An In Vitro Model of Blood-Brain Barrier for Studies on HIV Neuroinflammation and CNS Antibody Penetration.

The blood-brain barrier (BBB) is one of several barriers between the brain and the peripheral blood system to maintain homeostasis. Understanding the interactions between infectious agents such as human immunodeficiency virus type 1 (HIV-1), which are capable of traversing the BBB and causing neuroinflammation requires modeling an authentic BBB in vitro. Such an in vitro BBB model also helps develop means of targeting viruses that reside in the brain via natural immune effectors such as antibodies. The BBB consists of human brain microvascular endothelial cells (HBMECs), astrocytes, and pericytes. Here we report in vitro methods to establish a dual-cell BBB model consisting of primary HBMECs and primary astrocytes to measure the integrity of the BBB and antibody penetration of the BBB, as well as a method to establish a single cell BBB model to study the impact of HIV-1 infected medium on the integrity of such a BBB.

Development of rhesus macaque astrocyte cell lines supporting infection with a panel of viruses.

Non-human primate (NHP)-based model systems are highly relevant for biomedical research. However, only few NHP cell lines are available and the generation of additional cell lines is an urgent need to help in the refinement and replacement of these models. Using lentiviral transduction of c-Fos, we established cell lines from the brain of rhesus macaques (Macaca mulatta). Transcriptome analysis revealed that these cell lines are closely related to astrocytes, which was confirmed by immunoblot and immunofluorescence microscopy detecting expression of the astrocyte marker glial fibrillary acidic protein (GFAP). Quantitative real-time PCR (qRT-PCR) demonstrated that major pathways of the interferon (IFN) system are intact. Using retroviral pseudotypes we found that the cell lines are susceptible to entry driven by the glycoproteins of vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV) and to a lesser extent influenza A virus (IAV). Finally, these cells supported growth of Zika virus (ZIKV) and Papiine alphaherpesvirus 2 (PaHV2). In summary, we developed IFN-responsive cell lines from the rhesus macaque brain that allowed entry driven by several viral glycoproteins and were permissive to infection with ZIKV and a primate simplexvirus. These cell lines will be useful for efforts to analyze neurotropic viral infections in rhesus macaque models.

Cytosolic DNA sensors activation of human astrocytes inhibits herpes simplex virus through IRF1 induction.

Introduction: While astrocytes participate in the CNS innate immunity against herpes simplex virus type 1 (HSV-1) infection, they are the major target for the virus. Therefore, it is of importance to understand the interplay between the astrocyte-mediated immunity and HSV-1 infection. Methods: Both primary human astrocytes and the astrocyte line (U373) were used in this study. RT-qPCR and Western blot assay were used to measure IFNs, the antiviral IFN-stimulated genes (ISGs), IFN regulatory factors (IRFs) and HSV-1 DNA. IRF1 knockout or knockdown was performed with CRISPR/Cas9 and siRNA transfection techniques. Results: Poly(dA:dT) could inhibit HSV-1 replication and induce IFN-ß/IFN-λs production in human astrocytes. Poly(dA:dT) treatment of astrocytes also induced the expression of the antiviral ISGs (Viperin, ISG56 and MxA). Among IRFs members examined, poly(dA:dT) selectively unregulated IRF1 and IRF9, particularly IRF1 in human astrocytes. The inductive effects of poly(dA:dT) on IFNs and ISGs were diminished in the IRF1 knockout cells. In addition, IRF1 knockout attenuated poly(dA:dT)-mediated HSV-1 inhibition in the cells. Conclusion: The DNA sensors activation induces astrocyte intracellular innate immunity against HSV-1. Therefore, targeting the DNA sensors has potential for immune activation-based HSV-1 therapy.

Neuropathological findings in Down syndrome, Alzheimer's disease and control patients with and without SARS-COV-2: preliminary findings.

