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Although SARS-CoV-2 primarily targets the respiratory system, patients with and survivors of COVID-19 can suffer neurological symptoms 1 – 3 . However, an unbiased understanding of the cellular and molecular processes that are affected in the brains of patients with COVID-19 is missing. Here we profile 65,309 single-nucleus transcriptomes from 30 frontal cortex and choroid plexus samples across 14 control individuals (including 1 patient with terminal influenza) and 8 patients with COVID-19. Although our systematic analysis yields no molecular traces of SARS-CoV-2 in the brain, we observe broad cellular perturbations indicating that barrier cells of the choroid plexus sense and relay peripheral inflammation into the brain and show that peripheral T cells infiltrate the parenchyma. We discover microglia and astrocyte subpopulations associated with COVID-19 that share features with pathological cell states that have previously been reported in human neurodegenerative disease 4 – 6 . Synaptic signalling of upper-layer excitatory neurons—which are evolutionarily expanded in humans 7 and linked to cognitive function 8 —is preferentially affected in COVID-19. Across cell types, perturbations associated with COVID-19 overlap with those found in chronic brain disorders and reside in genetic variants associated with cognition, schizophrenia and depression. Our findings and public dataset provide a molecular framework to understand current observations of COVID-19-related neurological disease, and any such disease that may emerge at a later date. Single-nucleus transcriptomes of frontal cortex and choroid plexus samples from patients with COVID-19 reveal pathological cell states that are similar to those associated with human neurodegenerative diseases and chronic brain disorders.

The human polyomavirus, JCPyV, is the causative agent of progressive multifocal leukoencephalopathy (PML) in immunosuppressed and immunomodulated patients. Initial infection with JCPyV is common and the virus establishes a long-term persistent infection in the urogenital system of 50-70% of the human population worldwide. A major gap in the field is that we do not know how the virus traffics from the periphery to the brain to cause disease. Our recent discovery that human choroid plexus epithelial cells are fully susceptible to virus infection together with reports of JCPyV infection of choroid plexus in vivo has led us to hypothesize that the choroid plexus plays a fundamental role in this process. The choroid plexus is known to relay information between the blood and the brain by the release of extracellular vesicles. This is particularly important because human macroglia (oligodendrocytes and astrocytes), the major targets of virus infection in the central nervous system (CNS), do not express the known attachment receptors for the virus and do not bind virus in human tissue sections. In this report we show that JCPyV infected choroid plexus epithelial cells produce extracellular vesicles that contain JCPyV and readily transmit the infection to human glial cells. Transmission of the virus by extracellular vesicles is independent of the known virus attachment receptors and is not neutralized by antisera directed at the virus. We also show that extracellular vesicles containing virus are taken into target glial cells by both clathrin dependent endocytosis and macropinocytosis. Our data support the hypothesis that the choroid plexus plays a fundamental role in the dissemination of virus to brain parenchyma.

Choroid plexus (CP) inflammation can be quantified in vivo with MRI in people with multiple sclerosis (pwMS). It remains unknown whether Epstein Barr Virus (EBV) is related to CP changes. Total of 170 pwMS (116 relapsing-remitting; RRMS and 54 progressive MS; PMS) underwent MRI examination and measurement of humoral anti-EBV response. CP volume and CP pseudo-T2 (pT2), a relaxation time indicative of edema and neuroinflammation, were measured. Moreover, anti-EBV nuclear antigen-1 (EBNA-1) IgG and anti-EBV capsid antigen (VCA) IgG antibodies were measured. The PMS group had greater CP pT2 value when compared to RRMS (1120ms vs. 954ms, p  = 0.037). After adjusting for age and therapy effects, higher CP pT2 values were associated with higher anti-EBNA-1 IgG levels only in PMS ( r  = 0.352, p  = 0.015). Higher Anti-EBV humoral response in pwMS may be associated with increased CP neuroinflammatory changes and may be more relevant for the later chronic stage of the disease. Large-scale studies should investigate whether these findings are generalizable to all types of progressive MS.

Semliki Forest virus (SFV) is a neuropathogenic alphavirus which is of interest both as a model neurotropic alphavirus and as an oncolytic virus with proven potency in preclinical cancer models. In laboratory mice, peripherally administered SFV infiltrates the central nervous system (CNS) and causes encephalitis of varying severity. The route of SFV CNS entrance is poorly understood but has been considered to occur through the blood-brain barrier. Here we show that neuroinvasion of intravenously administered SFV is strictly dependent on very-low-density-lipoprotein receptor (VLDLR) which acts as an entry receptor for SFV. Moreover, SFV primarily enters the CNS through the blood-cerebrospinal fluid (B-CSF) barrier via infecting choroid plexus epithelial cells which show distinctly high expression of VLDLR. This is the first indication of neurotropic alphavirus utilizing choroid plexus for CNS entry, and VLDLR playing a specific and crucial role for mediating SFV entry through this pathway. The authors present that encephalitic Semliki Forest virus uses choroid plexus epithelial cells as neuroinvasion pathway in mice and show that entry is specifically dependent on expression of very low-density-lipoprotein receptor in these cells.

