Antibacterial and anti-coronavirus investigation of selected Senegalese plant species according to an ethnobotanical survey.

ETHNOPHARMACOLOGICAL RELEVANCE: In Senegal, upper and lower respiratory tract infections constitute a real health problem. To manage these disorders, most people rely on the use of local medicinal plants. This is particularly the case for species belonging to the botanical families, Combretaceae, Fabaceae, Myrtaceae and Rubiaceae, which are widely used to treat various respiratory problems such as colds, flu, rhinitis, sinusitis, otitis, angina, bronchitis, bronchiolitis and also pneumonia. AIM OF THE STUDY: The aim of this study was to identify medicinal plants traditionally used for the management of infectious diseases, in particular those of the respiratory tract. On the basis of these ethnopharmacological uses, this study made it possible to highlight the antibacterial, antiviral and cytotoxic activities of selected plant species. MATERIALS AND METHODS: An ethnobotanical survey was conducted in Senegal among informants, including herbalists, traditional healers, and households, using medicinal plants in the management of infectious diseases, with a focus on respiratory tract infections. The most cited plant species were evaluated in vitro on a panel of 18 human pathogenic bacteria may be involved in respiratory infections and against the human coronavirus HCoV-229E in Huh-7 cells. The antiviral activity of the most active extracts against HCoV-229E was also evaluated on COVID-19 causing agent, SARS-CoV-2 in Vero-81 cells. In parallel, cytotoxic activities were evaluated on Huh-7 cells. RESULTS: A total of 127 informants, including 100 men (78.74%) and 27 women (21.26%) participated in this study. The ethnobotanical survey led to the inventory of 41 plant species belonging to 19 botanical families used by herbalists and/or traditional healers and some households to treat infectious diseases, with a specific focus on upper respiratory tract disorders. Among the 41 plant species, the most frequently mentioned in the survey were Guiera senegalensis J.F. Gmel. (95.2%), Combretum glutinosum Perr. Ex DC. (93.9%) and Eucalyptus spp. (82.8%). Combretaceae (30.2%) represented the most cited botanical family with six species, followed by Fabaceae (29.3%, 12 species). A total of 33 crude methanolic extracts of the 24 plant species selected for their number of citations were evaluated in vitro for their antimicrobial and cytotoxic activities. Guiera senegalensis, Combretum glutinosum, Vachellia nilotica subsp. tomentosa (Benth.) Kyal. & Boatwr, Eucalyptus camaldulensis Dehnh., and Terminalia avicennioides Guill. & Perr., showed antibacterial activities. The most active plants against HCoV-229E were: Ficus sycomorus L., Mitragyna inermis (Willd.) Kuntze, Pterocarpus erinaceus Poir., and Spermacoce verticillata L. One of these plants, Mitragyna inermis, was also active against SARS-CoV-2. CONCLUSION: This work confirmed the anti-infective properties of plant species traditionally used in Senegal. Overall, the most frequently cited plant species showed the best antibacterial activities. Moreover, some of the selected plant species could be considered as a potential source for the management of coronavirus infections. This new scientific data justified the use of these plants in the management of some infectious pathologies, especially those of the respiratory tract.

Discovery of Anti-Coronavirus Cinnamoyl Triterpenoids Isolated from Hippophae rhamnoides during a Screening of Halophytes from the North Sea and Channel Coasts in Northern France.

