Novel high-yield potato protease inhibitor panels block a wide array of proteases involved in viral infection and crucial tissue damage.

Viruses critically rely on various proteases to ensure host cell entry and replication. In response to viral infection, the host will induce acute tissue inflammation pulled by granulocytes. Upon hyperactivation, neutrophil granulocytes may cause undue tissue damage through proteolytic degradation of the extracellular matrix. Here, we assess the potential of protease inhibitors (PI) derived from potatoes in inhibiting viral infection and reducing tissue damage. The original full spectrum of potato PI was developed into five fractions by means of chromatography and hydrolysis. Individual fractions showed varying inhibitory efficacy towards a panel of proteases including trypsin, chymotrypsin, ACE2, elastase, and cathepsins B and L. The fractions did not interfere with SARS-CoV-2 infection of Vero E6 cells in vitro. Importantly, two of the fractions fully inhibited elastin-degrading activity of complete primary human neutrophil degranulate. These data warrant further development of potato PI fractions for biomedical purposes, including tissue damage crucial to SARS-CoV-2 pathogenesis. KEY MESSAGES: Protease inhibitor fractions from potato differentially inhibit a series of human proteases involved in viral replication and in tissue damage by overshoot inflammation. Protease inhibition of cell surface receptors such as ACE2 does not prevent virus infection of Vero cells in vitro. Protease inhibitors derived from potato can fully inhibit elastin-degrading primary human neutrophil proteases. Protease inhibitor fractions can be produced at high scale (hundreds of thousands of kilograms, i.e., tons) allowing economically feasible application in lower and higher income countries.

Viral presence and immunopathology in a kidney transplant recipient with fatal COVID-19: a clinical autopsy report.

COVID-19 is of special concern to immunocompromised individuals, including organ transplant recipients. However, the exact implications of COVID-19 for the immunocompromised host remain unclear. Existing theories regarding this matter are controversial and mainly based on clinical observations. Here, the postmortem histopathology, immunopathology, and viral presence in various tissues of a kidney transplant recipient with COVID-19 were compared to those of 2 nontransplanted patients with COVID-19 matched for age, sex, length of intensive care unit stay, and admission period in the pandemic. None of the tissues of the kidney transplant recipient demonstrated the presence of SARS-CoV-2. In lung tissues of both controls, some samples showed viral positivity with high Ct values with quantitative reverse transcription polymerase chain reaction. The lungs of the kidney transplant recipient and controls demonstrated similar pathology, consisting of acute fibrinous and organizing pneumonia with thrombosis and an inflammatory response with T cells, B cells, and macrophages. The kidney allograft and control kidneys showed a similar pattern of interstitial lymphoplasmacytic infiltration. No myocarditis could be observed in the hearts of the kidney transplant recipient and controls, although all cases contained scattered lymphoplasmacytic infiltrates in the myocardium, pericardium, and atria. The brainstems of the kidney transplant recipient and controls showed a similar pattern of lymphocytic inflammation with microgliosis. This research report highlights the possibility that, based on the results obtained from this single case, at time of death, the immune response in kidney transplant recipients with long-term antirejection immunosuppression use prior to severe illness is similar to nontransplanted deceased COVID-19 patients.

Mitochondrial protein BNIP3 regulates Chikungunya virus replication in the early stages of infection.

Chikungunya virus (CHIKV) is a human pathogen causing outbreaks of febrile illness for which vaccines and specific treatments remain unavailable. Autophagy-related (ATG) proteins and autophagy receptors are a set of host factors that participate in autophagy, but have also shown to function in other unrelated cellular pathways. Although autophagy is reported to both inhibit and enhance CHIKV replication, the specific role of individual ATG proteins remains largely unknown. Here, a siRNA screen was performed to evaluate the importance of the ATG proteome and autophagy receptors in controlling CHIKV infection. We observed that 7 out of 50 ATG proteins impact the replication of CHIKV. Among those, depletion of the mitochondrial protein and autophagy receptor BCL2 Interacting Protein 3 (BNIP3) increased CHIKV infection. Interestingly, BNIP3 controls CHIKV independently of autophagy and cell death. Detailed analysis of the CHIKV viral cycle revealed that BNIP3 interferes with the early stages of infection. Moreover, the antiviral role of BNIP3 was found conserved across two distinct CHIKV genotypes and the closely related Semliki Forest virus. Altogether, this study describes a novel and previously unknown function of the mitochondrial protein BNIP3 in the control of the early stages of the alphavirus viral cycle.

