Mouse Hepatitis Virus Strain Research Articles

Neurotropic Murine β-Coronavirus Infection Causes Differential Expression of Connexin 47 in Oligodendrocyte Subpopulations Associated with Demyelination.

Gap junctions (GJs) play a crucial role in the survival of oligodendrocytes and myelination of the central nervous system (CNS). In this study, we investigated the spatiotemporal changes in the expression of oligodendroglial GJ protein connexin 47 (Cx47), its primary astroglial coupling partner, Cx43, and their association with demyelination following intracerebral infection with mouse hepatitis virus (MHV). Neurotropic strains of MHV, a β-coronavirus, induce an acute encephalomyelitis followed by a chronic demyelinating disease that shares similarities with the human disease multiple sclerosis (MS). Our results reveal that Cx47 GJs are persistently lost in mature oligodendrocytes, not only in demyelinating lesions but also in surrounding normal appearing white and gray matter areas, following an initial loss of astroglial Cx43 GJs during acute infection. At later stages after viral clearance, astroglial Cx43 GJs re-emerge but mature oligodendrocytes fail to fully re-establish GJs with astrocytes due to lack of Cx47 GJ expression. In contrast, at this later demyelinating stage, the increased oligodendrocyte precursor cells appear to exhibit Cx47 GJs. Our findings further highlight varying degrees of demyelination in distinct spinal cord regions, with the thoracic cord showing the most pronounced demyelination. The regional difference in demyelination correlates well with dynamic changes in the proportion of different oligodendrocyte lineage cells exhibiting differential Cx47 GJ expression, suggesting an important mechanism of progressive demyelination even after viral clearance.

Mouse hepatitis virus JHMV I protein is required for maximal virulence.

Betacoronaviruses encode a conserved accessory gene within the +1 open reading frame (ORF) of nucleocapsid called the internal N gene. This gene is referred to as "I" for mouse hepatitis virus (MHV), ORF9b for severe acute respiratory CoV (SARS-CoV) and SARS-CoV-2, and ORF8b for Middle East respiratory syndrome CoV (MERS-CoV). Previous studies have shown ORF8b and ORF9b have immunoevasive properties, while the only known information for MHV I is its localization within the virion of the hepatotropic/neurotropic A59 strain of MHV. Whether MHV I is an innate immune antagonist or has other functions has not been evaluated. In this report, we show that the I protein of the neurotropic JHM strain of MHV (JHMV) lacks a N terminal domain present in other MHV strains, has immunoevasive properties, and is a component of the virion. Genetic deletion of JHMV I (rJHMVIΔ57-137) resulted in a highly attenuated virus both in vitro and in vivo that displayed a post RNA replication/transcription defect that ultimately resulted in fewer infectious virions packaged compared with wild-type virus. This phenotype was only seen for rJHMVIΔ57-137, suggesting the structural changes predicted for A59 I altered its function, as genetic deletion of A59 I did not change viral replication or pathogenicity. Together, these data show that JHMV I both acts as a mild innate immune antagonist and aids in viral assembly and infectious virus production, and suggest that the internal N proteins from different betacoronaviruses have both common and virus strain-specific properties.IMPORTANCECoV accessory genes are largely studied in overexpression assays and have been identified as innate immune antagonists. However, functions identified after overexpression are often not confirmed in the infected animal host. Furthermore, some accessory proteins are components of the CoV virion, but their role in viral replication and release remains unclear. Here, we utilized reverse genetics to abrogate expression of a conserved CoV accessory gene, the internal N ("I") gene, of the neurotropic JHMV strain of MHV and found that loss of the I gene resulted in a post replication defect that reduced virion assembly and ultimately infectious virus production, while also increasing some inflammatory molecule expression. Thus, the JHMV I protein has roles in virion assembly that were previously underappreciated and in immunoevasion.