The limited availability of antiviral therapy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spurred the search for novel antiviral drugs. Here, we investigated the potential antiviral properties of plants adapted to high-salt environments collected in the north of France. Twenty-five crude methanolic extracts obtained from twenty-two plant species were evaluated for their cytotoxicity and antiviral effectiveness against coronaviruses HCoV-229E and SARS-CoV-2. Then, a bioguided fractionation approach was employed. The most active crude methanolic extracts were partitioned into three different sub-extracts. Notably, the dichloromethane sub-extract of the whole plant Hippophae rhamnoides L. demonstrated the highest antiviral activity against both viruses. Its chemical composition was evaluated by ultra-high performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS) and then it was fractionated by centrifugal partition chromatography (CPC). Six cinnamoyl triterpenoid compounds were isolated from the three most active fractions by preparative high-performance liquid chromatography (HPLC) and identified by high resolution MS (HR-MS) and mono- and bi-dimensional nuclear magnetic resonance (NMR). Specifically, these compounds were identified as 2-O-trans-p-coumaroyl-maslinic acid, 3ß-hydroxy-2α-trans-p-coumaryloxy-urs-12-en-28-oic acid, 3ß-hydroxy-2α-cis-p-coumaryloxy-urs-12-en-28-oic acid, 3-O-trans-caffeoyl oleanolic acid, a mixture of 3-O-trans-caffeoyl oleanolic acid/3-O-cis-caffeoyl oleanolic acid (70/30), and 3-O-trans-p-coumaroyl oleanolic acid. Infection tests demonstrated a dose-dependent inhibition of these triterpenes against HCoV-229E and SARS-CoV-2. Notably, cinnamoyl oleanolic acids displayed activity against both SARS-CoV-2 and HCoV-229E. Our findings suggest that Hippophae rhamnoides could represent a source of potential antiviral agents against coronaviruses.

Correction: Bilyy et al. Rapid Generation of Coronaviral Immunity Using Recombinant Peptide Modified Nanodiamonds. Pathogens 2021, 10, 861.

In the original publication [...].

Lichen or Associated Micro-Organism Compounds Are Active against Human Coronaviruses.

(1) Background: Since the emergence of SARS-CoV-2, responsible for the COVID-19 pandemic, efforts have been made to identify antiviral compounds against human coronaviruses. With the aim of increasing the diversity of molecule scaffolds, 42 natural compounds, of which 28 were isolated from lichens and 14 from their associated microorganisms (bacteria and fungi), were screened against human coronavirus HCoV-229E. (2) Methods: Antiviral assays were performed using HCoV-229E in Huh-7 and Huh-7/TMPRSS2 cells and SARS-CoV-2 in a Vero-81-derived clone with a GFP reporter probe. (3) Results: Four lichen compounds, including chloroatranol, emodin, perlatolic acid and vulpinic acid, displayed high activities against HCoV-229E (IC50 = 68.86, 59.25, 16.42 and 14.58 µM, respectively) and no toxicity at active concentrations. Kinetics studies were performed to determine their mode of action. The four compounds were active when added at the replication step. Due to their significant activity, they were further tested on SARS-CoV-2. Perlatolic acid was shown to be active against SARS-CoV-2. (4) Conclusions: Taken together, these results show that lichens are a source of interesting antiviral agents against human coronaviruses. Moreover, perlatolic acid might be further studied for its pan-coronavirus antiviral activity.

Removal of senescent cells reduces the viral load and attenuates pulmonary and systemic inflammation in SARS-CoV-2-infected, aged hamsters.

Older age is one of the strongest risk factors for severe COVID-19. In this study, we determined whether age-associated cellular senescence contributes to the severity of experimental COVID-19. Aged golden hamsters accumulate senescent cells in the lungs, and the senolytic drug ABT-263, a BCL-2 inhibitor, depletes these cells at baseline and during SARS-CoV-2 infection. Relative to young hamsters, aged hamsters had a greater viral load during the acute phase of infection and displayed higher levels of sequelae during the post-acute phase. Early treatment with ABT-263 lowered pulmonary viral load in aged (but not young) animals, an effect associated with lower expression of ACE2, the receptor for SARS-CoV-2. ABT-263 treatment also led to lower pulmonary and systemic levels of senescence-associated secretory phenotype factors and to amelioration of early and late lung disease. These data demonstrate the causative role of age-associated pre-existing senescent cells on COVID-19 severity and have clear clinical relevance.

New Phenolic Lipids from the Leaves of Clausena harmandiana Inhibit SARS-CoV-2 Entry into Host Cells.

Induced by the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 pandemic underlined the clear need for antivirals against coronaviruses. In an effort to identify new inhibitors of SARS-CoV-2, a screening of 824 extracts prepared from various parts of 400 plant species belonging to the Rutaceae and Annonaceae families was conducted using a cell-based HCoV-229E inhibition assay. Due to its significant activity, the ethyl acetate extract of the leaves of Clausena harmandiana was selected for further chemical and biological investigations. Mass spectrometry-guided fractionation afforded three undescribed phenolic lipids (1-3), whose structures were determined via spectroscopic analysis. The absolute configurations of 1 and 2 were determined by analyzing Mosher ester derivatives. The antiviral activity against SARS-CoV-2 was subsequently shown, with IC50 values of 0.20 and 0.05 µM for 2 and 3, respectively. The mechanism of action was further assessed, showing that both 2 and 3 are inhibitors of coronavirus entry by acting directly on the viral particle. Phenolic lipids from Clausena harmandiana might be a source of new antiviral agents against human coronaviruses.