Characterization of soluble TLR2 and CD14 levels during acute dengue virus infection.

Dengue virus infection results in a broad spectrum of diseases ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Hitherto, there is no consensus biomarker for the prediction of severe dengue disease in patients. Yet, early identification of patients who progress to severe dengue is pivotal for better clinical management. We have recently reported that an increased frequency of classical (CD14 ++CD16-) monocytes with sustained high TLR2 expression in acutely infected dengue patients correlates with severe dengue development. Here, we hypothesized that the relatively lower TLR2 and CD14 expression in mild dengue patients is due to the shedding of their soluble forms (sTLR2 and sCD14) and that these could be used as indicators of disease progression. Therefore, using commercial sandwich ELISAs, we evaluated the release of sTLR2 and sCD14 by peripheral blood mononuclear cells (PBMCs) in response to in vitro dengue virus (DENV) infection and assessed their levels in acute-phase plasma of 109 dengue patients. We show that while both sTLR2 and sCD14 are released by PBMCs in response to DENV infection in vitro, their co-circulation in an acute phase of the disease is not always apparent. In fact, sTLR2 was found only in 20% of patients irrespective of disease status. In contrast, sCD14 levels were detected in all patients and were significantly elevated in DF patients when compared to DHF patients and age-matched healthy donors. Altogether, our results suggest that sCD14 may help in identifying patients at risk of severe dengue at hospital admittance.

Colocalization of Chikungunya Virus with Its Receptor MXRA8 during Cell Attachment, Internalization, and Membrane Fusion.

Arthritogenic alphaviruses, including chikungunya virus (CHIKV), preferentially target joint tissues and cause chronic rheumatic disease that adversely impacts the quality of life of patients. Viruses enter target cells via interaction with cell surface receptor(s), which determine the viral tissue tropism and pathogenesis. Although MXRA8 is a recently identified receptor for several clinically relevant arthritogenic alphaviruses, its detailed role in the cell entry process has not been fully explored. We found that in addition to its localization on the plasma membrane, MXRA8 is present in acidic organelles, endosomes, and lysosomes. Moreover, MXRA8 is internalized into cells without a requirement for its transmembrane and cytoplasmic domains. Confocal microscopy and live cell imaging revealed that MXRA8 interacts with CHIKV at the cell surface and then enters cells along with CHIKV particles. At the moment of membrane fusion in the endosomes, many viral particles are still colocalized with MXRA8. These findings provide insight as to how MXRA8 functions in alphavirus internalization and suggest possible targets for antiviral development. IMPORTANCE The globally distributed arthritogenic alphaviruses have infected millions of humans and induce rheumatic disease, such as severe polyarthralgia/polyarthritis, for weeks to years. Alphaviruses infect target cells through receptor(s) followed by clathrin-mediated endocytosis. MXRA8 was recently identified as an entry receptor that shapes the tropism and pathogenesis for multiple arthritogenic alphaviruses, including chikungunya virus (CHIKV). Nonetheless, the exact functions of MXRA8 during the process of viral cell entry remain undetermined. Here, we have provided compelling evidence for MXRA8 as a bona fide entry receptor that mediates the uptake of alphavirus virions. Small molecules that disrupt MXRA8-dependent binding of alphaviruses or internalization steps could serve as a platform for unique classes of antiviral drugs.

