Since the onset of the COVID-19 (COronaVIrus Disease 19) pandemic, SARS-CoV-2 has exhibited a high transmission rate, further enhanced by new variants able to better adapt to humans. Addressing this issue has been challenging due to viral resistance and side effects associated with antiviral drugs and vaccines. As a result, there has been a growing interest in plant-derived compounds with antiviral properties. Our study revealed that pistachio extracts significantly inhibited SARS-CoV-2 viral entry. Employing pseudotyped particles bearing the S protein of SARS-CoV-2, we demonstrated that treatment with pistachio extracts inhibited binding of alpha (α) and omicron (ο) SARS-CoV-2 variants. Furthermore, our study revealed that the pistachio carotenoid zeaxanthin exhibited a different inhibitory activity against two SARS-CoV-2 variants. In silico analyses demonstrated a strong interaction between zeaxanthin and the receptor-binding domain (RBD) domain of the omicron spike (S) protein, thus reducing pseudovirus entry. However, zeaxanthin’s weaker interaction with the alpha variant’s RBD was insufficient to inhibit entry. Additionally, zeaxanthin suppressed the expression of the host protease TMPRSS2 at the protein level, thereby limiting the internalization of the alpha variant, which relies on TMPRSS2 for cellular entry.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has provoked a global health crisis due to the absence of a specific therapeutic agent. 3CLpro (also known as the main protease or Mpro) and PLpro are chymotrypsin-like proteases encoded by the SARS-CoV-2 genome, and play essential roles during the virus lifecycle. Therefore, they are recognized as a prospective therapeutic target in drug discovery against SARS-CoV-2 infection. Thus, this work aims to collectively present potential natural 3CLpro and PLpro inhibitors by in silico simulations and in vitro entry pseudotype-entry models. We screened luteolin-7-O-glucuronide (L7OG), cynarin (CY), folic acid (FA), and rosmarinic acid (RA) molecules against PLpro and 3CLpro through a luminogenic substrate assay. We only reported moderate inhibitory activity on the recombinant 3CLpro and PLpro by L7OG and FA. Afterward, the entry inhibitory activity of L7OG and FA was tested in cell lines transduced with the two different SARS-CoV-2 pseudotypes harboring alpha (α) and omicron (o) spike (S) protein. The results showed that both compounds have a consistent inhibitory activity on the entry for both variants. However, L7OG showed a greater degree of entry inhibition against α-SARS-CoV-2. Molecular modeling studies were used to determine the inhibitory mechanism of the candidate molecules by focusing on their interactions with residues recognized by the protease active site and receptor-binding domain (RBD) of spike SARS-CoV-2. This work allowed us to identify the binding sites of FA and L7OG within the RBD domain in the alpha and omicron variants, demonstrating how FA is active in both variants. We have confidence that future in vivo studies testing the safety and effectiveness of these natural compounds are warranted, given that they are effective against a variant of concerns.

Since the outbreak of COVID-19, researchers have been working tirelessly to discover effective ways to combat coronavirus infection. The use of computational drug repurposing methods and molecular docking has been instrumental in identifying compounds that have the potential to disrupt the binding between the spike glycoprotein of SARS-CoV-2 and human ACE2 (hACE2). Moreover, the pseudovirus approach has emerged as a robust technique for investigating the mechanism of virus attachment to cellular receptors and for screening targeted small molecule drugs. Pseudoviruses are viral particles containing envelope proteins, which mediate the virus’s entry with the same efficiency as that of live viruses but lacking pathogenic genes. Therefore, they represent a safe alternative to screen potential drugs inhibiting viral entry, especially for highly pathogenic enveloped viruses. In this review, we have compiled a list of antiviral plant extracts and natural products that have been extensively studied against enveloped emerging and re-emerging viruses by pseudovirus technology. The review is organized into three parts: (1) construction of pseudoviruses based on different packaging systems and applications; (2) knowledge of emerging and re-emerging viruses; (3) natural products active against pseudovirus-mediated entry. One of the most crucial stages in the life cycle of a virus is its penetration into host cells. Therefore, the discovery of viral entry inhibitors represents a promising therapeutic option in fighting against emerging viruses.