Luteolin Isolated from Juncus acutus L., a Potential Remedy for Human Coronavirus 229E.

The COVID-19 pandemic, caused by SARS-CoV-2, addressed the lack of specific antiviral drugs against coronaviruses. In this study, bioguided fractionation performed on both ethyl acetate and aqueous sub-extracts of Juncus acutus stems led to identifying luteolin as a highly active antiviral molecule against human coronavirus HCoV-229E. The apolar sub-extract (CH2Cl2) containing phenanthrene derivatives did not show antiviral activity against this coronavirus. Infection tests on Huh-7 cells, expressing or not the cellular protease TMPRSS2, using luciferase reporter virus HCoV-229E-Luc showed that luteolin exhibited a dose-dependent inhibition of infection. Respective IC50 values of 1.77 µM and 1.95 µM were determined. Under its glycosylated form (luteolin-7-O-glucoside), luteolin was inactive against HCoV-229E. Time of addition assay showed that utmost anti-HCoV-229E activity of luteolin was achieved when added at the post-inoculation step, indicating that luteolin acts as an inhibitor of the replication step of HCoV-229E. Unfortunately, no obvious antiviral activity for luteolin was found against SARS-CoV-2 and MERS-CoV in this study. In conclusion, luteolin isolated from Juncus acutus is a new inhibitor of alphacoronavirus HCoV-229E.

Eremoxylarins D-J, Antibacterial Eremophilane Sesquiterpenes Discovered from an Endolichenic Strain of Xylaria hypoxylon.

An endolichenic strain of the Ascomycetaceous Xylaria hypoxylon, cultivated alone or in coculture with another endolichenic fungus Dendrothyrium variisporum, produced seven new bioactive eremophilane sesquiterpenes eremoxylarins D-J (1-7). The isolated compounds disclosed a high similarity with the eremophilane core of the bioactive integric acid, and structures were elucidated by 1D and 2D NMR spectra and electronic circular dichroism (ECD) analyses. Eremoxylarins D, F, G, and I showed a selective activity against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus with minimum inhibitory concentration (MIC) values between 0.39 and 12.5 µg/mL. Eremoxylarin I, the most antibacterial active sesquiterpene, was also active against HCoV-229E at a concentration nontoxic to the hepatoma Huh-7 cell line with an 50% inhibitory concentration (IC50) of 18.1 µM and a 50% cytotoxic concentration (CC50) of 46.6 µM.

SARS-CoV-2 infection induces persistent adipose tissue damage in aged golden Syrian hamsters.

Coronavirus disease 2019 (COVID-19, caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)) is primarily a respiratory illness. However, various extrapulmonary manifestations have been reported in patients with severe forms of COVID-19. Notably, SARS-CoV-2 was shown to directly trigger white adipose tissue (WAT) dysfunction, which in turn drives insulin resistance, dyslipidemia, and other adverse outcomes in patients with COVID-19. Although advanced age is the greatest risk factor for COVID-19 severity, published data on the impact of SARS-CoV-2 infection on WAT in aged individuals are scarce. Here, we characterized the response of subcutaneous and visceral WAT depots to SARS-CoV-2 infection in young adult and aged golden hamsters. In both age groups, infection was associated with a decrease in adipocyte size in the two WAT depots; this effect was partly due to changes in tissue's lipid metabolism and persisted for longer in aged hamsters than in young-adult hamsters. In contrast, only the subcutaneous WAT depot contained crown-like structures (CLSs) in which dead adipocytes were surrounded by SARS-CoV-2-infected macrophages, some of them forming syncytial multinucleated cells. Importantly, older age predisposed to a unique manifestation of viral disease in the subcutaneous WAT depot during SARS-CoV-2 infection; the persistence of very large CLSs was indicative of an age-associated defect in the clearance of dead adipocytes by macrophages. Moreover, we uncovered age-related differences in plasma lipid profiles during SARS-CoV-2 infection. These data suggest that the WAT's abnormal response to SARS-CoV-2 infection may contribute to the greater severity of COVID-19 observed in elderly patients.

