New Highly Selective Antivirals for Chikungunya Virus identified from the Screening of a Drug-Like Compound Library.

Chikungunya fever is a mosquito-borne disease caused by Chikungunya virus (CHIKV). Treatment of CHIKV infections is currently supportive and does not limit viral replication or symptoms of persistent chronic arthritis. Although there are multiple compounds reported as antivirals active against CHIKV in vitro, there are still no effective and safe antivirals. Thus, active research aims at the identification of new chemical structures with antiviral activity. Here, we report the screen of the Pandemic Response Box library of small molecules against a fully infectious CHIKV reporter virus. Our screening approach successfully identified previously reported CHIKV antiviral compounds within this library and further expanded potentially active hits, supporting the use of reporter-virus-based assays in high-throughput screening format as a reliable tool for antiviral drug discovery. Four molecules were identified as potential drug candidates against CHIKV: MMV1634402 (Brilacidin) and MMV102270 (Diphyllin), which were previously shown to present broad-spectrum antiviral activities, in addition to MMV1578574 (Eravacycline), and the antifungal MMV689401 (Fluopicolide), for which their antiviral potential is uncovered here.

ß-enaminoester derivatives exhibit promising in vitro and in silico antiviral potential against Mayaro virus.

Mayaro virus (MAYV) is the causative agent of Mayaro fever, which is characterized mainly by acute fever and long-term severe arthralgia, common manifestations of other arbovirus infections, making the correct diagnosis a challenge. Besides, MAYV infections have been reported in South America, especially in Brazil. However, the lack of vaccines or specific antiviral drugs to control these infections makes the search for new antivirals an urgent need. Herein, we evaluated the antiviral potential of synthetic ß-enaminoesters derivatives against MAYV replication and their pharmacokinetic and toxicological (ADMET) properties using in vitro and in silico strategies. For this purpose, Vero cells were infected with MAYV at an MOI of 0.1, treated with compounds (50 µM) for 24 h, and virus titers were quantified by plaque reduction assays. Compounds 2b (83.33%) and 2d (77.53%) exhibited the highest activity with inhibition rates of 83.33% and 77.53%, respectively. The most active compounds 2b (EC50 = 18.92 µM; SI > 52.85), and 2d (EC50 = 14.52 µM; SI > 68.87) exhibited higher potency and selectivity than the control drug suramin (EC50 = 38.97 µM; SI > 25.66). Then, we investigated the mechanism of action of the most active compounds. None of the compounds showed virucidal activity, neither inhibited virus adsorption, but compound 2b inhibited virus entry (62.64%). Also, compounds 2b and 2d inhibited some processes involved with the release of new virus particles. Finally, in silico results indicated good ADMET parameters of the most active compounds and reinforced their promising profile as drug candidates against MAYV.

Characterization and Promising in vitro Antiherpetic Effect of Galactomannan from Delonix regia Seeds.

Herpes simplex virus (HSV) infections can occur throughout life, thereby allowing transmission to new hosts, with an impact on public health. Acyclovir remains the treatment of choice for these infections; however, an increase in resistant strains in recent years has been observed. In this study, the activity of a native Delonix regia galactomannan (NDr) against HSV-1 was investigated in vitro. NDr was characterized using infrared spectroscopy and NMR. Evaluation of cytotoxicity and the antiviral effect was determined, respectively, by MTT and plaque reduction assays. The NDr concentrations that inhibited cell viability (CC50) and viral infection (IC50) by 50% were above 2000 and 64 µg/mL, respectively. Thus, the polysaccharide showed a high selectivity index (> 31.25). When NDr was added at different stages of HSV-1 replication, a strong inhibitory effect was found by direct interaction with the virus (71-67%, virucidal effect) or previously with the cell, 6 h before infection (99.8-68.4%, prophylactic effect) at concentrations from 200 to 50 µg/mL. NDr showed similar effects in prophylactic 1 h (52%) and adsorption inhibition (55%) assays at 200 µg/mL. A reduction in the antiherpetic effect was observed after infection. These results suggest that NDr is effective in the early stages of HSV-1 infection and is a promising agent for controlling herpetic infections.