Epstein–Barr (EBV) is a human γ-herpesvirus that undergoes both a productive (lytic) cycle and a non-productive (latent) phase. The virus establishes enduring latent infection in B lymphocytes and productive infection in the oral mucosal epithelium. Like other herpesviruses, EBV expresses its genes in a coordinated pattern during acute infection. Unlike others, it replicates its DNA during latency to maintain the viral genome in an expanding pool of B lymphocytes, which are stimulated to divide upon infection. The reactivation from the latent state is associated with a productive gene expression pattern mediated by virus-encoded transcriptional activators BZLF-1 and BRLF-1. EBV is a highly transforming virus that contributes to the development of human lymphomas. Though viral vectors and mRNA platforms have been used to develop an EBV prophylactic vaccine, currently, there are no vaccines or antiviral drugs for the prophylaxis or treatment of EBV infection and EBV-associated cancers. Natural products and bioactive compounds are widely studied for their antiviral potential and capability to modulate intracellular signaling pathways. This review was intended to collect information on plant-derived products showing their antiviral activity against EBV and evaluate their feasibility as an alternative or adjuvant therapy against EBV infections and correlated oncogenesis in humans.

Increased global research is focused on the development of novel therapeutics to combat antimicrobial and antiviral resistance. Pistachio nuts represent a good source of protein, fiber, monounsaturated fatty acids, minerals, vitamins, and phytochemicals (carotenoids, phenolic acids, flavonoids and anthocyanins). The phytochemicals found in pistachios are structurally diverse compounds with antimicrobial and antiviral potential, demonstrated as individual compounds, extracts and complexed into nanoparticles. Synergistic effects have also been reported in combination with existing drugs. Here we report an overview of the antimicrobial and antiviral potential of pistachio nuts: studies show that Gram-positive bacterial strains, such as Staphylococcus aureus, are the most susceptible amongst bacteria, whereas antiviral effect has been reported against herpes simplex virus 1 (HSV-1). Amongst the known pistachio compounds, zeaxanthin has been shown to affect both HSV-1 attachment penetration of human cells and viral DNA synthesis. These data suggest that pistachio extracts and derivatives could be used for the topical treatment of S. aureus skin infections and ocular herpes infections.

The continuous emergence of SARS-CoV-2 variants caused the persistence of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. The viral proteases are the most attractive targets for developing antiviral drugs. In this scenario, our study explores the use of HIV-1 protease inhibitors against SARS-CoV-2. An in silico screening of a library of HIV-1 proteases identified four anti-HIV compounds able to interact with the 3CLpro of SARS-CoV-2. Thus, in vitro studies were designed to evaluate their potential antiviral effectiveness against SARS-CoV-2. We employed pseudovirus technology to simulate, in a highly safe manner, the adsorption of the alpha (α-SARS-CoV-2) and omicron (ο-SARS-CoV-2) variants of SARS-CoV-2 and study the inhibitory mechanism of the selected compounds for cell–virus interaction. The results reported a mild activity against the viral proteases 3CLpro and PLpro, but efficient inhibitory effects on the internalization of both variants mediated by cathepsin B/L. Our findings provide insights into the feasibility of using drugs exhibiting antiviral effects for other viruses against the viral and host SARS-CoV-2 proteases required for entry.