Clofoctol inhibits SARS-CoV-2 replication and reduces lung pathology in mice.

Drug repurposing has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to its favorable safety profile and pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC50 measured against SARS-CoV-2 in human pulmonary cells. This compound inhibits SARS-CoV-2 at a post-entry step. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and lowered pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients.

A Photoactivable Natural Product with Broad Antiviral Activity against Enveloped Viruses, Including Highly Pathogenic Coronaviruses.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has highlighted the need for broad-spectrum antivirals against coronaviruses (CoVs). Here, pheophorbide a (Pba) was identified as a highly active antiviral molecule against human CoV-229E after bioguided fractionation of plant extracts. The antiviral activity of Pba was subsequently shown for SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV), and its mechanism of action was further assessed, showing that Pba is an inhibitor of coronavirus entry by directly targeting the viral particle. Interestingly, the antiviral activity of Pba depends on light exposure, and Pba was shown to inhibit virus-cell fusion by stiffening the viral membrane, as demonstrated by cryoelectron microscopy. Moreover, Pba was shown to be broadly active against several other enveloped viruses and reduced SARS-CoV-2 and MERS-CoV replication in primary human bronchial epithelial cells. Pba is the first described natural antiviral against SARS-CoV-2 with direct photosensitive virucidal activity that holds potential for COVID-19 therapy or disinfection of SARS-CoV-2-contaminated surfaces.

Rapid Generation of Coronaviral Immunity Using Recombinant Peptide Modified Nanodiamonds.

Vaccination remains one of the most effective tools to prevent infectious diseases. To ensure that the best possible antigenic components are chosen to stimulate a cognitive immune response, boosting antigen presentation using adjuvants is common practice. Nanodiamond-based adjuvants are proposed here as a rapid and versatile platform for antigen conjugation, utilizing peptides common to different pathogenic strains and making this strategy a good candidate for a "ready-to-use" vaccine. Initiation of an inflammatory reaction with a resulting immune response is based on the ability of living organisms to entrap nanostructures such as nanodiamonds with neutrophil extracellular traps (NETs) formation. In this work, coronavirus peptide homological for MERS-CoV, fusion inhibitor, was conjugated to nanodiamonds and used to induce neutrophilic-driven self-limiting inflammation. The resulting adjuvant was safe and did not induce any tissue damage at the site of injection. Mice immunization resulted in IgG titers of »,000 within 28 days. Immunization of rabbits resulted in the formation of a high level of antibodies persistently present for up to 120 days after the first immunization (animal lifespan ~3 years). The peptide used for immunization proved to be reactive with sera of convalescent COVID patients, demonstrating the possibility of developing pancoronaviral vaccine candidates.

Ultrastructural modifications induced by SARS-CoV-2 in Vero cells: a kinetic analysis of viral factory formation, viral particle morphogenesis and virion release.

Many studies on SARS-CoV-2 have been performed over short-time scale, but few have focused on the ultrastructural characteristics of infected cells. We used TEM to perform kinetic analysis of the ultrastructure of SARS-CoV-2-infected cells. Early infection events were characterized by the presence of clusters of single-membrane vesicles and stacks of membrane containing nuclear pores called annulate lamellae (AL). A large network of host cell-derived organelles transformed into virus factories was subsequently observed in the cells. As previously described for other RNA viruses, these replication factories consisted of double-membrane vesicles (DMVs) located close to the nucleus. Viruses released at the cell surface by exocytosis harbored the typical crown of spike proteins, but viral particles without spikes were also observed in intracellular compartments, possibly reflecting incorrect assembly or a cell degradation process.

Anti-HCV Tannins From Plants Traditionally Used in West Africa and Extracted With Green Solvents.