The SARS-CoV-2 virus that led to COVID-19 is associated with significant and long-lasting neurologic symptoms in many patients, with an increased mortality risk for people with Alzheimer's disease (AD) and/or Down syndrome (DS). However, few studies have evaluated the neuropathological and inflammatory sequelae in postmortem brain tissue obtained from AD and people with DS with severe SARS-CoV-2 infections. We examined tau, beta-amyloid (Aß), inflammatory markers and SARS-CoV-2 nucleoprotein in DS, AD, and healthy non-demented controls with COVID-19 and compared with non-infected brain tissue from each disease group (total n = 24). A nested ANOVA was used to determine regional effects of the COVID-19 infection on arborization of astrocytes (Sholl analysis) and percent-stained area of Iba-1 and TMEM 119. SARS-CoV-2 antibodies labeled neurons and glial cells in the frontal cortex of all subjects with COVID-19, and in the hippocampus of two of the three DS COVID-19 cases. SARS-CoV-2-related alterations were observed in peri-vascular astrocytes and microglial cells in the gray matter of the frontal cortex, hippocampus, and para-hippocampal gyrus. Bright field microscopy revealed scattered intracellular and diffuse extracellular Aß deposits in the hippocampus of controls with confirmed SARS-CoV-2 infections. Overall, the present preliminary findings suggest that SARS-CoV-2 infections induce abnormal inflammatory responses in Down syndrome.

Modulation of Paracellular Permeability in SARS-CoV-2 Blood-to-Brain Transcytosis.

SARS-CoV-2 primarily infects the lungs via the ACE2 receptor but also other organs including the kidneys, the gastrointestinal tract, the heart, and the skin. SARS-CoV-2 also infects the brain, but the hematogenous route of viral entry to the brain is still not fully characterized. Understanding how SARS-CoV-2 traverses the blood-brain barrier (BBB) as well as how it affects the molecular functions of the BBB are unclear. In this study, we investigated the roles of the receptors ACE2 and DPP4 in the SARS-CoV-2 infection of the discrete cellular components of a transwell BBB model comprising HUVECs, astrocytes, and pericytes. Our results demonstrate that direct infection on the BBB model does not modulate paracellular permeability. Also, our results show that SARS-CoV-2 utilizes clathrin and caveolin-mediated endocytosis to traverse the BBB, resulting in the direct infection of the brain side of the BBB model with a minimal endothelial infection. In conclusion, the BBB is susceptible to SARS-CoV-2 infection in multiple ways, including the direct infection of endothelium, astrocytes, and pericytes involving ACE2 and/or DPP4 and the blood-to-brain transcytosis, which is an event that does not require the presence of host receptors.

SARS-CoV-2 infection exacerbates the cellular pathology of Parkinson's disease in human dopaminergic neurons and a mouse model.

While an association between Parkinson's disease (PD) and viral infections has been recognized, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on PD progression remains unclear. Here, we demonstrate that SARS-CoV-2 infection heightens the risk of PD using human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons and a human angiotensin-converting enzyme 2 (hACE2) transgenic (Tg) mouse model. Our findings reveal that SARS-CoV-2 infection exacerbates PD susceptibility and cellular toxicity in DA neurons pre-treated with human preformed fibrils (hPFFs). Additionally, nasally delivered SARS-CoV-2 infects DA neurons in hACE2 Tg mice, aggravating the damage initiated by hPFFs. Mice infected with SARS-CoV-2 display persisting neuroinflammation even after the virus is no longer detectable in the brain. A comprehensive analysis suggests that the inflammatory response mediated by astrocytes and microglia could contribute to increased PD susceptibility associated with SARS-CoV-2. These findings advance our understanding of the potential long-term effects of SARS-CoV-2 infection on the progression of PD.

[Changes and intervention of reactive astrocyte activation patterns in the progression of Japanese encephalitis].

