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1.
Viruses ; 14(4)2022 04 17.
Article in English | MEDLINE | ID: covidwho-1792411

ABSTRACT

Combined in silico, in vitro, and in vivo comparative studies between isogenic-recombinant Mouse-Hepatitis-Virus-RSA59 and its proline deletion mutant, revealed a remarkable contribution of centrally located two consecutive prolines (PP) from Spike protein fusion peptide (FP) in enhancing virus fusogenic and hepato-neuropathogenic potential. To deepen our understanding of the underlying factors, we extend our studies to a non-fusogenic parental virus strain RSMHV2 (P) with a single proline in the FP and its proline inserted mutant, RSMHV2 (PP). Comparative in vitro and in vivo studies between virus strains RSA59(PP), RSMHV2 (P), and RSMHV2 (PP) in the FP demonstrate that the insertion of one proline significantly resulted in enhancing the virus fusogenicity, spread, and consecutive neuropathogenesis. Computational studies suggest that the central PP in Spike FP induces a locally ordered, compact, and rigid structure of the Spike protein in RSMHV2 (PP) compared to RSMHV2 (P), but globally the Spike S2-domain is akin to the parental strain RSA59(PP), the latter being the most flexible showing two potential wells in the energy landscape as observed from the molecular dynamics studies. The critical location of two central prolines of the FP is essential for fusogenicity and pathogenesis making it a potential site for designing antiviral.


Subject(s)
Demyelinating Diseases , Spike Glycoprotein, Coronavirus , Animals , Mice , Peptides/metabolism , Proline , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Envelope Proteins/metabolism
2.
Front Cell Infect Microbiol ; 11: 729622, 2021.
Article in English | MEDLINE | ID: covidwho-1405404

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced COVID-19 has emerged as a defining global health crisis in current times. Data from the World Health Organization shows demographic variations in COVID-19 severity and lethality. Diet may play a significant role in providing beneficial host cell factors contributing to immunity against deadly SARS-CoV-2 pathogenesis. Spices are essential components of the diet that possess anti-inflammatory, antioxidant, and antiviral properties. Hyperinflammation, an aberrant systemic inflammation associated with pneumonia, acute respiratory failure, and multiorgan dysfunction, is a major clinical outcome in COVID-19. Knowing the beneficial properties of spices, we hypothesize that spice-derived bioactive components can modulate host immune responses to provide protective immunity in COVID-19. This study emphasizes that biologically active components of spices might alleviate the sustained pro-inflammatory condition by inhibiting the activity of tumor necrosis factor-alpha (TNF-α), interleukins (IL6, IL8), and chemokine (CCL2) known to be elevated in COVID-19. Spices may potentially prevent the tissue damage induced by oxidative stress and pro-inflammatory mediators during SARS-CoV-2 infection. The current study also highlights the effects of spices on the antioxidant pathways mediated by Nrf2 (nuclear factor erythroid 2-related factor 2) and Hmox1 (heme oxygenase 1) to restore oxidative homeostasis and protect from aberrant tissue damage. Taken together, the anti-inflammatory and antioxidant activities of bioactive components of spices may hold a promise to target the cellular pathways for developing antivirals against SARS-CoV-2 and pan ß-coronaviruses.


Subject(s)
COVID-19 , SARS-CoV-2 , Anti-Inflammatory Agents , Antiviral Agents , Humans , Immunity
3.
J Neurovirol ; 27(2): 197-216, 2021 04.
Article in English | MEDLINE | ID: covidwho-1080993

