) infection with infiltrated CD8+ T cells. Our data show that CD8+ T cells keep up with virus increases and decreases in immune-privileged organs. Furthermore, our study provides the first ex vivo comparative analyses of the composition and diversity related to TCRα/ß clonotypes across anatomical sites and ZIKV infection phases. We show that the vast majority of TCRα/ß clonotypes in tissues utilize TRAV9N-3 with conservation. Specific chemokine expression, including Ccr2 and Ccr5, was found to be selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of the virus-specific CD8+ T cells after the infection. Our study adds insights into the anti-viral immunological characterization and chemotaxis mechanism of virus-specific CD8+ T cells after ZIKV infection in different IPOs.

Fentanyl dysregulates neuroinflammation and disrupts blood-brain barrier integrity in HIV-1 Tat transgenic mice.

Opioid overdose deaths have dramatically increased by 781% from 1999 to 2021. In the setting of HIV, opioid drug abuse exacerbates neurotoxic effects of HIV in the brain, as opioids enhance viral replication, promote neuronal dysfunction and injury, and dysregulate an already compromised inflammatory response. Despite the rise in fentanyl abuse and the close association between opioid abuse and HIV infection, the interactive comorbidity between fentanyl abuse and HIV has yet to be examined in vivo. The HIV-1 Tat-transgenic mouse model was used to understand the interactive effects between fentanyl and HIV. Tat is an essential protein produced during HIV that drives the transcription of new virions and exerts neurotoxic effects within the brain. The Tat-transgenic mouse model uses a glial fibrillary acidic protein (GFAP)-driven tetracycline promoter which limits Tat production to the brain and this model is well used for examining mechanisms related to neuroHIV. After 7 days of fentanyl exposure, brains were harvested. Tight junction proteins, the vascular cell adhesion molecule, and platelet-derived growth factor receptor-ß were measured to examine the integrity of the blood brain barrier. The immune response was assessed using a mouse-specific multiplex chemokine assay. For the first time in vivo, we demonstrate that fentanyl by itself can severely disrupt the blood-brain barrier and dysregulate the immune response. In addition, we reveal associations between inflammatory markers and tight junction proteins at the blood-brain barrier.

HCMV Infection Reduces Nidogen-1 Expression, Contributing to Impaired Neural Rosette Development in Brain Organoids.

Human cytomegalovirus (HCMV) is a leading cause of birth defects in humans. These birth defects include microcephaly, sensorineural hearing loss, vision loss, and cognitive impairment. The process by which the developing fetus incurs these neurological defects is poorly understood. To elucidate some of these mechanisms, we have utilized HCMV-infected induced pluripotent stem cells (iPSCs) to generate in vitro brain organoids, modeling the first trimester of fetal brain development. Early during culturing, brain organoids generate neural rosettes. These structures are believed to model neural tube formation. Rosette formation was analyzed in HCMV-infected and mock-infected brain organoids at 17, 24, and 31 days postinfection. Histological analysis revealed fewer neural rosettes in HCMV-infected compared to mock-infected organoids. HCMV-infected organoid rosettes incurred multiple structural deficits, including increased lumen area, decreased ventricular zone depth, and decreased cell count. Immunofluorescent (IF) analysis found that nidogen-1 (NID1) protein expression in the basement membrane surrounding neural rosettes was greatly reduced by virus infection. IF analysis also identified a similar downregulation of laminin in basement membranes of HCMV-infected organoid rosettes. Knockdown of NID1 alone in brain organoids impaired their development, leading to the production of rosettes with increased lumen area, decreased structural integrity, and reduced laminin localization in the basement membrane, paralleling observations in HCMV-infected organoids. Our data strongly suggest that HCMV-induced downregulation of NID1 impairs neural rosette formation and integrity, likely contributing to many of HCMV's most severe birth defects. IMPORTANCE HCMV infection in pregnant women continues to be the leading cause of virus-induced neurologic birth defects. The mechanism through which congenital HCMV (cCMV) infection induces pathological changes to the developing fetal central nervous system (CNS) remains unclear. Our lab previously reproduced identified clinical defects in HCMV-infected infants using a three dimensional (3D) brain organoid model. In this new study, we have striven to discover very early HCMV-induced changes in developing brain organoids. We investigated the development of neural tube-like structures, neural rosettes. HCMV-infected rosettes displayed multiple structural abnormalities and cell loss. HCMV-infected rosettes displayed reduced expression of the key basement membrane protein, NID1. We previously found NID1 to be specifically targeted in HCMV-infected fibroblasts and endothelial cells. Brain organoids generated from NID1 knockdown iPSCs recapitulated the structural defects observed in HCMV-infected rosettes. Findings in this study revealed HCMV infection induced early and dramatic structural changes in 3D brain organoids. We believe our results suggest a major role for infection-induced NID1 downregulation in HCMV-induced CNS birth defects.