The present study aims to assess the antioxidant and antiviral effectiveness of leaf extracts obtained from Olea europaea L. var. sativa and Olea europaea L. var. sylvestris. The total antioxidant activity was determined via both an ammonium phosphomolybdate assay and a nitric oxide radical inhibition assay. Both extracts showed reducing abilities in an in vitro system and in human HeLa cells. Indeed, after oxidative stress induction, we found that exposition to olive leaf extracts protects human HeLa cells from lipid peroxidation and increases the concentration of enzyme antioxidants such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase. Additionally, OESA treatment affects viral DNA accumulation more than OESY, probably due to the exclusive oleuropein content. In fact, subtoxic concentrations of oleuropein inhibit HSV-1 replication, stimulating the phosphorylation of PKR, c-FOS, and c-JUN proteins. These results provide new knowledge about the potential health benefits and mechanisms of action of oleuropein and oleuropein-rich extracts.

The almond industry produces, by bleaching and stripping, two by-products: blanched skin (BS) and blanch water (BW). The aim of this study was to investigate the nutritional and polyphenolic profile, as well as the antioxidant, antimicrobial, antiviral, and potential prebiotic effects of BS and BW from three different Sicilian cultivars. The total phenols and flavonoids contents were ≥1.72 and ≥0.56 g gallic acid equivalents and ≥0.52 and ≥0.18 g rutin equivalents/100 g dry extract (DE) in BS and BW, respectively. The antioxidant activity, evaluated by 2,2-diphenyl-1-picrylhydrazyl scavenging ability, trolox equivalent antioxidant capacity, ferric-reducing antioxidant power, and oxygen radical absorbance capacity, was ≥3.07 and ≥0.83 g trolox equivalent/100 g DE in BS and BW, respectively. Isorhamnetin-3-O-glucoside was the most abundant flavonoid detected in both by-products. No antimicrobial effect was recorded, whereas BS samples exerted antiviral activity against herpes simplex virus 1 (EC50 160.96 μg/mL). BS also showed high fibre (≥52.67%) and protein (≥10.99) contents and low fat (≤15.35%) and sugars (≤5.55%), making it nutritionally interesting. The present study proved that the cultivar is not a discriminating factor in determining the chemical and biological properties of BS and BW.

The activation of the innate immune response during HSV-1 infection stimulates several transcription factors, such as NF-κB and IRF3, which are critical regulators of IFN-β expression. The released IFN-β activates the ISGs, which encode antiviral effectors such as the PKR. We found that HSV-1 triggers an antiviral transcriptional response during viral replication by activating TBK1-IRF3-NF-κB network kinetically. In contrast, we reported that infected PKR−/− cells fail to activate the transcription of TBK1. Downstream, TBK1 was unable to activate the transcription of IRF3 and NF-κB. These data suggested that in PKR−/− cells, HSV-1 replication counteracts TBK1-IRF3-NF-κB network. In this scenario, a combined approach of gene knockout and gene silencing was used to determine how the lack of PKR facilitates HSV-1 replication. We reported that in HEp-2-infected cells, PKR can influence the TBK1-IRF3-NF-κB network, consequently interfering with viral replication. Otherwise, an abrogated PKR-mediated signaling sustains the HSV-1 replication. Our result allows us to add additional information on the complex HSV-host interaction network by reinforcing the concept of the PKR role in the innate response-related networks during HSV replication in an in vitro model.

