Dietary Vitamin D Mitigates Coronavirus-Induced Lung Inflammation and Damage in Mice.

The COVID-19 pandemic caused by the SARS-CoV-2 (ß-CoV) betacoronavirus has posed a significant threat to global health. Despite the availability of vaccines, the virus continues to spread, and there is a need for alternative strategies to alleviate its impact. Vitamin D, a secosteroid hormone best known for its role in bone health, exhibits immunomodulatory effects in certain viral infections. Here, we have shown that bioactive vitamin D (calcitriol) limits in vitro replication of SARS-CoV-2 and murine coronaviruses MHV-3 and MHV-A59. Comparative studies involving wild-type mice intranasally infected with MHV-3, a model for studying ß-CoV respiratory infections, confirmed the protective effect of vitamin D in vivo. Accordingly, mice fed a standard diet rapidly succumbed to MHV-3 infection, whereas those on a vitamin D-rich diet (10,000 IU of Vitamin D3/kg) displayed increased resistance to acute respiratory damage and systemic complications. Consistent with these findings, the vitamin D-supplemented group exhibited lower viral titers in their lungs and reduced levels of TNF, IL-6, IL-1ß, and IFN-γ, alongside an enhanced type I interferon response. Altogether, our findings suggest vitamin D supplementation ameliorates ß-CoV-triggered respiratory illness and systemic complications in mice, likely via modulation of the host's immune response to the virus.

Medicinal herbal extracts of Sophorae radix, Acanthopanacis cortex, Sanguisorbae radix and Torilis fructus inhibit coronavirus replication in vitro.

BACKGROUND: Cimicifuga rhizome, Meliae cortex, Coptidis rhizome and Phellodendron cortex have been previously shown to exhibit anti-coronavirus activity. Here, an additional 19 traditional medicinal herbal extracts were evaluated for antiviral activities in vitro. METHODS: A plaque assay was used to evaluate the effects of 19 extracts, and the concentration of extract required to inhibit 50% of the replication (EC(50)) of mouse hepatitis virus (MHV) A59 strain (MHV-A59) was determined. The 50% cytotoxic concentration (CC(50)) of each extract was also determined. Northern and western blot analyses were conducted to evaluate antiviral activity on viral entry, viral RNA and protein expression, and release in MHV-infected DBT cells. RESULTS: Sophorae radix, Acanthopanacis cortex and Torilis fructus reduced intracellular viral RNA levels with comparable reductions in viral proteins and MHV-A59 production. The extracts also reduced the replication of the John Howard Mueller strain of MHV, porcine epidemic diarrhoea virus and vesicular stomatitis virus in vitro. Sanguisorbae radix reduced coronavirus production, partly as a result of decreased protein synthesis, but without a significant reduction in intracellular viral RNA levels. The EC(50) values of the four extracts ranged from 0.8 to 3.7 microg/ml, whereas the CC(50) values ranged from 156.5 to 556.8 microg/ml. Acanthopanacis cortex and Torilis fructus might exert their antiviral activities in MHV-A59-infected cells by inducing cyclooxygenase-2 expression via the activation of extracellular signal-related kinase (ERK) and p38 or ERK alone, respectively. CONCLUSIONS: Sophorae radix, Acanthopanacis cortex, Sanguisorbae radix and Torilis fructus might be considered as promising novel anti-coronavirus drug candidates.

In vitro inhibition of coronavirus replications by the traditionally used medicinal herbal extracts, Cimicifuga rhizoma, Meliae cortex, Coptidis rhizoma, and Phellodendron cortex.

BACKGROUND: A search for new anti-coronaviral drugs to treat coronaviral infections was motivated by an outbreak of severe acute respiratory syndrome (SARS). OBJECTIVES: In order to find drugs that treat coronavirus infections, including SARS, we screened traditional medicinal herbal extracts and evaluated their antiviral activities on coronavirus replication. STUDY DESIGN: We employed a plaque assay to evaluate the effect of 22 medicinal herbal extracts on virus replication. We determined the 50% effective concentration (EC50) of each extract that was necessary to inhibit the replication of mouse hepatitis virus A59 (MHV-A59); we also determined 50% cytotoxic concentrations (CC50) for each extract. Northern and Western blot analyzes were performed to investigate antiviral activity in MHV-infected DBT cells, including virus entry, viral RNA and protein expression, and virus release. Coronavirus specific inhibition was also demonstrated using porcine epidemic diarrhea virus (PEDV). RESULTS: Cimicifuga rhizoma, Meliae cortex, Coptidis rhizoma, Phellodendron cortex and Sophora subprostrata radix decreased the MHV production and the intracellular viral RNA and protein expression with EC50 values ranging from 2.0 to 27.5 microg/ml. These extracts also significantly decreased PEDV production and less dramatically decreased vesicular stomatitis virus (VSV) production in vitro. CONCLUSIONS: The extracts selected strongly inhibited MHV replication and could be potential candidates for new anti-coronavirus drugs.

Mitochondrial aconitase binds to the 3' untranslated region of the mouse hepatitis virus genome.

