Acacia mangium: A promising plant for isolating anti-hepatitis C virus agents.

Background: Medicinal plants are potential resources for isolating drug candidates. Various plants have been reported to possess pharmacological effects including anti-hepatitis C activities. The current study examined the anti-hepatitis C virus (HCV) activities of Acacia mangium extracts in solvents with various polarities and further evaluated the mechanism of action of the extracts using Western blotting and combination treatment models. Methods: The leaves of A. mangium were extracted in two phases, first in ethanol and then in solvents with different polarities (n-hexane, dichloromethane, and methanol). HCV-infected Huh7it-1 cells were treated with the extracts at concentrations of 0.01, 0.1, 1, 10, 50, and 100 µg/mL. Results: The results revealed the strong anti-HCV activities of the extracts. The 50% inhibition concentrations (IC 50s) of the ethanol, n-hexane, dichloromethane and methanol extracts were of 4.6 ± 0.3, 2.9 ± 0.2, 0.2 ± 0.3, and 2.8 ± 0.2 µg/mL, respectively, and no cytotoxic effect was detected. These extracts displayed stronger effects than the positive control ribavirin. The mode of action of the ethanol extract was evaluated at 30 µg/mL, revealing that the inhibitory effect was stronger on the post-entry step than on the entry step. Western blotting revealed that the extracts decreased NS3 protein expression, indicating that virus replication was suppressed. Further evaluation illustrated that combined treatment with the ethanol extract enhanced the anti-viral activity of simeprevir. Conclusions: These results indicated that A. mangium leaves could represent sources of anti-HCV agents.

Identification of natural compounds extracted from crude drugs as novel inhibitors of hepatitis C virus.

Natural product-derived crude drugs are expected to yield an abundance of new drugs to treat infectious diseases. Hepatitis C virus (HCV) is an oncogenic virus that significantly impacts public health. In this study, we sought to identify anti-HCV compounds in extracts of natural products. A total of 110 natural compounds extracted from several herbal medicine plants were examined for antiviral activity against HCV. Using a Huh7-mCherry-NLS-IPS reporter system for HCV infection, we first performed a rapid screening for anti-HCV compounds extracted from crude drugs. The compounds threo-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-butoxypropan-1-ol (#106) and medioresinol (#110), which were extracted from Crataegus cuneate, exhibited anti-HCV activity and significantly inhibited HCV production in a dose-dependent manner. Analyses using HCV pseudoparticle and subgenomic replicon systems indicated that compounds #106 and #110 specifically inhibit HCV RNA replication but not viral entry or translation. Interestingly, compound #106 also inhibited the replication and production of hepatitis A virus. Our findings suggest that C. cuneate is a new source for novel anti-hepatitis virus drug development.

Bioactive Natural Antivirals: An Updated Review of the Available Plants and Isolated Molecules.

Viral infections and associated diseases are responsible for a substantial number of mortality and public health problems around the world. Each year, infectious diseases kill 3.5 million people worldwide. The current pandemic caused by COVID-19 has become the greatest health hazard to people in their lifetime. There are many antiviral drugs and vaccines available against viruses, but they have many disadvantages, too. There are numerous side effects for conventional drugs, and active mutation also creates drug resistance against various viruses. This has led scientists to search herbs as a source for the discovery of more efficient new antivirals. According to the World Health Organization (WHO), 65% of the world population is in the practice of using plants and herbs as part of treatment modality. Additionally, plants have an advantage in drug discovery based on their long-term use by humans, and a reduced toxicity and abundance of bioactive compounds can be expected as a result. In this review, we have highlighted the important viruses, their drug targets, and their replication cycle. We provide in-depth and insightful information about the most favorable plant extracts and their derived phytochemicals against viral targets. Our major conclusion is that plant extracts and their isolated pure compounds are essential sources for the current viral infections and useful for future challenges.

Deguelin inhibits HCV replication through suppressing cellular autophagy via down regulation of Beclin1 expression in human hepatoma cells.