Objectives: The current study aimed to investigate the potential role in inhibiting the entry of both HSV-1 and Sars-Cov-2 viruses and the immunomodulatory effect of pistachio kernels on human monocytic cells during HSV-1 replication.Methods: Polyphenolic extracts from natural raw (NRRE) and roasted unsalted (RURE) pistachio kernels were obtained by two different extraction methods and characterized by RP-HPLC-DAD.The antiviral activity against HSV-1 replication was performed on VERO epithelial cells.THP-1 cells were employed to test the immunomodulatory role of NRRE and RURE by controlling the transcription factor NF-kB.Finally, in order to evaluate the inhibitory effect of NRRE and RURE on SARS-CoV-2, a pseudovirus generated on HEK293 T cells was used to test the inhibition of the related luciferase activity.Results: Both NRRE and RURE exhibited an antiviral effect against HSV-1 through i) partial block of the HSV-1 binding to the cell surface; ii) reduction of viral DNA accumulation and transcripts; iii) modulation of viral proteins expression.Similarly, HSV-1 replication was abrogated by both NRRE and RURE in THP-1 cell lines.In addition, NRRE and RURE downregulated the accumulation of NF-kB transcripts during HSV-1 replication/ inflammation.NRRE and RURE exhibited ≥ 50% viral entry inhibition activity at 0.6 mg/mL against SARS-CoV-2.Conclusions: These data suggest that the antiviral activity of polyphenol-rich extracts from pistachios may be relevant for the formulation of the adjuvant drug against acyclovir-resistant HSV-1 strains and anti-SARS-CoV-2, thus encouraging clinical studies.In the THP-1 cellular model, the control of NF-kB mediated by the pistachio extracts can be relevant as an immunomodulator effect for HSV-1 replication/inflammation.

The search for alternative clinical treatments to fight resistance and find alternative antiviral treatments for the herpes simplex virus (HSV) is of great interest. Plants are rich sources of novel antiviral, pharmacologically active agents that provide several advantages, including reduced side effects, less resistance, low toxicity, and different mechanisms of action. In the present work, the antiviral activity of Californian natural raw (NRRE) and roasted unsalted (RURE) pistachio polyphenols-rich extracts was evaluated against HSV-1 using VERO cells. Two different extraction methods, with or without n-hexane, were used. Results showed that n-hexane-extracted NRRE and RURE exerted an antiviral effect against HSV-1, blocking virus binding on the cell surface, affecting viral DNA synthesis as well as accumulation of ICP0, UL42, and Us11 viral proteins. Additionally, the identification and quantification of phenolic compounds by RP-HPLC-DAD confirmed that extraction with n-hexane exclusively accumulated tocopherols, carotenoids, and xanthophylls. Amongst these, zeaxanthin exhibited strong antiviral activity against HSV-1 (CC50: 16.1 µM, EC50 4.08 µM, SI 3.96), affecting both the viral attachment and penetration and viral DNA synthesis. Zeaxanthin is a dietary carotenoid that accumulates in the retina as a macular pigment. The use of pistachio extracts and derivates should be encouraged for the topical treatment of ocular herpetic infections.

Punica granatum is a rich source of bioactive compounds which exhibit various biological effects. In this study, pomegranate peel and leaf ethanolic crude extracts (PPE and PLE, respectively) were phytochemically characterized and screened for antioxidant, antimicrobial and antiviral activity. LC-PDA-ESI-MS analysis led to the identification of different compounds, including ellagitannins, flavonoids and phenolic acids. The low IC50 values, obtained by DPPH and FRAP assays, showed a noticeable antioxidant effect of PPE and PLE comparable to the reference standards. Both crude extracts and their main compounds (gallic acid, ellagic acid and punicalagin) were not toxic on Vero cells and exhibited a remarkable inhibitory effect on herpes simplex type 1 (HSV-1) viral plaques formation. Specifically, PPE inhibited HSV-1 adsorption to the cell surface more than PLE. Indeed, the viral DNA accumulation, the transcription of viral genes and the expression of viral proteins were significantly affected by PPE treatment. Amongst the compounds, punicalagin, which is abundant in PPE crude extract, inhibited HSV-1 replication, reducing viral DNA and transcripts accumulation, as well as proteins of all three phases of the viral replication cascade. In contrast, no antibacterial activity was detected. In conclusion, our findings indicate that Punica granatum peel and leaf extracts, especially punicalagin, could be a promising therapeutic candidate against HSV-1.