TLR2 axis on peripheral blood mononuclear cells regulates inflammatory responses to non-infectious immature dengue virus particles.

Severe dengue virus (DENV) infection is characterized by exacerbated inflammatory responses that lead to endothelial dysfunction and plasma leakage. We have recently demonstrated that Toll-like receptor 2 (TLR2) on blood monocytes senses DENV infection leading to endothelial activation. Here, we report that non-infectious immature DENV particles, which are released in large numbers by DENV-infected cells, drive endothelial activation via the TLR2 axis. We show that fully immature DENV particles induce a rapid, within 6 hours post-infection, inflammatory response in PBMCs. Furthermore, pharmacological blocking of TLR2/TLR6/CD14 and/or NF-kB prior to exposure of PBMCs to immature DENV reduces the initial production of inter alia TNF-α and IL-1ß by monocytes and prevents endothelial activation. However, prolonged TLR2 block induces TNF-α production and leads to exacerbated endothelial activation, indicating that TLR2-mediated responses play an important role not only in the initiation but also the resolution of inflammation. Altogether, these data indicate that the maturation status of the virus has the potential to influence the kinetics and extent of inflammatory responses during DENV infection.

Visualization of conformational changes and membrane remodeling leading to genome delivery by viral class-II fusion machinery.

Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKV's complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoprotein's post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.

Mediators of Obesity Do Not Influence SARS-CoV-2 Infection or Activation of Primary Human Lung Microvascular Endothelial Cells In Vitro.

Clinical observations have shown that obesity is associated with the severe outcome of SARS-CoV-2 infection hallmarked by microvascular dysfunction in the lungs and other organs. Excess visceral fat and high systemic levels of adipose tissue (AT) derived mediators such as leptin and other adipokines have also been linked to endothelial dysfunction. Consequently, we hypothesized that AT-derived mediators may exacerbate microvascular dysfunction during of SARS-CoV-2 infection and tested this in a primary human lung microvascular endothelial (HLMVEC) cell model. Our results indicate that HLMVEC are not susceptible to SARS-CoV-2 infection since no expression of viral proteins and no newly produced virus was detected. In addition, exposure to the virus did not induce endothelial activation as evidenced by a lack of adhesion molecule, E-selectin, VCAM-1, ICAM-1, and inflammatory cytokine IL-6 induction. Incubation of endothelial cells with the pro-inflammatory AT-derived mediator, leptin, prior to virus inoculation, did not alter the expression of endothelial SARS-CoV-2 entry receptors and did not alter their susceptibility to infection. Furthermore, it did not induce inflammatory activation of endothelial cells. To verify if the lack of activated phenotype in the presence of adipokines was not leptin-specific, we exposed endothelial cells to plasma obtained from critically ill obese COVID-19 patients. Plasma exposure did not result in E-selectin, VCAM-1, ICAM-1, or IL-6 induction. Together our results strongly suggest that aberrant inflammatory endothelial responses are not mounted by direct SARS-CoV-2 infection of endothelial cells, even in the presence of leptin and other mediators of obesity. Instead, endothelial activation associated with COVID-19 is likely a result of inflammatory responses initiated by other cells. Further studies are required to investigate the mechanisms regulating endothelial behavior in COVID-19 and the mechanisms driving severe disease in obese individuals.

Vitamin D-induced LL-37 modulates innate immune responses of human primary macrophages during DENV-2 infection.