Evaluation of Archival HIV DNA in Brain and Lymphoid Tissues.

HIV reservoirs persist in anatomic compartments despite antiretroviral therapy (ART). Characterizing archival HIV DNA in the central nervous system (CNS) and other tissues is crucial to inform cure strategies. We evaluated paired autopsy brain-frontal cortex (FC), occipital cortex (OCC), and basal ganglia (BG)-and peripheral lymphoid tissues from 63 people with HIV. Participants passed away while virally suppressed on ART at the last visit and without evidence of CNS opportunistic disease. We quantified total HIV DNA in all participants and obtained full-length HIV-envelope (FL HIV-env) sequences from a subset of 14 participants. We detected HIV DNA (gag) in most brain (65.1%) and all lymphoid tissues. Lymphoid tissues had higher HIV DNA levels than the brain (P < 0.01). Levels of HIV gag between BG and FC were similar (P > 0.2), while OCC had the lowest levels (P = 0.01). Females had higher HIV DNA levels in tissues than males (gag, P = 0.03; 2-LTR, P = 0.05), suggesting possible sex-associated mechanisms for HIV reservoir persistence. Most FL HIV-env sequences (n = 143) were intact, while 42 were defective. Clonal sequences were found in 8 out of 14 participants, and 1 participant had clonal defective sequences in the brain and spleen, suggestive of cell migration. From 10 donors with paired brain and lymphoid sequences, we observed evidence of compartmentalized sequences in 2 donors. Our data further the idea that the brain is a site for archival HIV DNA during ART where compartmentalized provirus may occur in a subset of people. Future studies assessing FL HIV-provirus and replication competence are needed to further evaluate the HIV reservoirs in tissues. IMPORTANCE HIV infection of the brain is associated with adverse neuropsychiatric outcomes, despite efficient antiretroviral treatment. HIV may persist in reservoirs in the brain and other tissues, which can seed virus replication if treatment is interrupted, representing a major challenge to cure HIV. We evaluated reservoirs and genetic features in postmortem brain and lymphoid tissues from people with HIV who passed away during suppressed HIV replication. We found a differential distribution of HIV reservoirs across brain regions which was lower than that in lymphoid tissues. We observed that most HIV reservoirs in tissues had intact envelope sequences, suggesting they could potentially generate replicative viruses. We found that women had higher HIV reservoir levels in brain and lymphoid tissues than men, suggesting possible sex-based mechanisms of maintenance of HIV reservoirs in tissues, warranting further investigation. Characterizing the archival HIV DNA in tissues is important to inform future HIV cure strategies.

Rabies virus infection is associated with variations in calbindin D-28K and calretinin mRNA expression levels in mouse brain tissue.