SARS-CoV-2 Modulation of HIV Latency Reversal in a Myeloid Cell Line: Direct and Bystander Effects.

Coronavirus disease 2019 (COVID-19) might impact disease progression in people living with HIV (PLWH), including those on effective combination antiretroviral therapy (cART). These individuals often experience chronic conditions characterized by proviral latency or low-level viral replication in CD4+ memory T cells and tissue macrophages. Pro-inflammatory cytokines, such as TNF-α, IL-1ß, IL-6, and IFN-γ, can reactivate provirus expression in both primary cells and cell lines. These cytokines are often elevated in individuals infected with SARS-CoV-2, the virus causing COVID-19. However, it is still unknown whether SARS-CoV-2 can modulate HIV reactivation in infected cells. Here, we report that exposure of the chronically HIV-1-infected myeloid cell line U1 to two different SARS-CoV-2 viral isolates (ancestral and BA.5) reversed its latent state after 24 h. We also observed that SARS-CoV-2 exposure of human primary monocyte-derived macrophages (MDM) initially drove their polarization towards an M1 phenotype, which shifted towards M2 over time. This effect was associated with soluble factors released during the initial M1 polarization phase that reactivated HIV production in U1 cells, like MDM stimulated with the TLR agonist resiquimod. Our study suggests that SARS-CoV-2-induced systemic inflammation and interaction with macrophages could influence proviral HIV-1 latency in myeloid cells in PLWH.

Exploring the antiviral activities of the FDA-approved drug sulfadoxine and its derivatives against Chikungunya virus.

BACKGROUND: Currently, there is no antiviral licensed to treat chikungunya fever, a disease caused by the infection with Alphavirus chikungunya (CHIKV). Treatment is based on analgesic and anti-inflammatory drugs to relieve symptoms. Our study aimed to evaluate the antiviral activity of sulfadoxine (SFX), an FDA-approved drug, and its derivatives complexed with silver(I) (AgSFX), salicylaldehyde Schiff base (SFX-SL), and with both Ag and SL (AgSFX-SL) against CHIKV. METHODS: The anti-CHIKV activity of SFX and its derivatives was investigated using BHK-21 cells infected with CHIKV-nanoluc, a marker virus-carrying nanoluciferase reporter. Dose-response and time of drug-addition assays were performed in order to assess the antiviral effects of the compounds, as well as in silico data and ATR-FTIR analysis for insights on their mechanisms of action. RESULTS: The SFX inhibited 34% of CHIKV replication, while AgSFX, SFX-SL, and AgSFX-SL enhanced anti-CHIKV activity to 84%, 89%, and 95%, respectively. AgSFX, SFX-SL, and AgSFX-SL significantly decreased viral entry and post-entry to host cells, and the latter also protected cells against infection. Additionally, molecular docking calculations and ATR-FTIR analysis demonstrated interactions of SFX-SL, AgSFX, and AgSFX-SL with CHIKV. CONCLUSIONS: Collectively, our findings suggest that the addition of metal ions and/or Schiff base to SFX improved its antiviral activity against CHIKV.

Evaluation of Thiazolidine Derivatives with Potential Anti-ZIKV Activity.