AIMS: Deguelin, a natural compound derived from Mundulea sericea (Leguminosae) and some other plants exhibits an activity to inhibit autophagy, a cellular machinery required for hepatitis C virus (HCV) replication. This study aimed to illuminate the impact of deguelin on HCV replication and mechanism(s) involved. METHODS: HCV JFH-1-Huh7 infectious system was used for the investigation. Real time RT-PCR, Western blot, fluorescent microscopy assay were used to measure the expression levels of viral or cellular factors. Overexpression and silencing expression techniques were used to determine the role of key cellular factors. RESULTS: Deguelin treatment of Huh7 cells significantly inhibited HCV JFH-1 replication in a dose- and time-dependent manner. Deguelin treatment suppressed autophagy in Huh7 cells, evidenced by the decrease of LC3B-II levels, the conversion of LC3B-I to LC3B-II, and the formation of GFP-LC3 puncta as well as the increase of p62 level in deguelin-treated cells compared with control cells. HCV infection could induce autophagy which was also suppressed by deguelin treatment. Mechanism research reveals that deguelin inhibited expression of Beclin1, which is a key cellular factor for the initiation of the autophagosome formation in autophagy. Overexpression or silencing expression of Beclin1 in deguelin-treated Huh7 cells could weaken or enhance the inhibitory effect on autophagy by deguelin, respectively, and thus partially recover or further inhibit HCV replication correspondingly. CONCLUSIONS: Deguelin may serve as a novel anti-HCV compound via its inhibitory effect on autophagy, which warrants further investigation as a potential therapeutic agent for HCV infection.

Outcomes of Direct-Acting Antiviral Treatment of Psychiatric Patients with Comorbid Hepatitis C Virus Infection.

BACKGROUND: The highest burden of hepatitis C virus (HCV) infection is seen in patients with psychiatric disorders who have been excluded from traditional treatments with Interferon due to treatment-emergent neuropsychiatric adverse effects. The goal of this study is to determine the tolerability, treatment retention, and efficacy of direct-acting antivirals with psychiatric disorders and comorbid substance use disorders in real-life settings. METHODS: This is a retrospective cohort observational study of HCV patients treated with direct-acting antivirals between January 2016 and December 2018. Patients were stratified and sub-stratified based on their psychiatric diagnosis and substance use. The primary assessment was the sustained virologic response at 12 weeks post-treatment (SVR12). RESULTS: Among the 291 patients analyzed, patients with psychiatric diagnosis and non-psychiatric patients made up 51.2% (n = 149) and 48.8% (n = 142) respectively. Majority of the patients included in the study were African-Americans (68.7%, n = 200). Overall, 95.3% (142/149) and 94.4% (134/142) of psychiatric and non-psychiatric patients, respectively, achieved SVR12 and treatment response was similar between the groups (p = 0.72). Among psychiatric patients, only the prior treatment status was identified as a predictor of treatment response (OR 0.153, 95% CI 0.03-0.79; p = 0.05). No statistical difference was observed among the patients with SVR12 based on their primary psychiatric diagnoses or by comorbid substance abuse. CONCLUSION: The results of our study show that direct-acting antiviral treatments are well tolerated in psychiatric patients, and an overwhelming majority of patients achieved SVR12. Our study highlights the need to integrate HCV screening with treatment linkage in psychiatry and primary care practice.

The role of iron in viral infections.

Crucial cellular processes such as DNA synthesis and the generation of ATP require iron. Viruses depend on iron in order to efficiently replicate within living host cells. Some viruses selectively infect iron - acquiring cells or influence the cellular iron metabolism via Human hemochromatosis protein (HFE) or hepcidin. During infection with human immunodeficiency virus (HIV), hepatitis B virus (HBV) or hepatitis C virus (HCV) iron overload is associated with poor prognosis for the patient and enhanced progression of the disease. Recent findings still lack to fully describe the viral interaction with the host iron metabolism during infection. This review summarizes the current knowledge of the viral regulation on the host cell iron metabolism in order to discuss the therapeutic option of iron chelation as a potential and beneficial adjuvant in antiviral therapy.