Epidemics of dengue, an acute and potentially severe disease caused by mosquito-borne dengue virus (DENV), pose a major challenge to clinicians and health care services across the sub(tropics). Severe disease onset is associated with a dysregulated inflammatory response to the virus, and there are currently no drugs to alleviate disease symptoms. LL-37 is a potent antimicrobial peptide with a wide range of immunoregulatory properties. In this study, we assessed the effect of LL-37 on DENV-2-induced responses in human monocyte-derived macrophages (MDMs). We show that simultaneous exposure of exogenous LL-37 and DENV-2 resulted in reduced replication of the virus in MDMs, while the addition of LL-37 postexposure to DENV-2 did not. Interestingly, the latter condition reduced the production of IL-6 and increased the expression of genes involved in virus sensing and antiviral response. Finally, we demonstrate that low endogenous levels and limited production of LL-37 in MDMs in response to DENV-2 infection can be increased by differentiating MDMs in the presence of Vitamin D (VitD3). Taken together, this study demonstrates that in addition to its antimicrobial properties, LL-37 has immunomodulatory properties in the curse of DENV infection and its production can be increased by VitD3.

Cooperative Chikungunya Virus Membrane Fusion and Its Substoichiometric Inhibition by CHK-152 Antibody.

Chikungunya virus (CHIKV) presents a major burden on healthcare systems worldwide, but specific treatment remains unavailable. Attachment and fusion of CHIKV to the host cell membrane is mediated by the E1/E2 protein spikes. We used an in vitro single-particle fusion assay to study the effect of the potent, neutralizing antibody CHK-152 on CHIKV binding and fusion. We find that CHK-152 shields the virions, inhibiting interaction with the target membrane and inhibiting fusion. The analysis of the ratio of bound antibodies to epitopes implied that CHIKV fusion is a highly cooperative process. Further, dissociation of the antibody at lower pH results in a finely balanced kinetic competition between inhibition and fusion, suggesting a window of opportunity for the spike proteins to act and mediate fusion, even in the presence of the antibody.

Moxidectin and Ivermectin Inhibit SARS-CoV-2 Replication in Vero E6 Cells but Not in Human Primary Bronchial Epithelial Cells.

Antiviral therapies are urgently needed to treat and limit the development of severe COVID-19 disease. Ivermectin, a broad-spectrum anti-parasitic agent, has been shown to have anti-SARS-CoV-2 activity in Vero cells at a concentration of 5 µM. These limited in vitro results triggered the investigation of ivermectin as a treatment option to alleviate COVID-19 disease. However, in April 2021, the World Health Organization stated the following: "The current evidence on the use of ivermectin to treat COVID-19 patients is inconclusive." It is speculated that the in vivo concentration of ivermectin is too low to exert a strong antiviral effect. Here, we performed a head-to-head comparison of the antiviral activity of ivermectin and the structurally related, but metabolically more stable moxidectin in multiple in vitro models of SARS-CoV-2 infection, including physiologically relevant human respiratory epithelial cells. Both moxidectin and ivermectin exhibited antiviral activity in Vero E6 cells. Subsequent experiments revealed that these compounds predominantly act on the steps following virus cell entry. Surprisingly, however, in human-airway-derived cell models, both moxidectin and ivermectin failed to inhibit SARS-CoV-2 infection, even at concentrations of 10 µM. These disappointing results call for a word of caution in the interpretation of anti-SARS-CoV-2 activity of drugs solely based on their activity in Vero cells. Altogether, these findings suggest that even using a high-dose regimen of ivermectin, or switching to another drug in the same class, is unlikely to be useful for treatment of SARS-CoV-2 in humans.

Posaconazole inhibits multiple steps of the alphavirus replication cycle.

Repurposing drugs is a promising strategy to identify therapeutic interventions against novel and re-emerging viruses. Posaconazole is an antifungal drug used to treat invasive aspergillosis and candidiasis. Recently, posaconazole and its structural analog, itraconazole were shown to inhibit replication of multiple viruses by modifying intracellular cholesterol homeostasis. Here, we show that posaconazole inhibits replication of the alphaviruses Semliki Forest virus (SFV), Sindbis virus and chikungunya virus with EC50 values ranging from 1.4 µM to 9.5 µM. Posaconazole treatment led to a significant reduction of virus entry in an assay using a temperature-sensitive SFV mutant, but time-of-addition and RNA transfection assays indicated that posaconazole also inhibits post-entry stages of the viral replication cycle. Virus replication in the presence of posaconazole was partially rescued by the addition of exogenous cholesterol. A transferrin uptake assay revealed that posaconazole considerably slowed down cellular endocytosis. A single point mutation in the SFV E2 glycoprotein, H255R, provided partial resistance to posaconazole as well as to methyl-ß-cyclodextrin, corroborating the effect of posaconazole on cholesterol and viral entry. Our results indicate that posaconazole inhibits multiple steps of the alphavirus replication cycle and broaden the spectrum of viruses that can be targeted in vitro by posaconazole, which could be further explored as a therapeutic agent against emerging viruses.