ABSTRACT

The pandemic caused by SARS-CoV-2 has caused widespread infection and significant mortality across the globe. Combined virology perspective of SARS-CoV-2 with a deep-rooted understanding of pathophysiological and immunological processes underlying the clinical manifestations of COVID-19 is of prime importance. The characteristic symptom of COVID-19 is respiratory distress with diffused alveolar damage, but emerging evidence suggests COVID-19 might also have neurologic consequences. Dysregulated homeostasis in the lungs has proven to be fatal, but one cannot ignore that the inability to breathe might be due to defects in the respiratory control center of the brainstem. While the mechanism of pulmonary distress has been documented in the literature, awareness of neurological features and their pathophysiology is still in the nascent state. This review makes references to the neuro-immune axis and neuro-invasive potential of SARS-CoV and SARS-CoV2, as well as the prototypic H-CoV strains in human brains. Simultaneously, considerable discussion on relevant experimental evidence of mild to severe neurological manifestations of fellow neurotropic murine-ß-CoVs (m-CoVs) in the mouse model will help understand the underpinning mechanisms of Neuro-COVID. In this review, we have highlighted the neuroimmunopathological processes in murine CoVs. While MHV infection in mice and SARS-CoV-2 infection in humans share numerous parallels, there are critical differences in viral recognition and viral entry. These similarities are highlighted in this review, while differences have also been emphasized. Though CoV-2 Spike does not favorably interact with murine ACE2 receptor, modification of murine SARS-CoV2 binding domain or development of transgenic ACE-2 knock-in mice might help in mediating consequential infection and understanding human CoV2 pathogenesis in murine models. While a global animal model that can replicate all aspects of the human disease remains elusive, prior insights and further experiments with fellow m-ß-CoV-induced cause-effect experimental models and current human COVID-19 patients data may help to mitigate the SARS-CoV-2-induced multifactorial multi-organ failure.


Subject(s)
COVID-19/pathology , Disease Models, Animal , Murine hepatitis virus/pathogenicity , Neuroimmunomodulation/physiology , Animals , COVID-19/immunology , Coronavirus Infections/immunology , Coronavirus Infections/pathology , Humans , Mice , Murine hepatitis virus/immunology , SARS-CoV-2
4.
PLoS Pathog ; 16(11): e1009034, 2020 11.
Article in English | MEDLINE | ID: covidwho-950851

ABSTRACT

The interferon-induced tetratricopeptide repeat protein (Ifit2) protects mice from lethal neurotropic viruses. Neurotropic coronavirus MHV-RSA59 infection of Ifit2-/- mice caused pronounced morbidity and mortality accompanied by rampant virus replication and spread throughout the brain. In spite of the higher virus load, induction of many cytokines and chemokines in the brains of infected Ifit2-/- mice were similar to that in wild-type mice. In contrast, infected Ifit2-/- mice revealed significantly impaired microglial activation as well as reduced recruitment of NK1.1 T cells and CD4 T cells to the brain, possibly contributing to the lack of viral clearance. These two deficiencies were associated with a lower level of microglial expression of CX3CR1, the receptor of the CX3CL1 (Fractalkine) chemokine, which plays a critical role in both microglial activation and leukocyte recruitment. The above results uncovered a new potential role of an interferon-induced protein in immune protection.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Cell Movement/immunology , Coronavirus Infections/virology , Leukocytes/virology , Murine hepatitis virus/pathogenicity , RNA-Binding Proteins/metabolism , Virus Replication/immunology , Animals , Apoptosis Regulatory Proteins/deficiency , Coronavirus Infections/immunology , Cytokines/metabolism , Interferons/metabolism , Leukocytes/cytology , Leukocytes/metabolism , Mice, Inbred C57BL , Microglia/metabolism , Murine hepatitis virus/metabolism
5.
Front Cell Neurosci ; 14: 116, 2020.
Article in English | MEDLINE | ID: covidwho-831021

ABSTRACT

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.

6.
J Biol Chem ; 295(20): 6926-6935, 2020 05 15.
Article in English | MEDLINE | ID: covidwho-830746

ABSTRACT

Mouse hepatitis virus (MHV; murine coronavirus) causes meningoencephalitis, myelitis, and optic neuritis followed by axonal loss and demyelination. This murine virus is used as a common model to study acute and chronic virus-induced demyelination in the central nervous system. Studies with recombinant MHV strains that differ in the gene encoding the spike protein have demonstrated that the spike has a role in MHV pathogenesis and retrograde axonal transport. Fusion peptides (FPs) in the spike protein play a key role in MHV pathogenesis. In a previous study of the effect of deleting a single proline residue in the FP of a demyelinating MHV strain, we found that two central, consecutive prolines are important for cell-cell fusion and pathogenesis. The dihedral fluctuation of the FP was shown to be repressed whenever two consecutive prolines were present, in contrast to the presence of a single proline in the chain. Using this proline-deleted MHV strain, here we investigated whether intracranial injection of this strain can induce optic neuritis by retrograde axonal transport from the brain to the retina through the optic nerve. We observed that the proline-deleted recombinant MHV strain is restricted to the optic nerve, is unable to translocate to the retina, and causes only minimal demyelination and no neuronal death. We conclude that an intact proline dyad in the FP of the recombinant demyelinating MHV strain plays a crucial role in translocation of the virus through axons and subsequent neurodegeneration.