Rabies virus (RABV) infection leads to a fatal neurological outcome in humans and animals and is associated with major alterations in cellular gene expression. In this study, we describe the effects of RABV infection on the mRNA expression levels of two genes, encoding the Ca2+-binding proteins (Ca-BPs) calbindin D-28K (Calb1) and calretinin (Calb2), in the brains of BALB/c mice. Sixty 4-week-old mice were divided into two test groups and one control group. Mice were inoculated intramuscularly with either a street rabies virus (SRV) strain or a challenge virus standard (CVS-11) strain and sacrificed at 3-day intervals up to day 18 postinfection. A direct fluorescent antibody test (DFAT) was used to verify the presence of RABV antigen in brain tissues, and real-time quantitative PCR (RT-PCR) was used to assess gene expression. Infection with both RABV strains resulted in significant (p < 0.05) increases in Calb1 and Calb2 expression in the test animals when compared with the controls at various time points in the study. Correlation analysis indicated very weak insignificant (p > 0.05) negative and positive relationships, respectively, between Calb1 expression (r = -0.04) and Calb2 expression (r = 0.08) with viral load (CVS-11 strain). Insignificant (p > 0.05) relationships were also observed Calb1 expression (r = -0.28) and Calb2 expression (r = 0.06) and viral load for the SRV strain.The observed alterations in Calb1 and Calb2 expression in this study indicate possible impairments in neuronal Ca2+ buffering and Ca2+ homeostasis as a result of RABV infection and, consequently, possible involvement of calbindin-D28K and calretinin in the neuropathogenesis of rabies.

Treatment with Granulocyte-Macrophage Colony-Stimulating Factor Reduces Viral Titers in the Brains of West Nile Virus-Infected Mice and Improves Survival.

West Nile virus (WNV) is the leading cause of epidemic arboviral encephalitis in the United States. As there are currently no proven antiviral therapies or licensed human vaccines, understanding the neuropathogenesis of WNV is critical for rational therapeutic design. In WNV-infected mice, the depletion of microglia leads to enhanced viral replication, increased central nervous system (CNS) tissue injury, and increased mortality, suggesting that microglia play a critical role in protection against WNV neuroinvasive disease. To determine if augmenting microglial activation would provide a potential therapeutic strategy, we administered granulocyte-macrophage colony-stimulating factor (GM-CSF) to WNV-infected mice. Recombinant human GM-CSF (rHuGMCSF) (sargramostim [Leukine]) is an FDA-approved drug used to increase white blood cells following leukopenia-inducing chemotherapy or bone marrow transplantation. Daily treatment of both uninfected and WNV-infected mice with subcutaneous injections of GM-CSF resulted in microglial proliferation and activation as indicated by the enhanced expression of the microglia activation marker ionized calcium binding adaptor molecule 1 (Iba1) and several microglia-associated inflammatory cytokines, including CCL2 (C-C motif chemokine ligand 2), interleukin 6 (IL-6), and IL-10. In addition, more microglia adopted an activated morphology as demonstrated by increased sizes and more pronounced processes. GM-CSF-induced microglial activation in WNV-infected mice was associated with reduced viral titers and apoptotic activity (caspase 3) in the brains of WNV-infected mice and significantly increased survival. WNV-infected ex vivo brain slice cultures (BSCs) treated with GM-CSF also showed reduced viral titers and caspase 3 apoptotic cell death, indicating that GM-CSF specifically targets the CNS and that its actions are not dependent on peripheral immune activity. Our studies suggest that stimulation of microglial activation may be a viable therapeutic approach for the treatment of WNV neuroinvasive disease. IMPORTANCE Although rare, WNV encephalitis poses a devastating health concern, with few treatment options and frequent long-term neurological sequelae. Currently, there are no human vaccines or specific antivirals against WNV infections, so further research into potential new therapeutic agents is critical. This study presents a novel treatment option for WNV infections using GM-CSF and lays the foundation for further studies into the use of GM-CSF as a treatment for WNV encephalitis as well as a potential treatment for other viral infections.

Nuclear accumulation of host transcripts during Zika Virus Infection.

Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these pathways remains unknown. Here we show that ZIKV infection disrupts subcellular partitioning of host transcripts critical for neurodevelopment in NPCs and functionally link this process to the up-frameshift protein 1 (UPF1). UPF1 is an RNA-binding protein known to regulate decay of cellular and viral RNAs and is less expressed in ZIKV-infected cells. Using infrared crosslinking immunoprecipitation and RNA sequencing (irCLIP-Seq), we show that a subset of mRNAs loses UPF1 binding in ZIKV-infected NPCs, consistent with UPF1's diminished expression. UPF1 target transcripts, however, are not altered in abundance but in subcellular localization, with mRNAs accumulating in the nucleus of infected or UPF1 knockdown cells. This leads to diminished protein expression of FREM2, a protein required for maintenance of NPC identity. Our results newly link UPF1 to the regulation of mRNA transport in NPCs, a process perturbed during ZIKV infection.