The human polyomavirus JCPyV is an opportunistic pathogen that infects greater than 60% of the world’s population. The virus establishes a persistent and asymptomatic infection in the urogenital system but can cause a fatal demyelinating disease in immunosuppressed or immunomodulated patients following invasion of the CNS. The mechanisms responsible for JCPyV invasion into CNS tissues are not known but direct invasion from the blood to the cerebral spinal fluid via the choroid plexus has been hypothesized. To study the potential of the choroid plexus as a site of neuroinvasion, we used an adult human choroid plexus epithelial cell line to model the blood-cerebrospinal fluid (B-CSF) barrier in a transwell system. We found that these cells formed a highly restrictive barrier to virus penetration either as free virus or as virus associated with extracellular vesicles (EV JC+ ). The restriction was not absolute and small amounts of virus or EV JC+ penetrated and were able to establish foci of infection in primary astrocytes. Disruption of the barrier with capsaicin did not increase virus or EV JC+ penetration leading us to hypothesize that virus and EV JC+ were highly cell-associated and crossed the barrier by an active process. An inhibitor of macropinocytosis increased virus penetration from the basolateral (blood side) to the apical side (CSF side). In contrast, inhibitors of clathrin and raft dependent transcytosis reduced virus transport from the basolateral to the apical side of the barrier. None of the drugs inhibited apical to basolateral transport suggesting directionality. Pretreatment with cyclosporin A, an inhibitor of P-gp, MRP2 and BCRP multidrug resistance transporters, restored viral penetration in cells treated with raft and clathrin dependent transcytosis inhibitors. Because choroid plexus epithelial cells are known to be susceptible to JCPyV infection both in vitro and in vivo we also examined the release of infectious virus from the barrier. We found that virus was preferentially released from the cells into the apical (CSF) chamber. These data show clearly that there are two mechanisms of penetration, direct transcytosis which is capable of seeding the CSF with small amounts of virus, and infection followed by directional release of infectious virions into the CSF compartment.

Epidemic Transient Neonatal Losses (ETNL) is a disease of piglets caused by Senecavirus A (SVA) in which the method of dissemination and associated lesions are not well-defined. This study investigated the possible SVA-induced lesions by examining spontaneous infections in newborn piglets. Histopathology revealed ballooning degeneration of transitional epithelium, nonsuppurative meningoencephalitis, plexus choroiditis, and atrophic enteritis. RT-PCR identified SVA in all tissues evaluated and sequencing confirmed these results. Positive immunoreactivity to SVA was observed in endothelial and epithelial tissues of all organs evaluated. Semithin analysis revealed vacuolization of apical enterocytes of the small intestine, balloon degeneration and necrosis of endothelial cells of the choroid plexus (CP) and nonsuppurative choroid plexitis. Ultrathin evaluation demonstrated hydropic degeneration of apical enterocytes, degeneration and necrosis of endothelium of CP fenestrated capillaries, degeneration of ependymocytes associated with intralesional viral particles. It is proposed that SVA initially infects apical enterocytes of newborn piglets and probably enters the circulatory system with entry to the brain via the CP, by first producing an initial inflammatory reaction, with subsequent encephalitic dissemination. Consequently, SVA probably uses an enteric-neurological method of dissemination.

Echovirus-30 (E-30) is responsible for the extensive global outbreaks of meningitis in children. To gain access to the central nervous system, E-30 first has to cross the epithelial blood–cerebrospinal fluid barrier. Several meningitis causing bacteria preferentially infect human choroid plexus papilloma (HIBCPP) cells in a polar fashion from the basolateral cell side. Here, we investigated the polar infection of HIBCPP cells with E-30. Both apical and basolateral infections caused a significant decrease in the transepithelial electrical resistance of HIBCPP cells. However, to reach the same impact on the barrier properties, the multiplicity of infection of the apical side had to be higher than that of the basolateral infection. Furthermore, the number of infected cells at respective time-points after basolateral infection was significantly higher compared to apical infection. Cytotoxic effects of E-30 on HIBCPP cells during basolateral infection were observed following prolonged infection and appeared more drastically compared to the apical infection. Gene expression profiles determined by massive analysis of cDNA ends revealed distinct regulation of specific genes depending on the side of HIBCPP cells’ infection. Altogether, our data highlights the polar effects of E-30 infection in a human in vitro model of the blood–cerebrospinal fluid barrier leading to central nervous system inflammation.