HSP70 contributes to pathogenesis of fulminant hepatitis induced by coronavirus

Fulminant viral hepatitis (FH) represents a significant clinical challenge, with its pathogenesis not yet fully elucidated. Heat shock protein (HSP)70, a molecular chaperone protein with a broad range of cytoprotective functions, is upregulated in response to stress. However, the role of HSP70 in FH remains to be investigated. Notably, HSP70 expression is upregulated in the livers of coronavirus-infected mice and patients. Therefore, we investigated the mechanistic role of HSP70 in coronavirus-associated FH pathogenesis. FH was induced in HSP70-deficient (HSP70 KO) mice or in WT mice treated with the HSP70 inhibitor VER155008 when infected with the mouse hepatitis virus strain A59 (MHV-A59). MHV-A59-infected HSP70 KO mice exhibited significantly reduced liver damage and mortality. This effect was attributed to decreased infiltration of monocyte-macrophages and neutrophils in the liver of HSP70 KO mice, resulting in lower levels of inflammatory cytokines such as IL-1β, TNFα, and IL-6, and a reduced viral load. Moreover, treatment with the HSP70 inhibitor VER155008 protected mice from MHV-A59-induced liver damage and FH mortality. In summary, HSP70 promotes coronavirus-induced FH pathogenesis by enhancing the infiltration of monocyte-macrophages and neutrophils and promoting the secretion of inflammatory cytokines. Therefore, HSP70 is a potential therapeutic target in viral FH intervention.

Cystatin F attenuates neuroinflammation and demyelination following murine coronavirus infection of the central nervous system

BackgroundCystatin F is a secreted lysosomal cysteine protease inhibitor that has been implicated in affecting the severity of demyelination and enhancing remyelination in pre-clinical models of immune-mediated demyelination. How cystatin F impacts neurologic disease severity following viral infection of the central nervous system (CNS) has not been well characterized and was the focus of this study. We used cystatin F null-mutant mice (Cst7-/-) with a well-established model of murine coronavirus-induced neurologic disease to evaluate the contributions of cystatin F in host defense, demyelination and remyelination.MethodsWildtype controls and Cst7-/- mice were intracranially (i.c.) infected with a sublethal dose of the neurotropic JHM strain of mouse hepatitis virus (JHMV), with disease progression and survival monitored daily. Viral plaque assays and qPCR were used to assess viral levels in CNS. Immune cell infiltration into the CNS and immune cell activation were determined by flow cytometry and 10X genomics chromium 3’ single cell RNA sequencing (scRNA-seq). Spinal cord demyelination was determined by luxol fast blue (LFB) and Hematoxylin/Eosin (H&E) staining and axonal damage assessed by immunohistochemical staining for SMI-32. Remyelination was evaluated by electron microscopy (EM) and calculation of g-ratios.ResultsJHMV-infected Cst7-/- mice were able to control viral replication within the CNS, indicating that cystatin F is not essential for an effective Th1 anti-viral immune response. Infiltration of T cells into the spinal cords of JHMV-infected Cst7-/- mice was increased compared to infected controls, and this correlated with increased axonal damage and demyelination associated with impaired remyelination. Single-cell RNA-seq of CD45 + cells enriched from spinal cords of infected Cst7-/- and control mice revealed enhanced expression of transcripts encoding T cell chemoattractants, Cxcl9 and Cxcl10, combined with elevated expression of interferon-g (Ifng) and perforin (Prf1) transcripts in CD8 + T cells from Cst7-/- mice compared to controls.ConclusionsCystatin F is not required for immune-mediated control of JHMV replication within the CNS. However, JHMV-infected Cst7-/- mice exhibited more severe clinical disease associated with increased demyelination and impaired remyelination. The increase in disease severity was associated with elevated expression of T cell chemoattractant chemokines, concurrent with increased neuroinflammation. These findings support the idea that cystatin F influences expression of proinflammatory gene expression impacting neuroinflammation, T cell activation and/or glia cell responses ultimately impacting neuroinflammation and neurologic disease.