Water-soluble extracts from edible mushrooms (Agaricus bisporus) as inhibitors of hepatitis C viral replication.

Hepatitis C virus (HCV) is the main agent responsible for chronic liver disease. Recent advances in anti-HCV treatment strategies have significantly increased the viral clearance rate (>90%). However, sustained antiviral responses vary in different cohorts, and high costs limit the broad use of direct-acting antivirals (DAAs). The goal of this study is to evaluate the inhibitory ability of well characterized (LC-QTOF-MS/MS) aqueous extracts obtained from edible mushrooms (Agaricus bisporus) to diminish HCV viral replication. Our data have demonstrated an in vitro inhibitory effect of A. bisporus extracts on NS3/4A protease and HCV replication. Fractionation by ultra-filtration and sequential liquid-liquid extraction showed that the compounds responsible for the inhibition are water-soluble with low molecular weights (<3 kDa) and that action could be through the following five compounds: ergothioneine, adenine, guanine, hypoxanthine, and xanthine, which are present in all fractions (UF-3, AqF-3 kDa and organic fractions) showing NS3/4A inhibition. Low molecular weight aqueous extracts (<3 kDa) from A. bisporus have potential applications in the prophylaxis and treatment of HCV, especially for patients who do not have access to the last generation of DAAs. They may be useful as well for other flaviviruses, which also possess a NS3 serine protease.

Development of a Method That Delivers Drugs to Enveloped Viruses.

Hepatitis C virus (HCV) infection leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in 50-80% of the cases. Interferons (IFNs) and the nucleoside analog ribavirin form the basis of the treatment of this infection but are not considered sufficiently effective and cause several side effects. In this study, we developed a novel viral-specific drug delivery method. Enveloped viruses, including HCV, expose an anionic phospholipid, phosphatidylserine (PS), on their surface to mediate their binding and entry into cells for infection. To target such exposed PS on HCV, we developed a chimeric recombinant protein containing human IFN and mouse lactadherin (also known as milk fat globule epidermal growth factor 8), which binds with high affinity to PS. The IFN-lactadherin fusion protein showed a high binding affinity toward PS and HCV and consequently blocked viral replication in the infected cells more efficiently than conventional IFN. Overall, these data suggest that conjugation with lactadherin facilitates the delivery of any protein drug to PS-exposing enveloped viruses.

Inactivating hepatitis C virus in donor lungs using light therapies during normothermic ex vivo lung perfusion.

Availability of organs is a limiting factor for lung transplantation, leading to substantial mortality rates on the wait list. Use of organs from donors with transmissible viral infections, such as hepatitis C virus (HCV), would increase organ donation, but these organs are generally not offered for transplantation due to a high risk of transmission. Here, we develop a method for treatment of HCV-infected human donor lungs that prevents HCV transmission. Physical viral clearance in combination with germicidal light-based therapies during normothermic ex-vivo Lung Perfusion (EVLP), a method for assessment and treatment of injured donor lungs, inactivates HCV virus in a short period of time. Such treatment is shown to be safe using a large animal EVLP-to-lung transplantation model. This strategy of treating viral infection in a donor organ during preservation could significantly increase the availability of organs for transplantation and encourages further clinical development.

Berberine inhibits hepatitis C virus entry by targeting the viral E2 glycoprotein.