Several classes of immunomodulators are used for treating relapsing-remitting multiple sclerosis (RRMS). Most of these disease-modifying therapies, except teriflunomide, carry the risk of progressive multifocal leukoencephalopathy (PML), a severely debilitating, often fatal virus-induced demyelinating disease. Because teriflunomide has been shown to have antiviral activity against DNA viruses, we investigated whether treatment of cells with teriflunomide inhibits infection and spread of JC polyomavirus (JCPyV), the causative agent of PML. Treatment of choroid plexus epithelial cells and astrocytes with teriflunomide reduced JCPyV infection and spread. We also used droplet digital PCR to quantify JCPyV DNA associated with extracellular vesicles isolated from RRMS patients. We detected JCPyV DNA in all patients with confirmed PML diagnosis (n = 2), and in six natalizumab-treated (n = 12), two teriflunomide-treated (n = 7), and two nonimmunomodulated (n = 2) patients. Of the 21 patients, 12 (57%) had detectable JCPyV in either plasma or serum. CSF was uniformly negative for JCPyV. Isolation of extracellular vesicles did not increase the level of detection of JCPyV DNA versus bulk unprocessed biofluid. Overall, our study demonstrated an effect of teriflunomide inhibiting JCPyV infection and spread in glial and choroid plexus epithelial cells. Larger studies using patient samples are needed to correlate these in vitro findings with patient data.

Many epidemiological studies have shown an association between infectious agents, particularly viruses, and psychiatric disorders including schizophrenia. However, evidence of a viral infection in the brain that associates with schizophrenia (SCZ), bipolar disorder (BPD), and major depression (MDD) has not been found. A potential reason for this discrepancy may be that viruses are more likely to infect the neuroepithelium than neural tissue. To test this hypothesis, we used viral sequence enrichment technology and performed RNA sequencing in postmortem choroid plexus (CP) isolated from 84 SCZ, 73 BPD, 23 MDD cases and 76 unaffected controls (CNT) from the Stanley Medical Research Institute brain collection. This approach enabled us to identify the presence of 13 viral species in the CP of 46 subjects. We discovered that CP samples collected from subjects with SCZ and BPD are more likely to contain viral sequences. In terms of individual viruses, the Hepatitis C Virus (HCV) was the only viral species that reached the threshold of statistical significance for an association with SCZ and BPD. Therefore, we focused on HCV to characterize the association between psychiatric disorders and viruses. Analysis of the TriNetX electronic health record database with data on 285 million patients revealed that the prevalence of chronic HCV was 3.6 and 3.9 percent in the SCZ and BPD populations respectively. The prevalence of chronic HCV in these populations was almost double that observed for MDD (1.8%) and approximately 7-fold higher than the control population (0.5%). These findings confirm previous studies that report higher HCV prevalence in SCZ and BPD and suggest that HCV infection may be associated with disease pathology rather than behaviors such as intravenous drug injection, since these behaviors are present in all three disorders. We analyzed hippocampus RNA sequencing data from the subjects identified to be HCV positive via sequence capture. We found that although the virus was absent in this tissue, HCV RNA in the CP was associated with consistent host transcriptional changes in the hippocampus that were potentially related to the innate immune response. Our results are consistent with previous studies and provide clues regarding the contribution of viruses to the pathology of psychiatric disorders.

Background Echovirus 30 (E-30) is one of the most frequently isolated pathogens in aseptic meningitis worldwide. To gain access to the central nervous system (CNS), E-30 and immune cells have to cross one of the two main barriers of the CNS, the epithelial blood–cerebrospinal fluid barrier (BCSFB) or the endothelial blood–brain barrier (BBB). In an in vitro model of the BCSFB, it has been shown that E-30 can infect human immortalized brain choroid plexus papilloma (HIBCPP) cells. Methods In this study we investigated the migration of different T cell subpopulations, naive and effector T cells, through HIBCPP cells during E-30 infection. Effects of E-30 infection and the migration process were evaluated via immunofluorescence and flow cytometry analysis, as well as transepithelial resistance and dextran flux measurement. Results Th1 effector cells and enterovirus-specific effector T cells migrated through HIBCPP cells more efficiently than naive CD4 + T cells following E-30 infection of HIBCPP cells. Among the different naive T cell populations, CD8 + T cells crossed the E-30-infected HIBCPP cell layer in a significantly higher number than CD4 + T cells. A large amount of effector T cells also remained attached to the basolateral side of the HIBCPP cells compared with naive T cells. Analysis of HIBCPP barrier function showed significant alteration after E-30 infection and trans- as well as paracellular migration of T cells independent of the respective subpopulation. Morphologic analysis of migrating T cells revealed that a polarized phenotype was induced by the chemokine CXCL12, but reversed to a round phenotype after E-30 infection. Further characterization of migrating Th1 effector cells revealed a downregulation of surface adhesion proteins such as LFA-1 PSGL-1, CD44, and CD49d. Conclusion Taken together these results suggest that naive CD8 + and Th1 effector cells are highly efficient to migrate through the BCSFB in an inflammatory environment. The T cell phenotype is modified during the migration process through HIBCPP cells.