Development of Mouse Hepatitis Virus Chimeric Reporter Viruses Expressing the 3CLpro Proteases of Human Coronaviruses HKU1 and OC43 Reveals Susceptibility to Inactivation by Natural Inhibitors Baicalin and Baicalein

The recent emergence of SARS-CoV-2 in 2019 has highlighted the necessity of antiviral therapeutics for current and future emerging coronaviruses. Recently, the traditional herbal medicines baicalein, baicalin, and andrographolide have shown inhibition against the main protease of SARS-CoV-2. This provides a promising new direction for COVID-19 therapeutics, but it remains unknown whether these three substances inhibit other human coronaviruses. In this study, we describe the development of novel chimeric mouse hepatitis virus (MHV) reporters that express firefly luciferase (FFL) and the 3CLpro proteases of human coronaviruses HKU1 and OC43. These chimeric viruses were used to determine if the phytochemicals baicalein, baicalin, and andrographolide are inhibitory against human coronavirus strains HKU1 and OC43. Our data show that both baicalein and baicalin exhibit inhibition towards the chimeric MHV strains. However, andrographolide induces cytotoxicity and failed to demonstrate selective toxicity towards the viruses. This study reports the development and use of a safe replicating reporter platform to investigate potential coronavirus 3CLpro inhibitors against common-cold human coronavirus strains HKU1 and OC43.

Retinoic Acid-Mediated Inhibition of Mouse Coronavirus Replication Is Dependent on IRF3 and CaMKK.

The ongoing COVID-19 pandemic has revealed the shortfalls in our understanding of how to treat coronavirus infections. With almost 7 million case fatalities of COVID-19 globally, the catalog of FDA-approved antiviral therapeutics is limited compared to other medications, such as antibiotics. All-trans retinoic acid (RA), or activated vitamin A, has been studied as a potential therapeutic against coronavirus infection because of its antiviral properties. Due to its impact on different signaling pathways, RA's mechanism of action during coronavirus infection has not been thoroughly described. To determine RA's mechanism of action, we examined its effect against a mouse coronavirus, mouse hepatitis virus strain A59 (MHV). We demonstrated that RA significantly decreased viral titers in infected mouse L929 fibroblasts and RAW 264.7 macrophages. The reduced viral titers were associated with a corresponding decrease in MHV nucleocapsid protein expression. Using interferon regulatory factor 3 (IRF3) knockout RAW 264.7 cells, we demonstrated that RA-induced suppression of MHV required IRF3 activity. RNA-seq analysis of wildtype and IRF3 knockout RAW cells showed that RA upregulated calcium/calmodulin (CaM) signaling proteins, such as CaM kinase kinase 1 (CaMKK1). When treated with a CaMKK inhibitor, RA was unable to upregulate IRF activation during MHV infection. In conclusion, our results demonstrate that RA-induced protection against coronavirus infection depends on IRF3 and CaMKK.

Microglia influence immune responses and restrict neurologic disease in response to central nervous system infection by a neurotropic murine coronavirus.

Intracranial (i.c.) inoculation of susceptible mice with a glial-tropic strain of mouse hepatitis virus (JHMV), a murine coronavirus, results in an acute encephalomyelitis followed by viral persistence in white matter tracts accompanied by chronic neuroinflammation and demyelination. Microglia serve numerous functions including maintenance of the healthy central nervous system (CNS) and are among the first responders to injury or infection. More recently, studies have demonstrated that microglia aid in tailoring innate and adaptive immune responses following infection by neurotropic viruses including flaviviruses, herpesviruses, and picornaviruses. These findings have emphasized an important role for microglia in host defense against these viral pathogens. In addition, microglia are also critical in optimizing immune-mediated control of JHMV replication within the CNS while restricting the severity of demyelination and enhancing remyelination. This review will highlight our current understanding of the molecular and cellular mechanisms by which microglia aid in host defense, limit neurologic disease, and promote repair following CNS infection by a neurotropic murine coronavirus.