OBJECTIVE: In this study, we have synthesized 19 Thiazolidine (TZD) derivatives to investigate their potential anti-ZIKV effects. METHODS: Nineteen thiazolidine derivatives were synthesized and evaluated for their cytotoxicity and antiviral activity against the ZIKA virus. RESULTS: Among them, six demonstrated remarkable selectivity against the ZIKV virus, exhibiting IC50 values of <5µM, and the other compounds did not demonstrate selectivity for the virus. Interestingly, several derivatives effectively suppressed the replication of ZIKV RNA copies, with derivatives significantly reducing ZIKV mRNA levels at 24 hours post-infection (hpi). Notably, two derivatives (ZKC-4 and -9) stood out by demonstrating a protective effect against ZIKV cell entry. Informed by computational analysis of binding affinity and intermolecular interactions within the NS5 domain's N-7 and O'2 positions, ZKC-4 and FT-39 displayed the highest predicted affinities. Intriguingly, ZKC-4 and ZKC-9 derivatives exhibited the most favorable predicted binding affinities for the ZIKV-E binding site. CONCLUSION: The significance of TZDs as potent antiviral agents is underscored by these findings, suggesting that exploring TZD derivatives holds promise for advancing antiviral therapeutic strategies.

The Mechanism of Action of L-Tyrosine Derivatives against Chikungunya Virus Infection In Vitro Depends on Structural Changes.

Although the disease caused by chikungunya virus (CHIKV) is of great interest to public health organizations around the world, there are still no authorized antivirals for its treatment. Previously, dihalogenated anti-CHIKV compounds derived from L-tyrosine (dH-Y) were identified as being effective against in vitro infection by this virus, so the objective of this study was to determine the mechanisms of its antiviral action. Six dH-Y compounds (C1 to C6) dihalogenated with bromine or chlorine and modified in their amino groups were evaluated by different in vitro antiviral strategies and in silico tools. When the cells were exposed before infection, all compounds decreased the expression of viral proteins; only C4, C5 and C6 inhibited the genome; and C1, C2 and C3 inhibited infectious viral particles (IVPs). Furthermore, C1 and C3 reduce adhesion, while C2 and C3 reduce internalization, which could be related to the in silico interaction with the fusion peptide of the E1 viral protein. Only C3, C4, C5 and C6 inhibited IVPs when the cells were exposed after infection, and their effect occurred in late stages after viral translation and replication, such as assembly, and not during budding. In summary, the structural changes of these compounds determine their mechanism of action. Additionally, C3 was the only compound that inhibited CHIKV infection at different stages of the replicative cycle, making it a compound of interest for conversion as a potential drug.

The GA-Hecate Peptide inhibits the ZIKV Replicative Cycle in Different Steps and can Inhibit the Flavivirus NS2B-NS3 Protease after Cell Infection.

BACKGROUND: Peptide drugs are advantageous because they are subject to rational design and exhibit highly diverse structures and broad biological activities. The NS2B-NS3 protein is a particularly promising flavivirus therapeutic target, with extensive research on the development of inhibitors as therapeutic candidates, and was used as a model in this work to determine the mechanism by which GA-Hecate inhibits ZIKV replication. OBJECTIVE: The present study aimed to evaluate the potential of GA-Hecate, a new antiviral developed by our group, against the Brazilian Zika virus and to evaluate the mechanism of action of this compound on the flavivirus NS2B-NS3 protein. METHODS: Solid-phase peptide Synthesis, High-Performance Liquid Chromatography, and Mass Spectrometry were used to obtain, purify, and characterize the synthesized compound. Real-time and enzymatic assays were used to determine the antiviral potential of GA-Hecate against ZIKV. RESULTS: The RT-qPCR results showed that GA-Hecate decreased the number of ZIKV RNA copies in the virucidal, pre-treatment, and post-entry assays, with 5- to 6-fold fewer RNA copies at the higher nontoxic concentration in Vero cells (HNTC: 10 µM) than in the control cells. Enzymatic and kinetic assays indicated that GA-Hecate acts as a competitive ZIKV NS2B-NS3 protease inhibitor with an IC50 of 32 nM and has activity against the yellow fever virus protease. CONCLUSION: The results highlight the antiviral potential of the GA-Hecate bioconjugate and open the door for the development of new antivirals.

Zika virus infection suppresses CYP24A1 and CAMP expression in human monocytes.