SARS-CoV-2 infection and persistence in the human body and brain at autopsy.

Coronavirus disease 2019 (COVID-19) is known to cause multi-organ dysfunction1-3 during acute infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients experiencing prolonged symptoms, termed post-acute sequelae of SARS-CoV-2 (refs. 4,5). However, the burden of infection outside the respiratory tract and time to viral clearance are not well characterized, particularly in the brain3,6-14. Here we carried out complete autopsies on 44 patients who died with COVID-19, with extensive sampling of the central nervous system in 11 of these patients, to map and quantify the distribution, replication and cell-type specificity of SARS-CoV-2 across the human body, including the brain, from acute infection to more than seven months following symptom onset. We show that SARS-CoV-2 is widely distributed, predominantly among patients who died with severe COVID-19, and that virus replication is present in multiple respiratory and non-respiratory tissues, including the brain, early in infection. Further, we detected persistent SARS-CoV-2 RNA in multiple anatomic sites, including throughout the brain, as late as 230 days following symptom onset in one case. Despite extensive distribution of SARS-CoV-2 RNA throughout the body, we observed little evidence of inflammation or direct viral cytopathology outside the respiratory tract. Our data indicate that in some patients SARS-CoV-2 can cause systemic infection and persist in the body for months.

Brain neuronal and glial damage during acute COVID-19 infection in absence of clinical neurological manifestations.

BACKGROUND: To assess whether SARS-CoV-2 infection may affect the central nervous system, specifically neurons and glia cells, even without clinical neurological involvement. METHODS: In this single centre prospective study, serum levels of neurofilament light chain (sNfL) and glial fibrillar acidic protein (sGFAp) were assessed using SimoaTM assay Neurology 2-Plex B Assay Kit, in 148 hospitalised patients with COVID-19 without clinical neurological manifestations and compared them to 53 patients with interstitial pulmonary fibrosis (IPF) and 108 healthy controls (HCs). RESULTS: Age and sex-corrected sNfL levels were higher in patients with COVID-19 (median log10-sNfL 1.41; IQR 1.04-1.83) than patients with IPF (median log10-sNfL 1.18; IQR 0.98-1.38; p<0.001) and HCs (median log10-sNfL 0.89; IQR 0.72-1.14; p<0.001). Likewise, age and sex-corrected sGFAP levels were higher in patients with COVID-19 (median log10-sGFAP 2.26; IQR 2.02-2.53) in comparison with patients with IPF (median log10-sGFAP 2.15; IQR 1.94-2.30; p<0.001) and HCs (median log10-sGFAP 1.87; IQR 0.64-2.09; p<0.001). No significant difference was found between patients with HCs and IPF (p=0.388 for sNfL and p=0.251 for sGFAp). In patients with COVID-19, a prognostic model with mortality as dependent variable (26/148 patients died during hospitalisation) and sNfl, sGFAp and age as independent variables, showed an area under curve of 0.72 (95% CI 0.59 to 0.84; negative predictive value (NPV) (%):80,positive predictive value (PPV)(%): 84; p=0.0008). CONCLUSION: The results of our study suggest that neuronal and glial degeneration can occur in patients with COVID-19 regardless of overt clinical neurological manifestations. With age, levels of sNfl and GFAp can predict in-hospital COVID-19-associated mortality and might be useful to assess COVID-19 patient prognostic profile.

First report on the detection of Japanese encephalitis virus in fruit bats from India.

Japanese encephalitis (JE) is a mosquito borne viral zoonotic disease and JE virus (JEV) is responsible for causing several children deaths every year in India. Since 1978, cases of JE have been reported from Gorakhpur district of Uttar Pradesh state annually. The knowledge on the role played by wildlife reservoirs in the sylvatic transmission and maintenance of JE virus remains limited. Bats are reservoir hosts for several emerging and re-emerging viral pathogens but their role in zoonotic cycle of JEV has not been elucidated yet. In Gorakhpur district of Uttar Pradesh, 52 fruit bats were found dead on 26 May 2020. The post-mortem report of the bat samples conducted at the Indian Veterinary Research Institute stated that the bats died due to brain hemorrhage, caused by excessive heat. The brain tissue samples of the bats were subjected to investigation using molecular techniques to determine the presence of JEV. The present work reports for the first time the detection of JEV in brain samples of bats from India. The viral load ranging from 8 to 18 copies/reaction was detected in brain samples by TaqMan real Time RT-PCR. The low viral load might be the reason for the absence of apparent clinical signs in bats and suggests the probable role of fruit bats in maintaining the JEV in nature.