Ablation of microglia following infection of the central nervous system with a neurotropic murine coronavirus infection leads to increased demyelination and impaired remyelination

Intracranial inoculation of susceptible mice with a glial-tropic strain of mouse hepatitis virus (JHMV), a murine coronavirus, results in an acute encephalomyelitis followed by viral persistence in white matter tracts accompanied by chronic neuroinflammation and demyelination. Microglia are the resident immune cell of the central nervous system (CNS) and are considered important in regulating events associated with neuroinflammation as well as influencing both white matter damage and remyelination. To better understand mechanisms by which microglia contribute to these immune-mediated events, JHMV-infected mice with established demyelination were treated with the small molecular inhibitor of colony stimulating factor 1 receptor (CSF1R), PLX5622, to deplete microglia. Treatment with PLX5622 did not affect viral replication within the CNS yet the severity of demyelination was increased and remyelination impaired compared to control mice. Gene expression analysis revealed that targeting microglia resulted in altered expression of genes associated with immune cell activation and phagocytosis of myelin debris. These findings indicate that microglia are not critical in viral surveillance in persistently JHMV-infected mice yet restrict white matter damage and remyelination, in part, by influencing phagocytosis of myelin debris.

Sustained Infiltration of Neutrophils Into the CNS Results in Increased Demyelination in a Viral-Induced Model of Multiple Sclerosis.

Intracranial inoculation of the neuroadapted JHM strain of mouse hepatitis virus (JHMV) into susceptible strains of mice results in acute encephalomyelitis followed by a cimmune-mediated demyelination similar to the human demyelinating disease multiple sclerosis (MS). JHMV infection of transgenic mice in which expression of the neutrophil chemoattractant chemokine CXCL1 is under the control of a tetracycline-inducible promoter active within GFAP-positive cells results in sustained neutrophil infiltration in the central nervous system (CNS) that correlates with an increase in spinal cord demyelination. We used single cell RNA sequencing (scRNAseq) and flow cytometry to characterize molecular and cellular changes within the CNS associated with increased demyelination in transgenic mice compared to control animals. These approaches revealed the presence of activated neutrophils as determined by expression of mRNA transcripts associated with neutrophil effector functions, including CD63, MMP9, S100a8, S100a9, and ASPRV1, as well as altered neutrophil morphology and protein expression. Collectively, these findings reveal insight into changes in the profile of neutrophils associated with increased white matter damage in mice persistently infected with a neurotropic coronavirus.

Priming With Rhinovirus Protects Mice Against a Lethal Pulmonary Coronavirus Infection.

Rhinoviruses (RV) have been shown to inhibit subsequent infection by heterologous respiratory viruses, including influenza viruses and severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). To better understand the mechanisms whereby RV protects against pulmonary coronavirus infection, we used a native murine virus, mouse hepatitis virus strain 1 (MHV-1), that causes severe disease in the lungs of infected mice. We found that priming of the respiratory tract with RV completely prevented mortality and reduced morbidity of a lethal MHV-1 infection. Replication of MHV-1 was reduced in RV-primed mouse lungs although expression of antiviral type I interferon, IFN-β, was more robust in mice infected with MHV-1 alone. We further showed that signaling through the type I interferon receptor was required for survival of mice given a non-lethal dose of MHV-1. RV-primed mice had reduced pulmonary inflammation and hemorrhage and influx of leukocytes, especially neutrophils, in the airways upon MHV-1 infection. Although MHV-1 replication was reduced in RV-primed mice, RV did not inhibit MHV-1 replication in coinfected lung epithelial cells in vitro. In summary, RV-mediated priming in the respiratory tract reduces viral replication, inflammation, and tissue damage, and prevents mortality of a pulmonary coronavirus infection in mice. These results contribute to our understanding of how distinct respiratory viruses interact with the host to affect disease pathogenesis, which is a critical step in understanding how respiratory viral coinfections impact human health.

Virus-Specific Regulatory T Cells Persist as Memory in a Neurotropic Coronavirus Infection.