Objective To clarify the relationship between astrocyte activation patterns and disease progression in epidemic encephalitis B (Japanese encephalitis). Methods First, a mouse model of epidemic encephalitis B was constructed by foot-pad injection of Japanese encephalitis virus (JEV), and the expression of viral protein NS3 in different brain regions was detected by immunofluorescence assay (IFA). Next, IFA, RNA sequencing (RNA-seq) and real-time quantitative PCR (qRT-PCR) were used to clarify the changes in the astrocyte activation patterns at different stages of epidemic encephalitis B. Finally, intracerebroventricular administration of irisin was conducted to regulate the proportion of activation in complement C3-positive A1 astrocytes and S100A10-positive A2 astrocytes, investigating whether it could improve the body mass, behavioral scores, and brain tissue damage in a mouse model. Results NS3 protein was detected by IFA predominantly in the M1/M2 region of the motor cortex and the hippocampus. The number and volume of GFAP-positive astrocytes significantly increased in JEV-infected brain regions, in which the expression of multiple genes associated with A1/A2 astrocyte activation was significantly enhanced. Although intracerebroventricular or intraperitoneal injection of irisin did not improve the prognosis of epidemic encephalitis B, it inhibited the activation of A1 astrocytes and ameliorate neuroinflammation. Conclusion Neurons in the M1/M2 motor cortex and hippocampus are susceptible to JEV infection, in which the abnormal astrocyte activation contributes to the neuroinflammatory injury. Irisin administration may restrain A1 astrocyte activation and alleviate neuroinflammation following JEV infection.

Differential Cytokine Responses of APOE3 and APOE4 Blood-brain Barrier Cell Types to SARS-CoV-2 Spike Proteins.

SARS-CoV-2 spike proteins have been shown to cross the blood-brain barrier (BBB) in mice and affect the integrity of human BBB cell models. However, the effects of SARS-CoV-2 spike proteins in relation to sporadic, late onset, Alzheimer's disease (AD) risk have not been extensively investigated. Here we characterized the individual and combined effects of SARS-CoV-2 spike protein subunits S1 RBD, S1 and S2 on BBB cell types (induced brain endothelial-like cells (iBECs) and astrocytes (iAstrocytes)) generated from induced pluripotent stem cells (iPSCs) harboring low (APOE3 carrier) or high (APOE4 carrier) relative Alzheimer's risk. We found that treatment with spike proteins did not alter iBEC integrity, although they induced the expression of several inflammatory cytokines. iAstrocytes exhibited a robust inflammatory response to SARS-CoV-2 spike protein treatment, with differences found in the levels of cytokine secretion between spike protein-treated APOE3 and APOE4 iAstrocytes. Finally, we tested the effects of potentially anti-inflammatory drugs during SARS-CoV-2 spike protein exposure in iAstrocytes, and discovered different responses between spike protein treated APOE4 iAstrocytes and APOE3 iAstrocytes, specifically in relation to IL-6, IL-8 and CCL2 secretion. Overall, our results indicate that APOE3 and APOE4 iAstrocytes respond differently to anti-inflammatory drug treatment during SARS-CoV-2 spike protein exposure with potential implications to therapeutic responses.

Zika virus infection impairs synaptogenesis, induces neuroinflammation, and could be an environmental risk factor for autism spectrum disorder outcome.

Zika virus (ZIKV) infection was first associated with Central Nervous System (CNS) infections in Brazil in 2015, correlated with an increased number of newborns with microcephaly, which ended up characterizing the Congenital Zika Syndrome (CZS). Here, we investigated the impact of ZIKV infection on the functionality of iPSC-derived astrocytes. Besides, we extrapolated our findings to a Brazilian cohort of 136 CZS children and validated our results using a mouse model. Interestingly, ZIKV infection in neuroprogenitor cells compromises cell migration and causes apoptosis but does not interfere in astrocyte generation. Moreover, infected astrocytes lost their ability to uptake glutamate while expressing more glutamate transporters and secreted higher levels of IL-6. Besides, infected astrocytes secreted factors that impaired neuronal synaptogenesis. Since these biological endophenotypes were already related to Autism Spectrum Disorder (ASD), we extrapolated these results to a cohort of children, now 6-7 years old, and found seven children with ASD diagnosis (5.14 %). Additionally, mice infected by ZIKV revealed autistic-like behaviors, with a significant increase of IL-6 mRNA levels in the brain. Considering these evidence, we inferred that ZIKV infection during pregnancy might lead to synaptogenesis impairment and neuroinflammation, which could increase the risk for ASD.

