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SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment. In this paper, the hepatitis C virus inhibitors Pibrentasvir and Ombitasvir are found to inhibit the SARS-CoV-2 exonuclease and are shown to have therapeutic potential when combined with SARS-CoV-2 polymerase inhibitors in viral cell cultures.

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro , SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.

The continuous emergence of SARS-CoV-2 variants underscores the need for novel antiviral candidates. Hypericin (HY), a compound derived from Hypericum perforatum, exhibited potent in vitro activity against SARS-CoV-2 in Vero E6 cells, with low cytotoxicity (CC50 > 200 nM). HY showed no significant activity against Influenza A (H1N1) or dengue virus serotype 2, supporting its selective action. Antiviral effects were most evident when HY was administered post-infection, in a concentration-dependent manner, while cellular pretreatment or viral pre-incubation produced limited effects. Notably, HY also displayed virucidal activity, significantly reducing viral titers at 4 °C, 22 °C, and 37 °C. Combination treatments with remdesivir or nirmatrelvir enhanced antiviral efficacy by 50–70% relative to monotherapy, depending on compound concentration. Molecular simulations revealed stable interactions with conserved residues in RdRp and 3CLpro, suggesting a low risk of resistance. Together, these findings highlight the potential of HY as a selective antiviral and virucidal agent against SARS-CoV-2, particularly in combination regimens.

Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with leukopenia and uncontrolled inflammatory response in critically ill patients. A better comprehension of SARS-CoV-2-induced monocyte death is essential for the identification of therapies capable to control the hyper-inflammation and reduce viral replication in patients with 2019 coronavirus disease (COVID-19). Here, we show that SARS-CoV-2 engages inflammasome and triggers pyroptosis in human monocytes, experimentally infected, and from patients under intensive care. Pyroptosis associated with caspase-1 activation, IL-1ß production, gasdermin D cleavage, and enhanced pro-inflammatory cytokine levels in human primary monocytes. At least in part, our results originally describe mechanisms by which monocytes, a central cellular component recruited from peripheral blood to respiratory tract, succumb to control severe COVID-19.

Despite the fast development of vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still circulating and generating variants of concern (VoC) that escape the humoral immune response. In this context, the search for anti-SARS-CoV-2 compounds is still essential. A class of natural polyphenols known as flavonoids, frequently available in fruits and vegetables, is widely explored in the treatment of different diseases and used as a scaffold for the design of novel drugs. Therefore, herein we evaluate seven flavonoids divided into three subclasses, isoflavone (genistein), flavone (apigenin and luteolin) and flavonol (fisetin, kaempferol, myricetin, and quercetin), for COVID-19 treatment using cell-based assays and in silico calculations validated with experimental enzymatic data. The flavonols were better SARS-CoV-2 inhibitors than isoflavone and flavones. The increasing number of hydroxyl groups in ring B of the flavonols kaempferol, quercetin, and myricetin decreased the 50% effective concentration (EC50) value due to their impact on the orientation of the compounds inside the target. Myricetin and fisetin appear to be preferred candidates; they are both anti-inflammatory (decreasing TNF-α levels) and inhibit SARS-CoV-2 mainly by targeting the processability of the main protease (Mpro) in a non-competitive manner, with a potency comparable to the repurposed drug atazanavir. However, fisetin and myricetin might also be considered hits that are amenable to synthetic modification to improve their anti-SARS-CoV-2 profile by inhibiting not only Mpro, but also the 3′–5′ exonuclease (ExoN).

Orally available antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are necessary because of the continuous circulation of new variants that challenge immunized individuals. Because severe COVID-19 is a virus-triggered immune and inflammatory dysfunction, molecules endowed with both antiviral and anti-inflammatory activity are highly desirable. We identified here that kinetin (MB-905) inhibits the in vitro replication of SARS-CoV-2 in human hepatic and pulmonary cell lines. On infected monocytes, MB-905 reduced virus replication, IL-6 and TNFα levels. MB-905 is converted into its triphosphate nucleotide to inhibit viral RNA synthesis and induce error-prone virus replication. Coinhibition of SARS-CoV-2 exonuclease, a proofreading enzyme that corrects erroneously incorporated nucleotides during viral RNA replication, potentiated the inhibitory effect of MB-905. MB-905 shows good oral absorption, its metabolites are stable, achieving long-lasting plasma and lung concentrations, and this drug is not mutagenic nor cardiotoxic in acute and chronic treatments. SARS-CoV-2-infected hACE-mice and hamsters treated with MB-905 show decreased viral replication, lung necrosis, hemorrhage and inflammation. Because kinetin is clinically investigated for a rare genetic disease at regimens beyond the predicted concentrations of antiviral/anti-inflammatory inhibition, our investigation suggests the opportunity for the rapid clinical development of a new antiviral substance for the treatment of COVID-19. The search for antivirals against SARS-CoV-2 continue due to the emergence of variants of concerns, able to escape the vaccinal humoral response. In this work, authors pre-clinically explore the potential of kinetin against SARS-CoV-2, which could be used alone or in combination with other antivirals.