Regulatory T cells (Tregs) are critical for regulating immunopathogenic responses in a variety of infections, including infection of mice with JHM strain of mouse hepatitis virus (JHMV), a neurotropic coronavirus that causes immune-mediated demyelinating disease. Although virus-specific Tregs are known to mitigate disease in this infection by suppressing pathogenic effector T cell responses of the same specificity, it is unclear whether these virus-specific Tregs form memory populations and persist similar to their conventional T cell counterparts of the same epitope specificity. Using congenically labeled JHMV-specific Tregs, we found that virus-specific Tregs persist long-term after murine infection, through at least 180 d postinfection and stably maintain Foxp3 expression. We additionally demonstrate that these cells are better able to proliferate and inhibit virus-specific T cell responses postinfection than naive Tregs of the same specificity, further suggesting that these cells differentiate into memory Tregs upon encountering cognate Ag. Taken together, these data suggest that virus-specific Tregs are able to persist long-term in the absence of viral Ag as memory Tregs.

Basic reproduction numbers of three strains of mouse hepatitis viruses in mice.

Mouse hepatitis virus (MHV) is a murine coronavirus and one of the most important pathogens in laboratory mice. Although various strains of MHV have been isolated, they are generally excreted in the feces and transmitted oronasally via aerosols and contaminated bedding. In this study, we attempted to determine the basic reproduction numbers (R0) of three strains of MHV to improve our understanding of MHV infections in mice. Five‐week‐old female C57BL/6J mice were inoculated intranasally with either the Y, NuU, or JHM variant strain of MHV and housed with two naïve mice. After 4 weeks, the presence or absence of anti‐MHV antibody in the mice was determined by ELISA. We also examined the distribution of MHV in the organs of Y, NuU, or JHM variant‐infected mice. Our data suggest that the transmissibility of MHV is correlated with viral growth in the gastrointestinal tract of infected mice. To the best of our knowledge, this is the first report to address the basic reproduction numbers among pathogens in laboratory animals.

Investigation of mouse hepatitis virus strain A59 inactivation under both ambient and cold environments reveals the mechanisms of infectivity reduction following UVC exposure

The surface contamination of SARS-CoV-2 is becoming a potential source of virus transmission during the pandemic of COVID-19. Under the cold environment, the infection incidents would be more severe with the increase of virus survival time. Thus, the disinfection of contaminated surfaces in both ambient and cold environments is a critical measure to restrain the spread of the virus. In our study, it was demonstrated that the 254 nm ultraviolet-C (UVC) is an efficient method to inactivate a coronavirus, mouse hepatitis virus strain A59 (MHV-A59). The inactivation rate to MHV-A59 coronavirus was up to 99.99% when UVC doses were 2.90 and 14.0 mJ/cm2 at room temperature (23 °C) and in cold environment (−20 °C), respectively. Further mechanistic study demonstrated that UVC could induce spike protein damage to partly impede virus attachment and genome penetration processes, which contributes to 12% loss of viral infectivity. Additionally, it can induce genome damage to significantly interrupt genome replication, protein synthesis, virus assembly and release processes, which takes up 88% contribution to viral inactivation. With these mechanistic understandings, it will greatly contribute to the prevention and control of the current SARS-CoV-2 transmissions in cold chains (low temperature-controlled product supply chains), public area such as airport, school, and warehouse.

Reviving chloroquine for anti-SARS-CoV-2 treatment with cucurbit[7]uril-based supramolecular formulation

The wide-spreading SARS-CoV-2 virus has put the world into boiling water for more than a year, however pharmacological therapies to act effectively against coronavirus disease 2019 (COVID-19) remain elusive. Chloroquine (CQ), an antimalarial drug, was found to exhibit promising antiviral activity in vitro and in vivo at a high dosage, thus CQ was approved by the FDA for the emergency use authorization (EUA) in the fight against COVID-19 in the US, but later was revoked the EUA status due to the severe clinical toxicity. Herein, we show that supramolecular formulation of CQ by a macrocyclic host, curcurbit[7]uril (CB[7]), reduced its non-specific toxicity and improved its antiviral activity against coronavirus, working in synergy with CB[7]. CB[7] was found to form 1:1 host-guest complexes with CQ, with a binding constant of ∼104 L/mol. The CQ-CB[7] formulation decreased the cytotoxicity of CQ against Vero E6 and L-02 cell lines. In particular, the cytotoxicity of CQ (60 μmol/L) against both Vero E6 cell line and L-02 cell lines was completely inhibited in the presence of 300 μmol/L and 600 μmol/L CB[7], respectively. Furthermore, the CB[7] alone showed astonishing antiviral activity in SARS-CoV-2 infected Vero E6 cells and mouse hepatitis virus strain A59 (MHV-A59) infected N2A cells, and synergistically improved the antiviral activity of CQ-CB[7], suggesting that CB[7]-based CQ formulation has a great potential as a safe and effective antiviral agent against SARS-CoV-2 and other coronavirus.