SARS-CoV-2 Infection of Human Neurons Is TMPRSS2 Independent, Requires Endosomal Cell Entry, and Can Be Blocked by Inhibitors of Host Phosphoinositol-5 Kinase.

2019 coronavirus disease (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to respiratory illness, COVID-19 patients exhibit neurological symptoms lasting from weeks to months (long COVID). It is unclear whether these neurological manifestations are due to an infection of brain cells. We found that a small fraction of human induced pluripotent stem cell (iPSC)-derived neurons, but not astrocytes, were naturally susceptible to SARS-CoV-2. Based on the inhibitory effect of blocking antibodies, the infection seemed to depend on the receptor angiotensin-converting enzyme 2 (ACE2), despite very low levels of its expression in neurons. The presence of double-stranded RNA in the cytoplasm (the hallmark of viral replication), abundant synthesis of viral late genes localized throughout infected cells, and an increase in the level of viral RNA in the culture medium (viral release) within the first 48 h of infection suggested that the infection was productive. Productive entry of SARS-CoV-2 requires the fusion of the viral and cellular membranes, which results in the delivery of the viral genome into the cytoplasm of the target cell. The fusion is triggered by proteolytic cleavage of the viral surface spike protein, which can occur at the plasma membrane or from endosomes or lysosomes. We found that SARS-CoV-2 infection of human neurons was insensitive to nafamostat and camostat, which inhibit cellular serine proteases, including transmembrane serine protease 2 (TMPRSS2). Inhibition of cathepsin L also did not significantly block infection. In contrast, the neuronal infection was blocked by apilimod, an inhibitor of phosphatidyl-inositol 5 kinase (PIK5K), which regulates early to late endosome maturation. IMPORTANCE COVID-19 is a disease caused by the coronavirus SARS-CoV-2. Millions of patients display neurological symptoms, including headache, impairment of memory, seizures, and encephalopathy, as well as anatomical abnormalities, such as changes in brain morphology. SARS-CoV-2 infection of the human brain has been documented, but it is unclear whether the observed neurological symptoms are linked to direct brain infection. The mechanism of virus entry into neurons has also not been characterized. Here, we investigated SARS-CoV-2 infection by using a human iPSC-derived neural cell model and found that a small fraction of cortical-like neurons was naturally susceptible to infection. The productive infection was ACE2 dependent and TMPRSS2 independent. We also found that the virus used the late endosomal and lysosomal pathway for cell entry and that the infection could be blocked by apilimod, an inhibitor of cellular PIK5K.

Evaluation of Baculoviruses as Gene Therapy Vectors for Brain Cancer.

We aimed to assess the potential of baculoviral vectors (BV) for brain cancer gene therapy. We compared them with adenoviral vectors (AdV), which are used in neuro-oncology, but for which there is pre-existing immunity. We constructed BVs and AdVs encoding fluorescent reporter proteins and evaluated their transduction efficiency in glioma cells and astrocytes. Naïve and glioma-bearing mice were intracranially injected with BVs to assess transduction and neuropathology. Transgene expression was also assessed in the brain of BV-preimmunized mice. While the expression of BVs was weaker than AdVs in murine and human glioma cell lines, BV-mediated transgene expression in patient-derived glioma cells was similar to AdV-mediated transduction and showed strong correlation with clathrin expression, a protein that interacts with the baculovirus glycoprotein GP64, mediating BV endocytosis. BVs efficiently transduced normal and neoplastic astrocytes in vivo, without apparent neurotoxicity. BV-mediated transgene expression was stable for at least 21 days in the brain of naïve mice, but it was significantly reduced after 7 days in mice systemically preimmunized with BVs. Our findings indicate that BVs efficiently transduce glioma cells and astrocytes without apparent neurotoxicity. Since humans do not present pre-existing immunity against BVs, these vectors may constitute a valuable tool for the delivery of therapeutic genes into the brain.