Tomatidine reduces Chikungunya virus progeny release by controlling viral protein expression.

Tomatidine, a natural steroidal alkaloid from unripe green tomatoes has been shown to exhibit many health benefits. We recently provided in vitro evidence that tomatidine reduces the infectivity of Dengue virus (DENV) and Chikungunya virus (CHIKV), two medically important arthropod-borne human infections for which no treatment options are available. We observed a potent antiviral effect with EC50 values of 0.82 µM for DENV-2 and 1.3 µM for CHIKV-LR. In this study, we investigated how tomatidine controls CHIKV infectivity. Using mass spectrometry, we identified that tomatidine induces the expression of p62, CD98, metallothionein and thioredoxin-related transmembrane protein 2 in Huh7 cells. The hits p62 and CD98 were validated, yet subsequent analysis revealed that they are not responsible for the observed antiviral effect. In parallel, we sought to identify at which step of the virus replication cycle tomatidine controls virus infectivity. A strong antiviral effect was seen when in vitro transcribed CHIKV RNA was transfected into Huh7 cells treated with tomatidine, thereby excluding a role for tomatidine during CHIKV cell entry. Subsequent determination of the number of intracellular viral RNA copies and viral protein expression levels during natural infection revealed that tomatidine reduces the RNA copy number and viral protein expression levels in infected cells. Once cells are infected, tomatidine is not able to interfere with active RNA replication yet it can reduce viral protein expression. Collectively, the results delineate that tomatidine controls viral protein expression to exert its antiviral activity. Lastly, sequential passaging of CHIKV in presence of tomatidine did not lead to viral resistance. Collectively, these results further emphasize the potential of tomatidine as an antiviral treatment towards CHIKV infection.

Regulation of innate immune responses in macrophages differentiated in the presence of vitamin D and infected with dengue virus 2.

A dysregulated or exacerbated inflammatory response is thought to be the key driver of the pathogenesis of severe disease caused by the mosquito-borne dengue virus (DENV). Compounds that restrict virus replication and modulate the inflammatory response could thus serve as promising therapeutics mitigating the disease pathogenesis. We and others have previously shown that macrophages, which are important cellular targets for DENV replication, differentiated in the presence of bioactive vitamin D (VitD3) are less permissive to viral replication, and produce lower levels of pro-inflammatory cytokines. Therefore, we here evaluated the extent and kinetics of innate immune responses of DENV-2 infected monocytes differentiated into macrophages in the presence (D3-MDMs) or absence of VitD3 (MDMs). We found that D3-MDMs expressed lower levels of RIG I, Toll-like receptor (TLR)3, and TLR7, as well as higher levels of SOCS-1 in response to DENV-2 infection. D3-MDMs produced lower levels of reactive oxygen species, related to a lower expression of TLR9. Moreover, although VitD3 treatment did not modulate either the expression of IFN-α or IFN-ß, higher expression of protein kinase R (PKR) and 2'-5'-oligoadenylate synthetase 1 (OAS1) mRNA were found in D3-MDMs. Importantly, the observed effects were independent of reduced infection, highlighting the intrinsic differences between D3-MDMs and MDMs. Taken together, our results suggest that differentiation of MDMs in the presence of VitD3 modulates innate immunity in responses to DENV-2 infection.