BACKGROUND: Despite the advent of direct-acting antivirals (DAAs), HCV remains an important public health problem globally. There is at present no effective vaccine against the virus, and the DAAs in current use cannot prevent de novo infection, including in liver transplant setting wherein donor livers inevitably become re-infected. Developing inhibitors to HCV entry using nature-derived small molecules may help to expand/complement the current treatment options. PURPOSE: In this study, we explored the effect of the plant alkaloid berberine (BBR) on HCV early viral entry. METHODS: Cell culture-derived HCV (HCVcc), viral pseudoparticles bearing HCV glycoproteins (HCVpp), and entry-related assays were employed to assess BBR's bioactivity. Molecular docking was used to predict BBR-HCV glycoproteins interaction, and the compound's antiviral activity was confirmed against HCVcc infection of primary human hepatocytes (PHHs). RESULTS: BBR specifically impeded HCVcc attachment and entry/fusion steps without inactivating the free virus particles or affecting the expression of host cell entry factors and post-entry viral replication. BBR also effectively inhibited infection by viral pseudoparticles expressing HCV E1/E2 glycoproteins and molecular docking analysis pointed at potential interaction with HCV E2. Finally, BBR could suppress HCVcc infection of PHHs. CONCLUSIONS: We identified BBR as a potent HCV entry inhibitor, which merits further evaluation particularly for use in transplant setting against graft re-infection by HCV.

Methanolic Extract of Rhizoma Coptidis Inhibits the Early Viral Entry Steps of Hepatitis C Virus Infection.

Hepatitis C Virus (HCV) remains an important public health threat with approximately 170 million carriers worldwide who are at risk of developing hepatitis C-associated end-stage liver diseases. Despite improvement of HCV treatment using the novel direct-acting antivirals (DAAs) targeting viral replication, there is a lack of prophylactic measures for protection against HCV infection. Identifying novel antivirals such as those that target viral entry could help broaden the therapeutic arsenal against HCV. Herein, we investigated the anti-HCV activity of the methanolic extract from Rhizoma coptidis (RC), a widely used traditional Chinese medicine documented by the WHO and experimentally reported to possess several pharmacological functions including antiviral effects. Using the cell culture-derived HCV system, we demonstrated that RC dose-dependently inhibited HCV infection of Huh-7.5 cells at non-cytotoxic concentrations. In particular, RC blocked HCV attachment and entry/fusion into the host cells without exerting any significant effect on the cell-free viral particles or modulating key host cell entry factors to HCV. Moreover, RC robustly suppressed HCV pseudoparticles infection of Huh-7.5 cells and impeded infection by several HCV genotypes. Collectively, our results identified RC as a potent antagonist to HCV entry with potential pan-genotypic properties, which deserves further evaluation for use as an anti-HCV agent.

Hypericin inhibits hepatitis C virus replication via deacetylation and down-regulation of heme oxygenase-1.

BACKGROUND: Hepatitis C virus (HCV) is a globally prevalent pathogen and a leading cause of death and morbidity. Traditional therapy with pegylated interferon- and ribavirin has had only limited success, with some adverse effects. Direct-acting antivirals (DAAs) are effective in suppressing HCV replication, but are expensive. PURPOSE: Hypericin has been reported to be a good antiviral agent for inhibiting HCV replication, however, little is known about its mechanisms of action. The aim of this study is to elucidate the mode of action of hypericin in Ava5 human hepatoma cell line (Huh7 derivative) harboring HCV subgenomic replicon RNA. METHODS: To determine the non-structure protein 5A (NS5A) mRNA and NS3 protein expression levels, real-time PCR and Western blot analysis were performed, respectively. To investigate how hypericin inhibits HCV replication, 5-aza-2'-deoxycytidine (5-Aza-dC) and chidamide were used for determining histone modification. Furthermore, shRNA was applied to confirm the role of heme oxygenase (HO-1) in HCV repression. RESULTS: Hypericin in experiment were tested and showed no cytotoxicity. Hypericin reduced HO-1 and NS5A in a time- and dose- dependent manner. Chidamide, but not 5-Aza-dc, restored hypericin-induced reduction in HCV NS3 expression and reversed HO-1 expression in Ava5 cells. LY294002 inhibited HCV replication via HO-1 down-regulation. Constitutive expressed p-AKT was not involved in hypericin-induced reduction in HCV replication. In addition, shHO-1 inhibited HCV replication. CONCLUSION: In conclusion, hypericin inhibits HCV replication via down-regulation of HO-1 expression and deacetylation.

Anti-hepatitis C virus activity and synergistic effect of Nymphaea alba extracts and bioactive constituents in liver infected cells.