Poly(I:C) promotes neurotoxic amyloid ß accumulation through reduced degradation by decreasing neprilysin protein levels in astrocytes.

Inflammation associated with viral infection of the nervous system has been involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD) and multiple sclerosis. Polyinosinic:polycytidylic acid (poly[I:C]) is a Toll-like receptor 3 (TLR3) agonist that mimics the inflammatory response to systemic viral infections. Despite growing recognition of the role of glial cells in AD pathology, their involvement in the accumulation and clearance of amyloid ß (Aß) in the brain of patients with AD is poorly understood. Neprilysin (NEP) and insulin-degrading enzyme (IDE) are the main Aß-degrading enzymes in the brain. This study investigated whether poly(I:C) regulated Aß degradation and neurotoxicity by modulating NEP and IDE protein levels through TLR3 in astrocytes. To this aim, primary rat primary astrocyte cultures were treated with poly(I:C) and inhibitors of the TLR3 signaling. Protein levels were assessed by Western blot. Aß toxicity to primary neurons was measured by lactate dehydrogenase release. Poly(I:C) induced a significant decrease in NEP levels on the membrane of astrocytes as well as in the culture medium. The degradation of exogenous Aß was markedly delayed in poly(I:C)-treated astrocytes. This delay significantly increased the neurotoxicity of exogenous Aß1-42. Altogether, these results suggest that viral infections induce Aß neurotoxicity by decreasing NEP levels in astrocytes and consequently preventing Aß degradation.

The NeuroinflammatoryPotential of HIV-1 NefVariants in Modulating the Gene Expression Profile of Astrocytes.

HIV-1 mediated neurotoxicity is thought to be associated with HIV-1 viral proteins activating astrocytes and microglia by inducing inflammatory cytokines leading to the development of HIV-associated neurocognitive disorder (HAND). In the current study, we observe how HIV-1 Nef upregulates the levels of IL-6, IP-10, and TNF-α around 6.0fold in normal human astrocytes (NHAs) compared to cell and empty vector controls. Moderate downregulation in the expression profile of inflammatory cytokines was observed due to RNA interference. Furthermore, we determine the impact of inflammatory cytokines in the upregulation of kynurenine pathway metabolites, such as indoleamine 2,3-dioxygenase (IDO), and 3-hydroxyanthranilic acid oxygenase (HAAO) in NHA, and found the same to be 3.0- and 3.2-fold, respectively. Additionally, the variation in the level of nitric oxide before and after RNA interference was significant. The upregulated cytokines and pathway-specific metabolites could be linked with the neurotoxic potential of HIV-1 Nef. Thus, the downregulation in cytokines and kynurenine metabolites observed after siRNA-Nef interference indicates the possibility of combining the RNA interference approach with current antiretroviral therapy to prevent neurotoxicity development.

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.

Tropism of SARS-CoV-2 for human cortical astrocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.

HIV-1 Tat endocytosis and retention in endolysosomes affects HIV-1 Tat-induced LTR transactivation in astrocytes.

The presence of latent HIV-1 reservoirs in the periphery and brain represents a major obstacle to curing HIV-1 infection. As an essential protein for HIV-1 viral replication, HIV-1 Tat, mostly intracellular, has been implicated in latent HIV-1 infection. From HIV-1 infected cells, HIV-1 Tat is actively secreted and bystander cells uptake the released Tat whereupon it is endocytosed and internalized into endolysosomes. However, to activate the HIV-1 LTR promoter and increase HIV-1 replication, HIV-1 Tat must first escape from the endolysosomes and then enter the nucleus. Here, we tested the hypothesis that HIV-1 Tat can accumulate in endolysosomes and contribute to the activation of latent HIV-1 in astrocytes. Using U87MG astrocytoma cells expressing HIV-1 LTR-driven luciferase and primary human astrocytes we found that exogenous HIV-1 Tat enters endolysosomes, resides in endolysosomes for extended periods of time, and induces endolysosome de-acidification as well as enlargement. The weak base chloroquine promoted the release of HIV-1 Tat from endolysosomes and induced HIV-1 LTR transactivation. Similar results were observed by activating endolysosome Toll-like receptor 3 (TLR3) and TLR7/8. Conversely, pharmacological block of TLRs and knocking down expression levels of TLR3 and TLR7, but not TLR8, prevented endolysosome leakage and attenuated HIV-1 Tat-mediated HIV-1 LTR transactivation. Our findings suggest that HIV-1 Tat accumulation in endolysosomes may play an important role in controlling HIV-1 transactivation.

Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection.

Hand, foot and mouth disease (HFMD) caused by Human Enterovirus A71 (HEVA71) infection is typically a benign infection. However, in minority of cases, children can develop severe neuropathology that culminate in fatality. Approximately 36.9% of HEVA71-related hospitalizations develop neurological complications, of which 10.5% are fatal. Yet, the mechanism by which HEVA71 induces these neurological deficits remain unclear. Here, we show that HEVA71-infected astrocytes release CXCL1 which supports viral replication in neurons by activating the CXCR2 receptor-associated ERK1/2 signaling pathway. Elevated CXCL1 levels correlates with disease severity in a HEVA71-infected mice model. In humans infected with HEVA71, high CXCL1 levels are only present in patients presenting neurological complications. CXCL1 release is specifically triggered by VP4 synthesis in HEVA71-infected astrocytes, which then acts via its receptor CXCR2 to enhance viral replication in neurons. Perturbing CXCL1 signaling or VP4 myristylation strongly attenuates viral replication. Treatment with AZD5069, a CXCL1-specific competitor, improves survival and lessens disease severity in infected animals. Collectively, these results highlight the CXCL1-CXCR2 signaling pathway as a potential target against HFMD neuropathogenesis.

Comparative pathogenesis of different phylogroup I bat lyssaviruses in a standardized mouse model.

A plethora of bat-associated lyssaviruses potentially capable of causing the fatal disease rabies are known today. Transmitted via infectious saliva, occasionally-reported spillover infections from bats to other mammals demonstrate the permeability of the species-barrier and highlight the zoonotic potential of bat-related lyssaviruses. However, it is still unknown whether and, if so, to what extent, viruses from different lyssavirus species vary in their pathogenic potential. In order to characterize and systematically compare a broader group of lyssavirus isolates for their viral replication kinetics, pathogenicity, and virus release through saliva-associated virus shedding, we used a mouse infection model comprising a low (102 TCID50) and a high (105 TCID50) inoculation dose as well as three different inoculation routes (intramuscular, intranasal, intracranial). Clinical signs, incubation periods, and survival were investigated. Based on the latter two parameters, a novel pathogenicity matrix was introduced to classify lyssavirus isolates. Using a total of 13 isolates from ten different virus species, this pathogenicity index varied within and between virus species. Interestingly, Irkut virus (IRKV) and Bokeloh bat lyssavirus (BBLV) obtained higher pathogenicity scores (1.14 for IRKV and 1.06 for BBLV) compared to rabies virus (RABV) isolates ranging between 0.19 and 0.85. Also, clinical signs differed significantly between RABV and other bat lyssaviruses. Altogether, our findings suggest a high diversity among lyssavirus isolates concerning survival, incubation period, and clinical signs. Virus shedding significantly differed between RABVs and other lyssaviruses. Our results demonstrated that active shedding of infectious virus was exclusively associated with two RABV isolates (92% for RABV-DogA and 67% for RABV-Insectbat), thus providing a potential explanation as to why sustained spillovers are solely attributed to RABVs. Interestingly, 3D imaging of a selected panel of brain samples from bat-associated lyssaviruses demonstrated a significantly increased percentage of infected astrocytes in mice inoculated with IRKV (10.03%; SD±7.39) compared to RABV-Vampbat (2.23%; SD±2.4), and BBLV (0.78%; SD±1.51), while only individual infected cells were identified in mice infected with Duvenhage virus (DUVV). These results corroborate previous studies on RABV that suggest a role of astrocyte infection in the pathogenicity of lyssaviruses.