Millions of people are still infected with hepatitis C virus (HCV) nowadays. Although recent antivirals targeting HCV proteins are very efficient, they are not affordable for many people infected with this virus. Therefore, new and more accessible treatments are needed. Several Ivorian medicinal plants are traditionally used to treat "yellow malaria", a nosological category including illness characterized by symptomatic jaundice such as hepatitis. Therefore, some of these plants might be active against HCV. An ethnobotanical survey in Côte d'Ivoire allowed us to select such medicinal plants. Those were first extracted with methanol and tested for their anti-HCV activity. The most active ones were further studied to specify their IC50 and to evaluate their toxicity in vitro. Greener solvents were tested to obtain extracts with similar activities. Following a phytochemical screening, tannins of the most active plants were removed before re-testing on HCV. Some of these tannins were identified by UPLC-MS and pure molecules were tested against HCV. Out of the fifteen Ivorian medicinal plants selected for their putative antiviral activities, Carapa procera DC. and Pericopsis laxiflora (Benth. ex Baker) Meeuwen were the most active against HCV (IC50: 0.71 and 0.23 µg/ml respectively) and not toxic for hepatic cells. Their crude extracts were rich in polyphenols, including tannins such as procyanidins A2 which is active against HCV. The same extracts without tannin lost their anti-HCV activity. Replacing methanol by hydro-ethanolic solvent led to tannins-rich extracts with similar antiviral activities, and higher than that of aqueous extracts.

Viral hepatitis in the Peruvian Amazon: Ethnomedical context and phytomedical resource.

ETHNOPHARMACOLOGICAL RELEVANCE: An extensive ethnopharmacological survey was carried out in the Peruvian Amazonian district of Loreto with informants of various cultural origins from the surroundings of Iquitos (capital city of Loreto) and from 15 isolated riverine Quechua communities of the Pastaza River. A close attention was paid to the medical context and plant therapy, leading to the selection of 35 plant species (45 extracts). The extracts were tested for antiviral activity against HCV with counting of Huh-7 cellular death in case of toxicity, and cytotoxicity was evaluated in HepG2 cells. AIM OF THE STUDY: The aim of the study was to inventory the plants used against hepatitis in Loreto, then to evaluate their antiviral activity and to suggest a way to improve local therapeutic strategy against viral hepatitis, which is a fatal disease that is still increasing in this area. MATERIALS AND METHODS: An ethnographic survey was carried out using "participant-observation" methodology and focusing on plant therapy against hepatitis including associated remedies. 45 parts of plant were extracted with methanol and tested in vitro for anti-HCV activity in 96-well plate, using HCV cell culture system with immunofluorescent detection assisted by automated confocal microscopy. Toxicity of plant extracts was also evaluated in microplates on hepatic cells by immunofluorescent detection, for the Huh-7 nuclei viability, and by UV-absorbance measurement of MTT formazan for cytotoxicity in HepG2 cells. RESULTS: In vitro assay revealed interesting activity of 18 extracts (50% infection inhibition at 25 µg/mL) with low cytotoxicity for 15 of them. Result analysis showed that at least 30% of HCV virus were inhibited at 25 µg/mL for 60% of the plant extracts. Moreover, the ethnomedical survey showed that remedies used with low and accurate dosing as targeted therapy against hepatitis are usually more active than species indicated with more flexible dosing to alleviate symptoms of hepatic diseases. CONCLUSION: Together with bibliographic data analysis, this study supported the traditional medicinal uses of many plants and contributed to a better understanding of the local medical system. It also permitted to refine the therapeutic plant indications regarding patients' liver injuries and vulnerability. Only 2 of the 15 most active plant species have already been studied for antiviral activity against hepatitis suggesting new avenues to be followed for the 13 other species.

Functional Carbon Quantum Dots as Medical Countermeasures to Human Coronavirus.

Therapeutic options for the highly pathogenic human coronavirus (HCoV) infections are urgently needed. Anticoronavirus therapy is however challenging, as coronaviruses are biologically diverse and rapidly mutating. In this work, the antiviral activity of seven different carbon quantum dots (CQDs) for the treatment of human coronavirus HCoV-229E infections was investigated. The first generation of antiviral CQDs was derived from hydrothermal carbonization of ethylenediamine/citric acid as carbon precursors and postmodified with boronic acid ligands. These nanostructures showed a concentration-dependent virus inactivation with an estimated EC50 of 52 ± 8 µg mL-1. CQDs derived from 4-aminophenylboronic acid without any further modification resulted in the second-generation of anti-HCoV nanomaterials with an EC50 lowered to 5.2 ± 0.7 µg mL-1. The underlying mechanism of action of these CQDs was revealed to be inhibition of HCoV-229E entry that could be due to interaction of the functional groups of the CQDs with HCoV-229E entry receptors; surprisingly, an equally large inhibition activity was observed at the viral replication step.

