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


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.

Demyelinating Diseases , Spike Glycoprotein, Coronavirus , Animals , Mice , Peptides/metabolism , Proline , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Envelope Proteins/metabolism
Virology ; 569: 13-28, 2022 04.
Article in English | MEDLINE | ID: covidwho-1740261


Emerging mutations in the SARS-CoV-2 genome pose a challenge for vaccine development and antiviral therapy. The antiviral efficacy of Azadirachta indica bark extract (NBE) was assessed against SARS-CoV-2 and m-CoV-RSA59 infection. Effects of in vivo intranasal or oral NBE administration on viral load, inflammatory response, and histopathological changes were assessed in m-CoV-RSA59-infection. NBE administered inhibits SARS-CoV-2 and m-CoV-RSA59 infection and replication in vitro, reducing Envelope and Nucleocapsid gene expression. NBE ameliorates neuroinflammation and hepatitis in vivo by restricting viral replication and spread. Isolated fractions of NBE enriched in Nimbin isomers shows potent inhibition of m-CoV-RSA59 infection in vitro. In silico studies revealed that NBE could target Spike and RdRp of m-CoV and SARS-CoV-2 with high affinity. NBE has a triterpenoids origin that may allow them to competitively target panoply of viral proteins to inhibit mouse and different strains of human coronavirus infections, suggesting its potential as an antiviral against pan-ß-Coronaviruses.

Azadirachta , COVID-19 , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , Limonins , Mice , Plant Bark , Plant Extracts/pharmacology , SARS-CoV-2 , Virus Replication
Front Cell Neurosci ; 14: 116, 2020.
Article in English | MEDLINE | ID: covidwho-831021


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.