BACKGROUND: Without an effective vaccine, hepatitis C virus (HCV) remains a global threat, inflicting 170-300 million carriers worldwide at risk of cirrhosis and hepatocellular carcinoma (HCC). Though various direct acting antivirals have been redeemed the hepatitis C treatment, a few restraints persist including possible side effects, viral resistance emergence, excessive cost which restricts its availability to a common person. HYPOTHESIS: There is no preventive HCV vaccine available today so the discovery of potent antiviral natural flora and their bioactive constituents may help to develop preventive cures against HCV infection. STUDY DESIGN: In current study, we aim to clarify anti-HCV activity of methanol and acetone extracts along with the purified fractions of Pakistani local plant, Nymphaea alba L (N. alba) using Huh-7 cell line as transfection model. Synergistic study of purified fractions with interferon was performed using MDBK cell line (expressing interferon receptors) as transfection model. MATERIALS AND METHODS: Recent study by our research group has observed potent anti-HCV NS3 protease activity of methanol and acetone extracts of N. alba. Effect of N. alba extracts, its fractions precisely, the N1 and N8 fractions on HCV replication was demonstrated by analyzing viral gene expression using in vitro transfection model. Considering NS3 protease as a dynamic drug target, fourteen phytochemicals of N. alba were selected as ligands for interaction with NS3 protein using Molecular Operating Environment (MOE) software. Boceprevir, FDA approved NS3 protease inhibitor, was used as standard for comparative study in docking screening. RESULTS: Herein we report 84% and 94% reduction of 3a genotype of HCV NS3/4A gene expression at mRNA level at non-toxic concentration. Specifically, two fractions 'N1' & 'N8' isolated from acetone extract suppressed HCV NS3 gene expression in transfected target cells with an EC50 value of 37 ±â€¯0.03 µg/ml and 20 ±â€¯0.02 µg/ml respectively. Similarly, viral genotype 1a replication is strongly suppressed in target cells by N. alba flower extracts and purified fractions. Moreover, combination of fractions with standard antiviral drug displayed synergistic effects for inhibition of HCV replication. Phytochemicals including Isoquercetin, Hyperoside, Quercetin, Reynoutrin, Apigenin and Isokaempferide displayed minimum binding energies as compared to standard protease inhibitor. CONCLUSION: N. alba and its purified phytochemicals with new scaffolds might significantly serve as valuable and alternative regimen against HCV either alone or in combination with other potential anti-HCV agents.

Inhibitory Effects of Amentoflavone and Orobol on Daclatasvir-Induced Resistance-Associated Variants of Hepatitis C Virus.

Hepatitis C virus (HCV) is recognized as a major causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite rapid progress in the development of direct-acting antivirals (DAA) against HCV infection in recent years, cost-effective antiviral drugs with more affordable prices still need to be developed. In this study, we screened a library of natural compounds to identify natural HCV inhibitors. The library of the pure compounds extracted from Chinese herbs deposited in the chemical bank of National Research Institute of Chinese Medicine (NRICM), Taiwan was screened in the cell culture-derived HCV (HCVcc) system. We identified the flavone or flavan-based compounds amentoflavone, 7,4[Formula: see text]-dihydroxyflavanone, and orobol with the inhibition of viral entry, replication, and translation of the HCV life cycle. Amentoflavone and orobol also showed inhibitory effects on resistant-associated variants to the NS5A inhibitor daclatasvir. The results of this study have the potential to benefit patients who are intolerant to the adverse effect of pegylated interferon or who harbor resistant strains refractory to treatment by current direct-acting antiviral agents.

Vitamin D deficiency and hepatitis viruses-associated liver diseases: A literature review.