Monocytes are the primary targets of Zika virus (ZIKV) and are associated with ZIKV pathogenesis. Currently, there is no effective treatment for ZIKV infection. It is known that 1,25-dihydroxy vitamin D3 (VitD3) has strong antiviral activity in dengue virus-infected macrophages, but it is unknown whether VitD3 inhibits ZIKV infection in monocytes. We investigated the relationship between ZIKV infection and the expression of genes of the VitD3 pathway, as well as the inflammatory response of infected monocytes in vitro. ZIKV replication was evaluated using a plaque assay, and VitD3 pathway gene expression was analyzed by RT-qPCR. Pro-inflammatory cytokines/chemokines were quantified using ELISA. We found that VitD3 did not suppress ZIKV replication. The results showed a significant decrease in the expression of vitamin D3 receptor (VDR), cytochrome P450 family 24 subfamily A member 1 (CYP24A1), and cathelicidin antimicrobial peptide (CAMP) genes upon ZIKV infection. Treatment with VitD3 was unable to down-modulate production of pro-inflammatory cytokines, except TNF-α, and chemokines. This suggests that ZIKV infection inhibits the expression of VitD3 pathway genes, thereby preventing VitD3-dependent inhibition of viral replication and the inflammatory response. This is the first study to examine the effects of VitD3 in the context of ZIKV infection, and it has important implications for the role of VitD3 in the control of viral replication and inflammatory responses during monocyte infection.

3-Chymotrypsin-like Protease (3CLpro) of SARS-CoV-2: Validation as a Molecular Target, Proposal of a Novel Catalytic Mechanism, and Inhibitors in Preclinical and Clinical Trials.

Proteases represent common targets in combating infectious diseases, including COVID-19. The 3-chymotrypsin-like protease (3CLpro) is a validated molecular target for COVID-19, and it is key for developing potent and selective inhibitors for inhibiting viral replication of SARS-CoV-2. In this review, we discuss structural relationships and diverse subsites of 3CLpro, shedding light on the pivotal role of dimerization and active site architecture in substrate recognition and catalysis. Our analysis of bioinformatics and other published studies motivated us to investigate a novel catalytic mechanism for the SARS-CoV-2 polyprotein cleavage by 3CLpro, centering on the triad mechanism involving His41-Cys145-Asp187 and its indispensable role in viral replication. Our hypothesis is that Asp187 may participate in modulating the pKa of the His41, in which catalytic histidine may act as an acid and/or a base in the catalytic mechanism. Recognizing Asp187 as a crucial component in the catalytic process underscores its significance as a fundamental pharmacophoric element in drug design. Next, we provide an overview of both covalent and non-covalent inhibitors, elucidating advancements in drug development observed in preclinical and clinical trials. By highlighting various chemical classes and their pharmacokinetic profiles, our review aims to guide future research directions toward the development of highly selective inhibitors, underscore the significance of 3CLpro as a validated therapeutic target, and propel the progression of drug candidates through preclinical and clinical phases.

Antiviral Activities of Mastoparan-L-Derived Peptides against Human Alphaherpesvirus 1.

Human alphaherpesvirus 1 (HSV-1) is a significantly widespread viral pathogen causing recurrent infections that are currently incurable despite available treatment protocols. Studies have highlighted the potential of antimicrobial peptides sourced from Vespula lewisii venom, particularly those belonging to the mastoparan family, as effective against HSV-1. This study aimed to demonstrate the antiviral properties of mastoparans, including mastoparan-L [I5, R8], mastoparan-MO, and [I5, R8] mastoparan, against HSV-1. Initially, Vero cell viability was assessed in the presence of these peptides, followed by the determination of antiviral activity, mechanism of action, and dose-response curves through plaque assays. Structural analyses via circular dichroism and nuclear magnetic resonance were conducted, along with evaluating membrane fluidity changes induced by [I5, R8] mastoparan using fluorescence-labeled lipid vesicles. Cytotoxic assays revealed high cell viability (>80%) at concentrations of 200 µg/mL for mastoparan-L and mastoparan-MO and 50 µg/mL for [I5, R8] mastoparan. Mastoparan-MO and [I5, R8] mastoparan exhibited over 80% HSV-1 inhibition, with up to 99% viral replication inhibition, particularly in the early infection stages. Structural analysis indicated an α-helical structure for [I5, R8] mastoparan, suggesting effective viral particle disruption before cell attachment. Mastoparans present promising prospects for HSV-1 infection control, although further investigation into their mechanisms is warranted.