Siparuna glycycarpa occurs in the Amazon region, and some species of this genus are used in Brazilian folk medicine. A recent study showed the inhibitory effect of this species against influenza A(H1N1)pdm09 virus, and in order to acquire active fractions, a polar solvent system n-butanol-methanol-water (9:1:10, v/v) was selected and used for bioassay-guided fractionation of n-butanol extract by centrifugal partition chromatography (CPC). The upper phase was used as stationary phase and the lower phase as mobile (descending mode). Among the collected fractions, the ones coded SGA, SGC, SGD, and SGO showed the highest antiviral inhibition levels (above 74%) at 100 µg·mL−1 after 24 h of infection. The bioactive fractions chemical profiles were investigated by LC-HRMS/MS data in positive and negative ionization modes exploring the Global Natural Products Social Molecular Networking (GNPS) platform to build a molecular network. Benzylisoquinoline alkaloids were annotated in the fractions coded SGA, SGC, and SGD collected during elution step. Aporphine alkaloids, O-glycosylated flavonoids, and dihydrochalcones in SGO were acquired with the change of mobile phase from lower aqueous to upper organic. Benzylisoquinolinic and aporphine alkaloids as well as glycosylated flavonoids were annotated in the most bioactive fractions suggesting this group of compounds as responsible for antiviral activity.

SARS-CoV-2 induces major cellular lipid rearrangements, exploiting the host’s metabolic pathways to replicate. Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that control lipid metabolism. SREBP1 is associated with the regulation of fatty acids, whereas SREBP2 controls cholesterol metabolism, and both isoforms are associated with lipid droplet (LD) biogenesis. Here, we evaluated the effect of SREBP in a SARS-CoV-2–infected lung epithelial cell line (Calu-3). We showed that SARS-CoV-2 infection induced the activation of SREBP1 and SREBP2 and LD accumulation. Genetic knockdown of both SREBPs and pharmacological inhibition with the dual SREBP activation inhibitor fatostatin promote the inhibition of SARS-CoV-2 replication, cell death, and LD formation in Calu-3 cells. In addition, we demonstrated that SARS-CoV-2 induced inflammasome-dependent cell death by pyroptosis and release of IL-1β and IL-18, with activation of caspase-1, cleavage of gasdermin D1, was also reduced by SREBP inhibition. Collectively, our findings help to elucidate that SREBPs are crucial host factors required for viral replication and pathogenesis. These results indicate that SREBP is a host target for the development of antiviral strategies.

Influenza A virus (IAV) is one of the leading causes of respiratory tract infections in humans, representing a major public health concern. The various types of cell death have a crucial role in IAV pathogenesis because this virus may trigger both apoptosis and necroptosis in airway epithelial cells in parallel. Macrophages play an important role in the clearance of virus particles, priming the adaptive immune response in influenza. However, the contribution of macrophage death to pathogenesis of IAV infection remains unclear. In this work, we investigated IAV-induced macrophage death, along with potential therapeutic intervention. We conducted in vitro and in vivo experiments to evaluate the mechanism and the contribution of macrophages death to the inflammatory response induced by IAV infection. We found that IAV or its surface glycoprotein hemagglutinin (HA) triggers inflammatory programmed cell death in human and murine macrophages in a Toll-like receptor-4 (TLR4)- and TNF-dependent manner. Anti-TNF treatment in vivo with the clinically approved drug etanercept prevented the engagement of the necroptotic loop and mouse mortality. Etanercept impaired the IAV-induced proinflammatory cytokine storm and lung injury. In summary, we demonstrated a positive feedback loop of events that led to necroptosis and exacerbated inflammation in IAV-infected macrophages. Our results highlight an additional mechanism involved in severe influenza that could be attenuated with clinically available therapies.

Ilhéus Virus (ILHV) was first detected in 1944 in Ilhéus, state of Bahia, northeast Brazil. During cellular infection, orthoflaviviruses induce cellular changes related both to the replication process, the formation of replication complexes, and to structures resulting from cellular damage. Although more detailed data are available in the literature for other orthoflaviviruses, the relationship between ILHV, the formation of these structures, its replication cycle, and cellular changes remains unknown. One of the main objectives of this study is to characterize the primary ultrastructural changes in green monkey kidney epithelial cell lineage (Vero cell) infected with ILHV, as well as to map its replication cycle, virion structure, and genome. To achieve these objectives, Vero cell monolayers were infected with an MOI of 0.01 and collected at different times post-infection. Cell monolayers were evaluated under bright-field microscopy and transmission electron microscopy. Ultrastructural analyses confirmed that ILHV can induce the formation of double-membrane vesicles, convoluted membranes, and vesicular packets. These structures, like those observed in zika (ZIKV) and dengue (DENV) viruses, form replication complexes that aid ILHV’s replication process in cells. Our preliminary results reveal that ILHV infection induces cytopathogenesis like that observed in vitro studies for other arboviruses.