Mouse hepatitis virus (MHV), a member of the Coronaviridae, contains a polyadenylated positive-sense single-stranded genomic RNA which is 31 kb long. MHV replication and transcription take place via the synthesis of negative-strand RNA intermediates from a positive-strand genomic template. A cis-acting element previously identified in the 3' untranslated region binds to trans-acting host factors from mouse fibroblasts and forms at least three RNA-protein complexes. The largest RNA-protein complex formed by the cis-acting element and the lysate from uninfected mouse fibroblasts has a molecular weight of about 200 kDa. The complex observed in gel shift assays has been resolved by second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis into four proteins of approximately 90, 70, 58, and 40 kDa after RNase treatment. Specific RNA affinity chromatography also has revealed the presence of a 90-kDa protein associated with RNA containing the cis-acting element bound to magnetic beads. The 90-kDa protein has been purified from uninfected mouse fibroblast crude lysates. Protein microsequencing identified the 90-kDa protein as mitochondrial aconitase. Antibody raised against purified mitochondrial aconitase recognizes the RNA-protein complex and the 90-kDa protein, which can be released from the complex by RNase digestion. Furthermore, UV cross-linking studies indicate that highly purified mitochondrial aconitase binds specifically to the MHV 3' protein-binding element. Increasing the intracellular level of mitochondrial aconitase by iron supplementation resulted in increased RNA-binding activity in cell extracts and increased virus production as well as viral protein synthesis at early hours of infection. These results are particularly interesting in terms of identification of an RNA target for mitochondrial aconitase, which has a cytoplasmic homolog, cytoplasmic aconitase, also known as iron regulatory protein 1, a well-recognized RNA-binding protein. The binding properties of mitochondrial aconitase and the functional relevance of RNA binding appear to parallel those of cytoplasmic aconitase.

Control of virus-induced cell fusion by host cell lipid composition.

Virus-induced cell fusion has been examined in a series of stable cell lines which were originally selected for resistance to the fusogenic effects of polyethylene glycol (PEG). For a wide variety of viruses, including murine hepatitis virus (a coronavirus), vesicular stomatitis virus (a rhabdovirus), and two paramyxoviruses (Sendai virus and SV5), susceptibility to virus-induced fusion was found to be inversely correlated with susceptibility to PEG-induced fusion. This phenomenon was observed both for cell fusion occurring in the course of viral infection and for fusion induced "from without" by the addition of high titers of noninfectious or inactivated virus. The fusion-altered cell lines (fusible by virus but not by PEG) are characterized by their unusual lipid composition, including marked elevation of saturated fatty acids and the presence of an unusual ether-linked neutral lipid. To test the association between lipid composition and fusion, acyl chain saturation was manipulated by supplementing the culture medium with exogenous fatty acids. In such experiments, it was possible to control the responses of these cells to both viral and chemical fusogens. Increasing the cellular content of saturated fatty acyl chains increased the susceptibility of cells to viral fusion and decreased susceptibility to PEG-induced fusion, whereas lowering fatty acid saturation had the opposite effect. Thus, parallel cultures of cells can be either driven toward the PEG-fusible/virus-fusion-resistant phenotype of the parental cells or rendered susceptible to viral fusion but resistant to PEG-induced fusion, solely by the alteration of cellular lipids. The ability of cellular lipid composition to regulate virus-induced membrane fusion suggests a possible role for lipids in viral infection and pathogenesis.

Cholesterol enhances mouse hepatitis virus-mediated cell fusion.

Mouse hepatitis virus (MHV) infection of the L-2 subline of mouse fibroblasts results in acute infection characterized by extensive cell fusion. In contrast, infection of the LM-K subline leads to virus persistence with reduced cell fusion. We undertook studies designed to elucidate the role of host cell membrane lipid composition and the cytoskeleton in modulating the fusion process and the resultant effect(s) on virus persistence. MHV-induced cell fusion proceeded normally in cells treated with cytoskeleton-disrupting drugs, cytochalasin B and colchicine. Modification of cell membrane fatty acid composition by supplementation of LM-K cells with arachidonic (C-20:4) or palmitic (C-16:0) acids had little effect on the extent of MHV-induced cell fusion or on virus replication. However, supplementation of both cell types with cholesterol (resulting in increased membrane cholesterol/fatty acid ratio) resulted in marked enhancement of virus-mediated cell fusion. The increase in cell membrane cholesterol did not enhance internalization of MHV suggesting that cholesterol primarily modulates a later event. This suggestion was confirmed by demonstrating cholesterol-enhancement of fusion in a contact fusion assay. Cholesterol-supplemented L-2 cells were less productive for virus replication than unsupplemented cells, in agreement with our previous observations that MHV replication is compromised by extensive cytopathic effect. Although cholesterol-supplemented LM-K cells showed increased susceptibility to MHV-mediated cell fusion, the extent of such susceptibility did not approach that observed in L-2 cells. Also, the property of LM-K cells to support MHV persistence was not abolished by cholesterol supplementation. Thus membrane fusion resistance and MHV persistence are modulated but not alleviated by cell membrane cholesterol content.

Characterization of small plaque mutants of mouse hepatitis virus, JHM strain.

Two small plaque mutants designated as 1a and 2c were isolated from DBT cells persistently infected with the JHM strain of mouse hepatitis virus. Unlike the wild type JHM, these two mutant viruses grew more slowly with no prominent cell fusion. The buoyant densities of the mutants were slightly lower and 2c was revealed to have fewer peplomers than JHM by electron microscopy. The purified JHM contained five polypeptides with molecular weights (M.W.) of 260,000, 105,000 (GP105), 65,000, 60,000 (P60), and 23,000 (GP23). In addition to two polypeptides, P60 and GP23, which were common to JHM and the mutants, 1a was found to contain three other specific polypeptides with M.W. of 180,000 (GP180), 110,000, and 95,000 (GP95), while 2c had GP180, GP105, GP95, and one with a M.W. of 175,000. All of these polypeptides were shown to be glycosylated except for P60. After bromelain treatment, all these viruses lost the peplomers and contained P60 and another new 18,000 dalton polypeptide.