Antiviral Activity of Kappaphycus alvarezii Seaweed against ZIKV.

INTRODUCTION: Zika virus (ZIKV) is a flavivirus transmitted through the bites of infected Aedes mosquitoes. These viruses can also be transmitted through sexual contact, vertical transmission, and possibly transfusion. Most cases are asymptomatic, but symptoms can include rash, conjunctivitis, fever, and arthralgia, which are characteristic of other arboviruses. Zika infection can lead to complications such as microcephaly, miscarriage, brain abnormalities, and Guillain-Barré syndrome (GBS). OBJECTIVE: The aim is to determine the inhibitory potential of the algae Kappaphycus alvarezii (K. alvarezii) on ZIKV replication. METHODOLOGY: Cytotoxicity experiments were performed using Vero cells to determine the CC50, and ZIKV replication inhibition assays (ATCC® VR-1839™) were conducted to determine the EC50. The mechanism of action was also studied to assess any synergistic effect with Ribavirin. RESULTS: K. alvarezii demonstrated low toxicity with a CC50 of 423 µg/mL and a potent effect on ZIKV replication with an EC50 of 0.65 µg/mL and a Selectivity Index (SI) of 651, indicating the extract's safety. Virucidal effect assays were carried out to evaluate the possible mechanism of action, and the compound addition time was studied, showing the potential to delay the treatment of infected cells by up to 6 hours. A potential synergistic effect was observed when K. alvarezii extract was combined with suboptimal concentrations of Ribavirin, resulting in 99% inhibition of viral replication. CONCLUSION: Our data demonstrate the significant potential of K. alvarezii extract and highlight the need for further studies to investigate its mechanism of action. We propose this extract as a potential anti-Zika compound.

The infusion of Baccharis spicata (Lam. ) Baill (Asteraceae) has antiviral activity against hepatitis B virus without cytotoxic effects against several cell lines / La infusión de Baccharis spicata (Lam. ) Baill (Asteraceae) posee actividad antiviral contra el virus de la hepatitis B sin efectos citotóxicos contra varias líneas celulares

Medicinal plants are used to cure diseases, and their replacement is frequent and affects public health. The genus Baccharis has representatives within the medicinal flora of Argentina, although the replacement of the species of this genus known under the vulgar name of "carqueja" by Baccharis spicata has been detected i n herbalists or markets of herbal products. The genotoxic safety of this species has been established in previous work of our group. The aim of this study was to evaluate the antiviral activity of an infusion made from B. spicata leaves against hepatitis B virus with the HepG2.2.15 cellular system and to determine cytotoxicity in HepG2.2,15, A549 and Vero cell lines. Infusion of B. spicata was active to inhibit HBV replication with an EC 50 of 22.54 µg/mL and a CC 50 of 190 µg/mL.

Las plantas medicinales son empleadas para la cura de enfermedades, y su sustituc ión es frecuente y afecta a la salud pública. El género Baccharis posee representantes dentro de la flora medicinal de Argentina, aunque se ha detectado la sustitución de las especies de dicho género conocidas bajo el nombre vulgar de "carqueja" por Baccha ris spicata en herboristerías o mercados de productos herb arios . Se ha establecido la seguridad genotóxica de esta especie en trabajos previos de nuestro grupo. Este estudio buscó evaluar la actividad antiviral de una infusión elaborada a partir de hojas de B. spicata frente al virus de la hepatitis B con el sistema celular HepG2.2.15 y determinar la citotoxicidad en las líneas celulares HepG2.2.15, A549 y Vero. La infusión de B. spicata fue activa para inhibir la replicación del virus con un EC 50 de 22.54 µg/mL y un CC 50 de 190 µg/mL.