Subject(s)
Axonal Transport/genetics , Murine hepatitis virus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Animals , Axonal Transport/physiology , Axons/metabolism , Axons/virology , Brain/metabolism , Coronavirus Infections/pathology , Demyelinating Diseases/metabolism , Male , Mice , Mice, Inbred C57BL , Murine hepatitis virus/metabolism , Optic Nerve/metabolism , Optic Nerve/virology , Peptides/metabolism , Proline/metabolism , Sequence Deletion/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Envelope Proteins/metabolism
7.
J Virol ; 94(14)2020 07 01.
Article in English | MEDLINE | ID: covidwho-823496

ABSTRACT

Mouse hepatitis virus (MHV) is a murine betacoronavirus (m-CoV) that causes a wide range of diseases in mice and rats, including hepatitis, enteritis, respiratory diseases, and encephalomyelitis in the central nervous system (CNS). MHV infection in mice provides an efficient cause-effect experimental model to understand the mechanisms of direct virus-induced neural-cell damage leading to demyelination and axonal loss, which are pathological features of multiple sclerosis (MS), the most common disabling neurological disease in young adults. Infiltration of T lymphocytes, activation of microglia, and their interplay are the primary pathophysiological events leading to disruption of the myelin sheath in MS. However, there is emerging evidence supporting gray matter involvement and degeneration in MS. The investigation of T cell function in the pathogenesis of deep gray matter damage is necessary. Here, we employed RSA59 (an isogenic recombinant strain of MHV-A59)-induced experimental neuroinflammation model to compare the disease in CD4-/- mice with that in CD4+/+ mice at days 5, 10, 15, and 30 postinfection (p.i.). Viral titer estimation, nucleocapsid gene amplification, and viral antinucleocapsid staining confirmed enhanced replication of the virions in the absence of functional CD4+ T cells in the brain. Histopathological analyses showed elevated susceptibility of CD4-/- mice to axonal degeneration in the CNS, with augmented progression of acute poliomyelitis and dorsal root ganglionic inflammation rarely observed in CD4+/+ mice. Depletion of CD4+ T cells showed unique pathological bulbar vacuolation in the brain parenchyma of infected mice with persistent CD11b+ microglia/macrophages in the inflamed regions on day 30 p.i. In summary, the current study suggests that CD4+ T cells are critical for controlling acute-stage poliomyelitis (gray matter inflammation), chronic axonal degeneration, and inflammatory demyelination due to loss of protective antiviral host immunity.IMPORTANCE The current trend in CNS disease biology is to attempt to understand the neural-cell-immune interaction to investigate the underlying mechanism of neuroinflammation, rather than focusing on peripheral immune activation. Most studies in MS are targeted toward understanding the involvement of CNS white matter. However, the importance of gray matter damage has become critical in understanding the long-term progressive neurological disorder. Our study highlights the importance of CD4+ T cells in safeguarding neurons against axonal blebbing and poliomyelitis from murine betacoronavirus-induced neuroinflammation. Current knowledge of the mechanisms that lead to gray matter damage in MS is limited, because the most widely used animal model, experimental autoimmune encephalomyelitis (EAE), does not present this aspect of the disease. Our results, therefore, add to the existing limited knowledge in the field. We also show that the microglia, though important for the initiation of neuroinflammation, cannot establish a protective host immune response without the help of CD4+ T cells.


Subject(s)
Axons/immunology , Axons/metabolism , CD4 Antigens/deficiency , Coronavirus Infections/immunology , Coronavirus Infections/virology , Murine hepatitis virus/physiology , Poliomyelitis/etiology , Animals , Axons/pathology , Brain/immunology , Brain/metabolism , Brain/pathology , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Coronavirus Infections/pathology , Cytokines/metabolism , Disease Models, Animal , Disease Susceptibility/immunology , Ganglia, Spinal/immunology , Ganglia, Spinal/metabolism , Ganglia, Spinal/pathology , Immunohistochemistry , Inflammation Mediators/metabolism , Mice
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