Morphological, cellular, and molecular basis of brain infection in COVID-19 patients.

Although increasing evidence confirms neuropsychiatric manifestations associated mainly with severe COVID-19 infection, long-term neuropsychiatric dysfunction (recently characterized as part of "long COVID-19" syndrome) has been frequently observed after mild infection. We show the spectrum of cerebral impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, ranging from long-term alterations in mildly infected individuals (orbitofrontal cortical atrophy, neurocognitive impairment, excessive fatigue and anxiety symptoms) to severe acute damage confirmed in brain tissue samples extracted from the orbitofrontal region (via endonasal transethmoidal access) from individuals who died of COVID-19. In an independent cohort of 26 individuals who died of COVID-19, we used histopathological signs of brain damage as a guide for possible SARS-CoV-2 brain infection and found that among the 5 individuals who exhibited those signs, all of them had genetic material of the virus in the brain. Brain tissue samples from these five patients also exhibited foci of SARS-CoV-2 infection and replication, particularly in astrocytes. Supporting the hypothesis of astrocyte infection, neural stem cell-derived human astrocytes in vitro are susceptible to SARS-CoV-2 infection through a noncanonical mechanism that involves spike-NRP1 interaction. SARS-CoV-2-infected astrocytes manifested changes in energy metabolism and in key proteins and metabolites used to fuel neurons, as well as in the biogenesis of neurotransmitters. Moreover, human astrocyte infection elicits a secretory phenotype that reduces neuronal viability. Our data support the model in which SARS-CoV-2 reaches the brain, infects astrocytes, and consequently, leads to neuronal death or dysfunction. These deregulated processes could contribute to the structural and functional alterations seen in the brains of COVID-19 patients.

CD4dim CD8bright T Cells Home to the Brain and Mediate HIV Neuroinvasion.

CD4dim CD8bright T cells are a mature population of CD8+ T cells that upon activation upregulate CD4 dimly on their surface. Expression of CD4 on these cells suggests that they can be an additional source of HIV neuroinvasion and persistence in the brain. We used HIV-infected NOD/SCID/IL-2rcγ-/- (NSG) humanized mice to track CD4dim CD8bright T cell homing to the brain and define their role in HIV dissemination into the brain. We report here that CD4dim CD8bright T cells are found in the brain at a median frequency of 2.6% and in the spleen at median frequency of 7.6% of CD3+ T cells. In the brain, 10 to 20% of CD4dim CD8bright T cells contain integrated provirus, which is infectious as demonstrated by viral outgrowth assay. CD4dim CD8bright T cells in the brain exhibited significantly higher expression of the brain homing receptors CX3CR1 and CXCR3 in comparison to their single-positive CD8+ T cell counterpart. Blocking lymphocyte trafficking into the brain of humanized mice via anti-VLA4 and anti-LFA1 antibodies reduced CD4dim CD8bright T cell trafficking into the brain by 60% and diminished brain HIV proviral DNA by 72%. Collectively, our findings demonstrate that CD4dim CD8bright T cells can home to the brain and support productive HIV replication. These studies also reveal for the first time that CD4dim CD8bright T cells are capable of HIV neuroinvasion and are a reservoir for HIV. IMPORTANCE We report here a seminal finding of a novel population of T cells, termed CD4dim CD8bright T cells, that plays a role in HIV neuroinvasion and is a reservoir for HIV in the brain.

Prevalence of individual brain and eye defects potentially related to Zika virus in pregnancy in 22 U.S. states and territories, January 2016 to June 2017.