The combination of cyclodextrins (CD) with graphene-based nanomaterials (GBN) has been widely investigated to obtain graphene/cyclodextrin nanomaterials (GCD) endowed with unique relationships with biomolecules and/or living cells. However, their development in nanomedicine is still confined in the laboratory arena due to the lack of in vivo studies and safety data. Here, we report the synthesis and physicochemical properties of a new fluorescent graphene platform obtained by supramolecular assembly of cyclodextrin-functionalized graphene (GCD) and adamantanyl-rhodamine derivative (AdaRhod-2). The safety issues of GCD were assessed by in vitro cell culture and in vivo by a zebrafish embryo toxicity test (ZFET) to evaluate possible toxicity and pathological lesions of zebrafish (ZF) exposed to GCD@AdaRhod-2 at two selected concentrations 4 mg/L and 80 mg/L. GCD@AdaRhod-2 showed a reduced potential risk with respect to other GBN with a survival rate percentage more than 80% for both investigated concentrations. Finally, the fate of GCD@AdaRhod-2 during ZF embryo development was monitored by confocal microscopy images.

The HSV-1 tegument protein Us11 counteracts the antiviral defense mechanisms by precluding the host protein shutoff. Previous works demonstrated that Us11 prevents heat-and staurosporine-induced apoptosis and inhibits autophagy. Therefore, in the present study, we investigated the hypothesis that HSV-1, through Us11, could recruit caspase-8, a key enzyme regulating programmed cell death. We first show that HSV-1 promotes the accumulation of caspase-8-p18 active fragments in both semi permissive THP-1 cells and fully permissive HEp-2 cells to HSV-1 replication. Using a recombinant virus R3630 (ΔUs11/ΔUs12) and a plasmid encoding Us11-recombinant protein we have proven that Us11 promotes p18 accumulation, which does not trigger the apoptotic signaling. Additional, in an in vitro model, we demonstrated that Us11-recombinant protein induces caspase-8-p18 cleavage by physically interacting with the caspase-8 recombinant protein. Finally, we found that, during HSV-1 replication, activated-caspase-8 cleaves Atg3 protein to potentially block autophagy and support its replication.

In the last decade, nanotechnological progress has generated new opportunities to improve the safety and efficacy of conventional anticancer therapies. Compared with other carriers, graphene nanoplatforms possess numerous tunable functionalities for the loading of multiple bioactive compounds, although their biocompatibility is still a debated concern. Recently, we have investigated the modulation of genes involved in cancer-associated canonical pathways induced by graphene engineered with cyclodextrins (GCD). Here, we investigated the GCD impact on cells safety, the HEp-2 responsiveness to Doxorubicin (DOX) and the cancer-related intracellular signalling pathways modulated by over time exposure to DOX loaded on GCD (GCD@DOX). Our studies evidenced that both DOX and GCD@DOX induced p53 and p21 signalling resulting in G0/G1 cell cycle arrest. A genotoxic behaviour of DOX was reported via detection of CDK (T14/Y15) activation and reduction of Wee-1 expression. Similarly, we found a cleavage of PARP by DOX within 72 h of exposure. Conversely, GCD@DOX induced a late cleavage of PARP, which could be indicative of less toxic effect due to controlled release of the drug from the GCD nanocarrier. Finally, the induction of the autophagy process supports the potential recycling of DOX with the consequent limitation of its toxic effects. Together, these findings demonstrate that GCD@DOX is a biocompatible drug delivery system able to evade chemoresistance and doxorubicin toxicity.

Olea europaea L. var. sativa (OESA) preparations are widely used in traditional medicine in the Mediterranean region to prevent and treat different diseases. In this research, olive extracts derived from the leaves of the OESA tree have been screened for antioxidant activity by two methods: the DPPH free radical scavenging assay (DPPH) and the Ferric reducing antioxidant power (FRAP) assay. The DPPH assay showed that OESA possesses a stronger antioxidant activity (84%) at 1 mg/mL while the FRAP method showed a strong metal ion chelating activity (90%) at 1 mg/mL. The low IC50 values, obtained by two different methods, implies that OESA has a noticeable effect on scavenging free radicals comparable to standards. During EBV infection, the free radicals increased triggering lipid oxidation. Therefore, the monitoring of the secondary lipid peroxidation products was done by measuring malonaldehyde (MDA) and conjugated dienes (DC). The simultaneous treatment of Raji cells with OESA and TPA, as an inductorof the lytic cycle, generated a significant decrease in MDA levels and DC (p < 0.05). Besides, Raji cells simultaneously exposed to TPA and OESA exhibited a percentage of EBV-positive fluorescence cells lower than TPA treated cells (**** p < 0.0001). This suggests that OESA treatment has a protective effect against EBV lytic cycle induction.