Essential oil of Cordia curassavica (Jacq) Roem. & Schult: in vitro and in silico evaluation of effects on dengue virus replication and cytokines production / Aceite esencial de Cordia curassavica (Jacq) Roem. & Schult: evaluación in vitro e in silico del efecto sobre la replicación del virus dengue y la producción de citoquinas

The lack of effective conventional therapie s against dengue has created an interest in herbal preparations as alternative therapies. In the present study, in vitro effects of Cordia curassavica essential oil (EO) on both dengue virus replication and cytokine production were examined. Predictions of molecular interactions between EO compounds and virus and cell proteins were performed with AutoDock Vina. The EO inhibited replication of dengue virus serotypes at IC 50 < 30 µg/mL, and it reduced 87% TNF - α, 67% IL - 8 and 46% IFN - α in LPS - stimulated PBMCs. The main EO compounds were trans - ß - caryophyllene (21.4%), germacrene D (17.8%), α - copaene (16.5%), trans - ß - guaiene (8.2%), and α - pinene (6.0%). The first two compounds, δ - cadinene, α - muurolene, α - cubebene and ß - burbonene were coupled to proteins involved in the TLR - 4 cytokine effector pathway. 3,7 - Guaiadiene was coupled to the viral E and C proteins. This study demonstrates the potential of C. curassavica EO as a starting point for discovering novel therapeutic for dengue.

La falta de terapias eficaces para el dengue ha suscitado interés por preparados herbales como terapias alternativas. En el presente estudio se examinaron efectos in vitro del aceite e sencial (AE) de Cordia curassavica sobre la replicación del virus dengue y producción de citoquinas. Se realizaron predicciones de interacciones moleculares entre los compuestos del AE y proteínas virales y celulares con AutoDock Vina. El AE inhibió la rep licación de serotipos del virus a CI 50 < 30 µg/mL y redujo 87% TNF - α, 67% IL - 8 y 46% IFN - α en MNCP. Los principales compuestos del AE fueron trans - ß - cariofileno, germacreno D, α - copaeno, trans - ß - guaieno y α - pineno. Los dos primeros compuestos, el δ - cadineno, el α - muuroleno, el α - cubebeno y el ß - burboneno se acoplaron a proteínas implicadas en la vía efectora de citoquinas TLR - 4. El 3,7 - guaiadiene se acopló a las proteínas virales E y C. Este estudio demuestra el potencial del AE de C. curassavica como punto de partida para descubrir nuevas tera pias para el dengue.

Larrea divaricata a native Argentine plant with potential activity against SARS-CoV-2? / ¿Larrea divaricata una planta nativa argentina con actividad potencial sobre SARS-CoV-2?

Larrea divaricata Cav. is an autochthonous South American plant popularly used in inflammatory and infectious diseases with reported anti - inflammatory, immunomodulatory, antimicrobial and antioxidant activities. Covid - 19 is an infection ca used by the severe acute respiratory syndrome coronavirus 2 (SARS - CoV - 2). This virus can cause pneumonia and even death in about 5% of the cases. The objective of the article was to demonstrate, through a literature review, that L. divaricata has sufficie nt attributes to be assayed against SARS - CoV - 2. For this, the chemical composition, reported activities and docking studies were taken into account. This review demonstrated that the plant extracts are capable of inhibiting the proliferation of fungi, bact eria and viruses and that they exert anti - inflammatory and immunomodulatory actions in different " in vitro " and " in vivo " models. These results suggest that the plant is a good candidate to be studied for the prevention and/or treatment of SARS - CoV - 2.