Dehydrojuncusol, a Natural Phenanthrene Compound Extracted from Juncus maritimus, Is a New Inhibitor of Hepatitis C Virus RNA Replication.

Recent emergence of direct-acting antivirals (DAAs) targeting hepatitis C virus (HCV) proteins has considerably enhanced the success of antiviral therapy. However, the appearance of DAA-resistant-associated variants is a cause of treatment failure, and the high cost of DAAs renders the therapy not accessible in countries with inadequate medical infrastructures. Therefore, the search for new inhibitors with a lower cost of production should be pursued. In this context, the crude extract of Juncus maritimus Lam. was shown to exhibit high antiviral activity against HCV in cell culture. Bio-guided fractionation allowed the isolation and identification of the active compound, dehydrojuncusol. A time-of-addition assay showed that dehydrojuncusol significantly inhibited HCV infection when added after virus inoculation of HCV genotype 2a (50% effective concentration [EC50] = 1.35 µM). This antiviral activity was confirmed with an HCV subgenomic replicon, and no effect on HCV pseudoparticle entry was observed. Antiviral activity of dehydrojuncusol was also demonstrated in primary human hepatocytes. No in vitro toxicity was observed at active concentrations. Dehydrojuncusol is also efficient on HCV genotype 3a and can be used in combination with sofosbuvir. Interestingly, dehydrojuncusol was able to inhibit RNA replication of two frequent daclatasvir-resistant mutants (L31M or Y93H in NS5A). Finally, mutants resistant to dehydrojuncusol were obtained and showed that the HCV NS5A protein is the target of the molecule. In conclusion, dehydrojuncusol, a natural compound extracted from J. maritimus, inhibits infection of different HCV genotypes by targeting the NS5A protein and is active against resistant HCV variants frequently found in patients with treatment failure.IMPORTANCE Tens of millions of people are infected with hepatitis C virus (HCV) worldwide. Recently marketed direct-acting antivirals (DAAs) targeting HCV proteins have enhanced the efficacy of treatment. However, due to its high cost, this new therapy is not accessible to the vast majority of infected patients. Furthermore, treatment failures have also been reported due to the appearance of viral resistance. Here, we report on the identification of a new HCV inhibitor, dehydrojuncusol, that targets HCV NS5A and is able to inhibit RNA replication of replicons harboring resistance mutations to anti-NS5A DAAs used in current therapy. Dehydrojuncusol is a natural compound isolated from Juncus maritimus, a halophilic plant species that is very common in coastlines worldwide. This molecule might serve as a lead for the development of a new therapy that is more accessible to hepatitis C patients in the future.

Theaflavins, polyphenols of black tea, inhibit entry of hepatitis C virus in cell culture.

The treatment of hepatitis C virus (HCV) infection by combination of direct acting antivirals (DAA), with different mode of action, has made substantial progress in the past few years. However, appearance of resistance and high cost of the therapy is still an obstacle in the achievement of the therapy, more specifically in developing countries. In this context, search for affordable antivirals with new mechanisms of action is still needed. Tea, after water, is the most popular drink worldwide. Polyphenols extracted from green tea have already shown anti-HCV activity as entry inhibitors. Here, three different theaflavins, theaflavin (TF1), theaflavin-3'-monogallate (TF2), and theaflavin-3-3'-digallate (TF3), which are major polyphenols from black tea, were tested against HCV in cell culture. The results showed that all theaflavins inhibit HCV infection in a dose-dependent manner in an early step of infection. Results obtained with HCV pseudotyped virions confirmed their activity on HCV entry and demonstrated their pan-genotypic action. No effect on HCV replication was observed by using HCV replicon. Investigation on the mechanism of action of black tea theaflavins showed that they act directly on the virus particle and are able to inhibit cell-to-cell spread. Combination study with inhibitors most widely used in anti-HCV treatment regimen demonstrated that TF3 exerts additive effect. In conclusion, theaflavins, that are present in high quantity in black tea, are new inhibitors of HCV entry and hold promise for developing in therapeutic arsenal for HCV infection.