Insights into coronavirus immunity taught by the murine coronavirus.

Coronaviruses (CoVs) represent enveloped, ss RNA viruses with the ability to infect a range of vertebrates causing mainly lung, CNS, enteric, and hepatic disease. While the infection with human CoV is commonly associated with mild respiratory symptoms, the emergence of SARS‐CoV, MERS‐CoV, and SARS‐CoV‐2 highlights the potential for CoVs to cause severe respiratory and systemic disease. The devastating global health burden caused by SARS‐CoV‐2 has spawned countless studies seeking clinical correlates of disease severity and host susceptibility factors, revealing a complex network of antiviral immune circuits. The mouse hepatitis virus (MHV) is, like SARS‐CoV‐2, a beta‐CoV and is endemic in wild mice. Laboratory MHV strains have been extensively studied to reveal coronavirus virulence factors and elucidate host mechanisms of antiviral immunity. These are reviewed here with the aim to identify translational insights for SARS‐CoV‐2 learned from murine CoVs.

RT-PCR Assay for Detection of Enterotropic Strains of Mouse Hepatitis Virus in Faecal Samples

Background: Mouse Hepatitis Virus (MHV) is one of the most important and common viral infections of laboratory mice, due to its highly contagious and subclinical nature, posing threat to the research outcomes. Periodic screening of laboratory mice for MHV is mandatory. Hence, this study was intended to develop sensitive faecal based RT-PCR assays to detect active infection of enterotropic MHV in laboratory mice. Methods: Primers targeting N-gene of MHV were selected and their sensitivity was analysed in tenfold serially diluted gene template in the presence of negative mouse faecal cDNA. Thirty-six weaned mice at the age of 6 to 18 weeks were randomly selected at different periods and the blood and faecal sample were collected for serology and RT-PCR assay respectively. RT-PCR assay in colon samples was carried out for comparison. Result: PCR assay of MHV detected as low as 4 fg of plasmid DNA. Seroprevalence of MHV is very high than the prevalence by RT-PCR assay showing its retrospective nature and also seroprevalence includes both enterotropic and polytropic strain. RT-PCR results in faecal samples are analogous with that of the colon samples, showing the reliability of antemortem testing of mice for enterotropic strain of MHV.

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.

Oligodendrocytes that survive acute coronavirus infection induce prolonged inflammatory responses in the CNS

Neurotropic strains of mouse hepatitis virus (MHV), a coronavirus, cause acute and chronic demyelinating encephalomyelitis with similarities to the human disease multiple sclerosis. Here, using a lineage-tracking system, we show that some cells, primarily oligodendrocytes (OLs) and oligodendrocyte precursor cells (OPCs), survive the acute MHV infection, are associated with regions of demyelination, and persist in the central nervous system (CNS) for at least 150 d. These surviving OLs express major histocompatibility complex (MHC) class I and other genes associated with an inflammatory response. Notably, the extent of inflammatory cell infiltration was variable, dependent on anatomic location within the CNS, and without obvious correlation with numbers of surviving cells. We detected more demyelination in regions with larger numbers of T cells and microglia/macrophages compared to those with fewer infiltrating cells. Conversely, in regions with less inflammation, these previously infected OLs more rapidly extended processes, consistent with normal myelinating function. Together, these results show that OLs are inducers as well as targets of the host immune response and demonstrate how a CNS infection, even after resolution, can induce prolonged inflammatory changes with CNS region-dependent impairment in remyelination.