Owing to the richness of bioactive compounds, Olea europea leaf extracts exhibit a range of health effects. The present research evaluated the antibacterial and antiviral effect of leaf extracts obtained from Olea europea L. var. sativa (OESA) and Olea europea var. sylvestris (OESY) from Tunisia. LC-DAD-ESI-MS analysis allowed the identification of different compounds that contributed to the observed biological properties. Both OESA and OESY were active against Gram-positive bacteria (MIC values between 7.81 and 15.61 μg/mL and between 15.61 and 31.25 μg/mL against Staphylococcus aureus ATCC 6538 for OESY and OESA, respectively). The antiviral activity against the herpes simplex type 1 (HSV-1) was assessed on Vero cells. The results of cell viability indicated that Olea europea leaf extracts were not toxic to cultured Vero cells. The half maximal cytotoxic concentration (CC50) values for OESA and OESY were 0.2 mg/mL and 0.82 mg/mL, respectively. Furthermore, both a plaque reduction assay and viral entry assay were used to demonstrate the antiviral activity. In conclusion, Olea europea leaf extracts demonstrated a bacteriostatic effect, as well as remarkable antiviral activity, which could provide an alternative treatment against resistant strains.

Viral infections are responsible for several chronic and acute diseases in both humans and animals. Despite the incredible progress in human medicine, several viral diseases, such as acquired immunodeficiency syndrome, respiratory syndromes, and hepatitis, are still associated with high morbidity and mortality rates in humans. Natural products from plants or other organisms are a rich source of structurally novel chemical compounds including antivirals. Indeed, in traditional medicine, many pathological conditions have been treated using plant-derived medicines. Thus, the identification of novel alternative antiviral agents is of critical importance. In this review, we summarize novel phytochemicals with antiviral activity against human viruses and their potential application in treating or preventing viral disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

To replicate, spread and persist in the host environment, viruses have evolved several immunological escape mechanisms via the action of specific viral proteins. The model “host shut off” adopted by virion host shut off (VHS) protein of Herpes simplex type 1 (HSV-1) represents an immune evasion mechanism which affects the best-characterized component of the innate immunological response, protein kinase R (PKR). However, up to now, the real mechanism employed by VHS to control PKR is still unknown. In this paper, we implement and extend our previous findings reporting that wild-type HSV-1 is able to control PKR, whereas a VHS mutant virus (R2621) clearly induces an accumulation of phosphorylated PKR in several cell types in a VHS-RNase activity-dependent manner. Furthermore, we demonstrate for the first time a new PKR-regulatory mechanism based on the involvement of Us3 and UL13 tegument viral proteins. The combined approach of transfection and infection assay was useful to discover the new role of both viral proteins in the immunological escape and demonstrate that Us3 and UL13 control the accumulation of the phosphorylated form (ph-PKR). Lastly, since protein kinases are tightly regulated by phosphorylation events and, at the same time, phosphorylate other proteins by inducing post-translational modifications, the interplay between Us3 and VHS during HSV-1 infection has been investigated. Interestingly, we found that VHS protein accumulates at higher molecular weight following Us3 transfection, suggesting an Us3-mediated phosphorylation of VHS. These findings reveal a new intriguing interplay between viral proteins during HSV-1 infection involved in the regulation of the PKR-mediated immune response.