Larrea divaricata Cav. es una planta autóctona Sudamericana, utilizada popularmente en enfermedades inflamatorias e infecciosas, con activida d anti - inflamatoria, inmunomoduladora, antimicrobiana y antioxidante reportada. El Covid - 19 es una infección causada por una cepa de coronavirus, SARS - CoV - 2 (coronavirus tipo 2 causante del síndrome respiratorio agudo severo). Este virus puede originar neu monía e incluso la muerte en alrededor del 5% de los casos. Nuestro objetivo fue demostrar, a través de una revisión bibliográfica, que esta planta tiene atributos suficientes para ser ensayada en estudios contra SARS - CoV - 2. Se tuvo en cuenta la composici ón química, los antecedentes científicos y los estudios de acoplamiento molecular. Esta revisión permitió demostrar que extractos de la planta son capaces de inhibir la proliferación de hongos, bacterias y virus y que presentan acción anti - inflamatoria en diferentes modelos " in vitro " e " in vivo ", lo que los hace candidatos a ser estudiados en la prevención y/o tratamiento de la infección contra SARS - CoV - 2.

Deubiquitinating Enzyme Inhibitors Block Chikungunya Virus Replication.

Ubiquitination and deubiquitination processes are widely involved in modulating the function, activity, localization, and stability of multiple cellular proteins regulating almost every aspect of cellular function. Several virus families have been shown to exploit the cellular ubiquitin-conjugating system to achieve a productive infection: enter the cell, promote genome replication, or assemble and release viral progeny. In this study, we analyzed the role of deubiquitinating enzymes (DUBs) during chikungunya virus (CHIKV) infection. HEK293T, Vero-E6, and Huh-7 cells were treated with two DUB inhibitors (PR619 or WP1130). Then, infected cells were evaluated by flow cytometry, and viral progeny was quantified using the plaque assay method. The changes in viral proteins and viral RNA were analyzed using Western blotting and RT-qPCR, respectively. Results indicate that treatment with DUB inhibitors impairs CHIKV replication due to significant protein and viral RNA synthesis deregulation. Therefore, DUB activity may be a pharmacological target for blocking CHIKV infection.

Simvastatin Downregulates the SARS-CoV-2-Induced Inflammatory Response and Impairs Viral Infection Through Disruption of Lipid Rafts.

Coronavirus disease 2019 (COVID-19) is currently a worldwide emergency caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In observational clinical studies, statins have been identified as beneficial to hospitalized patients with COVID-19. However, experimental evidence of underlying statins protection against SARS-CoV-2 remains elusive. Here we reported for the first-time experimental evidence of the protective effects of simvastatin treatment both in vitro and in vivo. We found that treatment with simvastatin significantly reduced the viral replication and lung damage in vivo, delaying SARS-CoV-2-associated physiopathology and mortality in the K18-hACE2-transgenic mice model. Moreover, simvastatin also downregulated the inflammation triggered by SARS-CoV-2 infection in pulmonary tissue and in human neutrophils, peripheral blood monocytes, and lung epithelial Calu-3 cells in vitro, showing its potential to modulate the inflammatory response both at the site of infection and systemically. Additionally, we also observed that simvastatin affected the course of SARS-CoV-2 infection through displacing ACE2 on cell membrane lipid rafts. In conclusion, our results show that simvastatin exhibits early protective effects on SARS-CoV-2 infection by inhibiting virus cell entry and inflammatory cytokine production, through mechanisms at least in part dependent on lipid rafts disruption.

Infection of Endothelial Cells by Dengue Virus Induces ROS Production by Different Sources Affecting Virus Replication, Cellular Activation, Death and Vascular Permeability.