Microglia influence host defense, disease, and repair following murine coronavirus infection of the central nervous system.

The present study examines functional contributions of microglia in host defense, demyelination, and remyelination following infection of susceptible mice with a neurotropic coronavirus. Treatment with PLX5622, an inhibitor of colony stimulating factor 1 receptor (CSF1R) that efficiently depletes microglia, prior to infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in increased mortality compared with control mice that correlated with impaired control of viral replication. Single cell RNA sequencing (scRNASeq) of CD45+ cells isolated from the CNS revealed that PLX5622 treatment resulted in muted CD4+ T cell activation profile that was associated with decreased expression of transcripts encoding MHC class II and CD86 in macrophages but not dendritic cells. Evaluation of spinal cord demyelination revealed a marked increase in white matter damage in PLX5622‐treated mice that corresponded with elevated expression of transcripts encoding disease‐associated proteins Osteopontin (Spp1), Apolipoprotein E (Apoe), and Triggering receptor expressed on myeloid cells 2 (Trem2) that were enriched within macrophages. In addition, PLX5622 treatment dampened expression of Cystatin F (Cst7), Insulin growth factor 1 (Igf1), and lipoprotein lipase (Lpl) within macrophage populations which have been implicated in promoting repair of damaged nerve tissue and this was associated with impaired remyelination. Collectively, these findings argue that microglia tailor the CNS microenvironment to enhance control of coronavirus replication as well as dampen the severity of demyelination and influence repair.

Azadirachta indica A. Juss Ameliorates Mouse Hepatitis Virus-Induced Neuroinflammatory Demyelination by Modulating Cell-to-Cell Fusion in an Experimental Animal Model of Multiple Sclerosis.

Mouse hepatitis virus (MHV)-induced murine neuroinflammation serves as a model to study acute meningoencephalomyelitis, hepatitis, and chronic neuroinflammatory demyelination; which mimics certain pathologies of the human neurologic disease, multiple sclerosis (MS). MHV-induced acute neuroinflammation occurs due to direct glial cell dystrophy instigated by central nervous system (CNS)-resident microglia and astrocytes, in contrast to peripheral CD4+T cell-mediated myelin damage prevalent in the experimental autoimmune encephalomyelitis (EAE) model of MS. Viral envelope Spike glycoprotein-mediated cell-to-cell fusion is an essential mechanistic step for MHV-induced CNS pathogenicity. Although Azadirachta indica (Neem), a traditional phytomedicine, is known for its anti-inflammatory, anti-fungal, and spermicidal activities, not much is known about anti-neuroinflammatory properties of its bark (NBE) in MHV-induced acute neuroinflammation and chronic demyelination. Recombinant demyelinating MHV strain (RSA59) was preincubated with NBE to arrest the infection-initiation event, and its effect on viral replication, viral transcription, cytokine expression, and successive pathogenicity were investigated in vitro and in vivo. Virus-free Luciferase assay explained NBE’s anti-virus-to-cell fusion activity in vitro. Intracranial inoculation of RSA59 preincubated with NBE into the mouse brain significantly reduces acute hepatitis, meningoencephalomyelitis, and chronic progressive demyelination. Additionally, NBE effectively restricts viral entry, dissemination in CNS, viral replication, viral transcription, and expression of the viral nucleocapsid and inflammatory cytokines. From mechanistic standpoints, RSA59 preincubated with NBE reduced viral entry, viral replication and cell-to-cell fusion, as a mode of viral dissemination. Moreover, intraperitoneal injection with NBE (25 mg/kg B.W.) into mice revealed a significant reduction in viral Nucleocapsid protein expression in vivo. Conclusively, A. indica bark extract may directly bind to the virus-host attachment Spike glycoprotein and suppresses MHV-induced neuroinflammation and neuropathogenesis by inhibiting cell-to-cell fusion and viral replication. Further studies will focus on combining bioanalytical assays to isolate potential NBE bioactive compound(s) that contribute towards the anti-viral activity of NBE.