During the Centers for Disease Control and Prevention's Zika Virus Response, birth defects surveillance programs adapted to monitor birth defects potentially related to Zika virus (ZIKV) infection during pregnancy. Pregnancy outcomes occurring during January 2016 to June 2017 in 22 U.S. states and territories were used to estimate the prevalence of those brain and eye defects potentially related to ZIKV. Jurisdictions were divided into three groups: areas with widespread ZIKV transmission, areas with limited local ZIKV transmission, and areas without local ZIKV transmission. Prevalence estimates for selected brain and eye defects and microcephaly per 10,000 live births were estimated. Prevalence ratios (PRs) and 95% confidence intervals (CIs) were estimated using Poisson regression for areas with widespread and limited ZIKV transmission compared with areas without local ZIKV transmission. Defects with significantly higher prevalence in areas of widespread transmission were pooled, and PRs were calculated by quarter, comparing subsequent quarters to the first quarter (January-March 2016). Nine defects had significantly higher prevalence in areas of widespread transmission. The highest PRs were seen in intracranial calcifications (PR = 12.6, 95% CI [7.4, 21.3]), chorioretinal abnormalities (12.5 [7.1, 22.3]), brainstem abnormalities (9.3 [4.7, 18.4]), and cerebral/cortical atrophy (6.7 [4.2, 10.8]). The PR of the nine pooled defects was significantly higher in three quarters in areas with widespread transmission. The largest difference in prevalence was observed for defects consistently reported in infants with congenital ZIKV infection. Birth defects surveillance programs could consider monitoring a subset of birth defects potentially related to ZIKV in pregnancy.

Neurodegenerative phagocytes mediate synaptic stripping in Neuro-HIV.

Glial cell activation is a hallmark of several neurodegenerative and neuroinflammatory diseases. During HIV infection, neuroinflammation is associated with cognitive impairment, even during sustained long-term suppressive antiretroviral therapy. However, the cellular subsets contributing to neuronal damage in the CNS during HIV infection remain unclear. Using post-mortem brain samples from eight HIV patients and eight non-neurological disease controls, we identify a subset of CNS phagocytes highly enriched in LGALS3, CTSB, GPNMB and HLA-DR, a signature identified in the context of ageing and neurodegeneration. In HIV patients, the presence of this phagocyte phenotype was associated with synaptic stripping, suggesting an involvement in the pathogenesis of HIV-associated neurocognitive disorder. Taken together, our findings elucidate some of the molecular signatures adopted by CNS phagocytes in HIV-positive patients and contribute to the understanding of how HIV might pave the way to other forms of cognitive decline in ageing HIV patient populations.

Efficiency, sensitivity and specificity of a quantitative real-time PCR assay for Tilapia Lake virus (TiLV).

Tilapia lake virus (TiLV) is an emerging viral pathogen of tilapiines worldwide in wild and farmed tilapia. TiLV is an orthomyxo-like, negative sense segmented RNA virus, belonging to genus Tilapinevirus, family Amnoonviridae. Here we developed a quantitative real-time PCR (qRT-PCR) assay testing primer sets targeting the 10 segments of TiLV. Sensitivity, specificity, efficiency and reproducibility of these assays were examined. Detection sensitivity was equivalent to 2 TCID50/ml when tested on supernatants from cell culture-grown TiLV. Specificity tests showed that all primer sets amplified their respective TiLV segments, and standard curves showed linear correlation of R2 > 0.998 and amplification efficiencies between 93 % and 98 %. Intra- and inter-assay coefficients of variation (CV %) were in the range of 0.0 %- 2.6 % and 0.0 %- 5.9 %, respectively. Sensitivity tests showed that primer sets targeting segments 1, 2, 3 and 4 had the highest detection sensitivities (100.301 TCID50/ml). The qRT-PCR used for detection of viral genome in TiLV infected organs gave virus titers equivalent to 3.80 log10, 3.94 log10 and 3.52 log10 TCID50/ml for brain, kidney and liver tissues, respectively as calculated on the basis of Ct values. These findings suggest that primer optimization for qPCR should not only focus on attaining high amplification efficiency but also sensitivity comparison of primer sets targeting different viral segments in order to develop a method with the highest sensitivity.