The secosteroid hormone vitamin D has, in addition to its effects in bone metabolism also functions in the modulation of immune responses against infectious agents and in inhibiting tumorigenesis. Thus, deficiency of vitamin D is associated with several malignancies, but also with a plethora of infectious diseases. Among other communicable diseases, vitamin D deficiency is involved in the pathogenesis of chronic liver diseases caused by hepatitis B and C viruses (HBV, HCV) and high prevalence of vitamin D deficiency with serum levels below 20 mg/mL in patients with HBV and HCV infection are found worldwide. Several studies have assessed the effects of vitamin D supplementation on the sustained virological response (SVR) to interferon (IFN) plus ribavirin (RBV) therapy in HBV and HCV infection. In these studies, inconsistent results were reported. This review addresses general aspects of vitamin D deficiency and, in particular, the significance of vitamin D hypovitaminosis in the outcome of HBV- and HCV-related chronic liver diseases. Furthermore, current literature was reviewed in order to understand the effects of vitamin D supplementation in combination with IFN-based therapy on the virological response in HBV and HCV infected patients.

A natural small molecule inhibitor corilagin blocks HCV replication and modulates oxidative stress to reduce liver damage.

Hepatitis C virus (HCV) infection causes chronic liver disease, which often leads to hepatocellular carcinoma. Earlier, we have demonstrated anti-HCV property of the methanolic extract of Phyllanthus amarus, an age-old folk-medicine against viral hepatitis. Here, we report identification of a principal bioactive component 'corilagin', which showed significant inhibition of the HCV key enzymes, NS3 protease and NS5B RNA-dependent-RNA-polymerase. This pure compound could effectively inhibit viral replication in the infectious cell culture system, displayed strong antioxidant activity by blocking HCV induced generation of reactive oxygen species and suppressed up-regulation of NOX4 and TGF-ß mRNA levels. Oral administration of corilagin in BALB/c mice demonstrated its better tolerability and systemic bioavailability. More importantly, corilagin could restrict serum HCV RNA levels, decrease collagen deposition and hepatic cell denaturation in HCV infected chimeric mice harbouring human hepatocytes. Taken together, results provide a basis towards developing a pure natural drug as an alternate therapeutic strategy for restricting viral replication and prevent liver damage towards better management of HCV induced pathogenesis.

(-)-Epigallocatechin-3-gallate enhances poly I:C-induced interferon-λ1 production and inhibits hepatitis C virus replication in hepatocytes.

AIM: To investigate the effect of (-)-epigallocatechin-3-gallate (EGCG) on polyinosinic-polycytidylic acid (poly I:C)-triggered intracellular innate immunity against hepatitis C virus (HCV) in hepatocytes. METHODS: A cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain (JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular mRNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon (IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells. RESULTS: Our recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFN-stimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2'-5'-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway. CONCLUSION: Our observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.

Pentagalloylglucose, a highly bioavailable polyphenolic compound present in Cortex moutan, efficiently blocks hepatitis C virus entry.

Approximately 142 million people worldwide are infected with hepatitis C virus (HCV). Although potent direct acting antivirals are available, high costs limit access to treatment. Chronic hepatitis C virus infection remains a major cause of orthotopic liver transplantation. Moreover, re-infection of the graft occurs regularly. Antivirals derived from natural sources might be an alternative and cost-effective option to complement therapy regimens for global control of hepatitis C virus infection. We tested the antiviral properties of a mixture of different Chinese herbs/roots named Zhi Bai Di Huang Wan (ZBDHW) and its individual components on HCV. One of the ZBDHW components, Penta-O-Galloyl-Glucose (PGG), was further analyzed for its mode of action in vitro, its antiviral activity in primary human hepatocytes as well as for its bioavailability and hepatotoxicity in mice. ZBDHW, its component Cortex Moutan and the compound PGG efficiently block entry of HCV of all major genotypes and also of the related flavivirus Zika virus. PGG does not disrupt HCV virion integrity and acts primarily during virus attachment. PGG shows an additive effect when combined with the well characterized HCV inhibitor Daclatasvir. Analysis of bioavailability in mice revealed plasma levels above tissue culture IC50 after a single intraperitoneal injection. In conclusion, PGG is a pangenotypic HCV entry inhibitor with high bioavailability. The low cost and wide availability of this compound make it a promising candidate for HCV combination therapies, and also emerging human pathogenic flaviviruses like ZIKV.