Exacerbated inflammatory response and altered vascular function are hallmarks of dengue disease. Reactive oxygen species (ROS) production has been associated to endothelial barrier disturbance and microvascular alteration in distinct pathological conditions. Increased ROS has been reported in in vitro models of dengue virus (DENV) infection, but its impact for endothelial cell physiology had not been fully investigated. Our group had previously demonstrated that infection of human brain microvascular endothelial cells (HBMEC) with DENV results in the activation of RNA sensors and production of proinflammatory cytokines, which culminate in cell death and endothelial permeability. Here, we evaluated the role of mitochondrial function and NADPH oxidase (NOX) activation for ROS generation in HBMEC infected by DENV and investigated whether altered cellular physiology could be a consequence of virus-induced oxidative stress. DENV-infected HBMECs showed a decrease in the maximal respiratory capacity and altered membrane potential, indicating functional mitochondrial alteration, what might be related to mtROS production. Indeed, mtROS was detected at later time points after infection. Specific inhibition of mtROS diminished virus replication, cell death, and endothelial permeability, but did not affect cytokine production. On the other hand, inhibition of NOX-associated ROS production decreased virus replication and cell death, as well as the secretion of inflammatory cytokines, including IL-6, IL-8, and CCL5. These results demonstrated that DENV replication in endothelial cells induces ROS production by different pathways, which impacts biological functions that might be relevant for dengue pathogenesis. Those data also indicate oxidative stress events as relevant therapeutical targets to avoid vascular permeability, inflammation, and neuroinvasion during DENV infection.

In Vitro Inhibition of Replication of Dengue Virus Serotypes 1-4 by siRNAs Bound to Non-Toxic Liposomes.

Dengue virus is a ssRNA+ flavivirus, which produces the dengue disease in humans. Currently, no specific treatment exists. siRNAs regulate gene expression and have been used systematically to silence viral genomes; however, they require controlled release. Liposomes show favorable results encapsulating siRNA for gene silencing. The objective herein was to design and evaluate in vitro siRNAs bound to liposomes that inhibit DENV replication. siRNAs were designed against DENV1-4 from conserved regions using siDirect2.0 and Web-BLOCK-iT™ RNAiDesigner; the initial in vitro evaluation was carried out through transfection into HepG2 cells. siRNA with silencing capacity was encapsulated in liposomes composed of D-Lin-MC3-DMA, DSPC, Chol. Cytotoxicity, hemolysis, pro-inflammatory cytokine release and antiviral activity were evaluated using plaque assay and RT-qPCR. A working concentration of siRNA was established at 40 nM. siRNA1, siRNA2, siRNA3.1, and siRNA4 were encapsulated in liposomes, and their siRNA delivery through liposomes led to a statistically significant decrease in viral titers, yielded no cytotoxicity or hemolysis and did not stimulate release of pro-inflammatory cytokines. Finally, liposomes were designed with siRNA against DENV, which proved to be safe in vitro.

Targeting Host PIM Protein Kinases Reduces Mayaro Virus Replication.

Mayaro virus (MAYV) manipulates cell machinery to successfully replicate. Thus, identifying host proteins implicated in MAYV replication represents an opportunity to discover potential antiviral targets. PIM kinases are enzymes that regulate essential cell functions and also appear to be critical factors in the replication of certain viruses. In this study we explored the consequences of PIM kinase inhibition in the replication of MAYV and other arboviruses. Cytopathic effects or viral titers in samples from MAYV-, Chikungunya-, Una- or Zika-infected cells treated with PIM kinase inhibitors were evaluated using an inverted microscope or plaque-forming assays. The expression of viral proteins E1 and nsP1 in MAYV-infected cells was assessed using an immunofluorescence confocal microscope or Western blot. Our results revealed that PIM kinase inhibition partially prevented MAYV-induced cell damage and also promoted a decrease in viral titers for MAYV, UNAV and ZIKV. The inhibitory effect of PIM kinase blocking was observed for each of the MAYV strains tested and also occurred as late as 8 h post infection (hpi). Finally, PIM kinase inhibition suppressed the expression of MAYV E1 and nsP1 proteins. Taken together, these findings suggest that PIM kinases could represent an antiviral target for MAYV and other arboviruses.