Resveratrol and Pterostilbene Inhibit SARS-CoV-2 Replication in Air-Liquid Interface Cultured Human Primary Bronchial Epithelial Cells.

The current COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has an enormous impact on human health and economy. In search for therapeutic options, researchers have proposed resveratrol, a food supplement with known antiviral, anti-inflammatory, and antioxidant properties as an advantageous antiviral therapy for SARS-CoV-2 infection. Here, we provide evidence that both resveratrol and its metabolically more stable structural analog, pterostilbene, exhibit potent antiviral properties against SARS-CoV-2 in vitro. First, we show that resveratrol and pterostilbene antiviral activity in African green monkey kidney cells. Both compounds actively inhibit virus replication within infected cells as reduced virus progeny production was observed when the compound was added at post-inoculation conditions. Without replenishment of the compound, antiviral activity was observed up to roughly five rounds of replication, demonstrating the long-lasting effect of these compounds. Second, as the upper respiratory tract represents the initial site of SARS-CoV-2 replication, we also assessed antiviral activity in air-liquid interface (ALI) cultured human primary bronchial epithelial cells, isolated from healthy volunteers. Resveratrol and pterostilbene showed a strong antiviral effect in these cells up to 48 h post-infection. Collectively, our data indicate that resveratrol and pterostilbene are promising antiviral compounds to inhibit SARS-CoV-2 infection. Because these results represent laboratory findings in cells, we advocate evaluation of these compounds in clinical trials before statements are made whether these drugs are advantageous for COVID-19 treatment.

Recent advances in antiviral drug development towards dengue virus.

Despite the high disease burden of dengue virus, there is no approved antiviral treatment or broadly applicable vaccine to treat or prevent dengue virus infection. In the last decade, many antiviral compounds have been identified but only few have been further evaluated in pre-clinical or clinical trials. This review will give an overview of the direct-acting and host-directed antivirals identified to date. Furthermore, important parameters for further development that is, drug properties including efficacy, specificity and stability, pre-clinical animal testing, and combinational drug therapy will be discussed.

Chikungunya virus requires an intact microtubule network for efficient viral genome delivery.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus, which has rapidly spread around the globe thereby causing millions of infections. CHIKV is an enveloped virus belonging to the Togaviridae family and enters its host cell primarily via clathrin-mediated endocytosis. Upon internalization, the endocytic vesicle containing the virus particle moves through the cell and delivers the virus to early endosomes where membrane fusion is observed. Thereafter, the nucleocapsid dissociates and the viral RNA is translated into proteins. In this study, we examined the importance of the microtubule network during the early steps of infection and dissected the intracellular trafficking behavior of CHIKV particles during cell entry. We observed two distinct CHIKV intracellular trafficking patterns prior to membrane hemifusion. Whereas half of the CHIKV virions remained static during cell entry and fused in the cell periphery, the other half showed fast-directed microtubule-dependent movement prior to delivery to Rab5-positive early endosomes and predominantly fused in the perinuclear region of the cell. Disruption of the microtubule network reduced the number of infected cells. At these conditions, membrane hemifusion activity was not affected yet fusion was restricted to the cell periphery. Furthermore, follow-up experiments revealed that disruption of the microtubule network impairs the delivery of the viral genome to the cell cytosol. We therefore hypothesize that microtubules may direct the particle to a cellular location that is beneficial for establishing infection or aids in nucleocapsid uncoating.

Strategies employed by viruses to manipulate autophagy.

Autophagy, originally described as a conserved bulk degradation pathway important to maintain cellular homeostasis during starvation, has also been implicated in playing a central role in multiple physiological processes. For example, autophagy is part of our innate immunity by targeting intracellular pathogens to lysosomes for degradation in a process called xenophagy. Coevolution and adaptation between viruses and autophagy have armed viruses with a multitude of strategies to counteract the antiviral functions of the autophagy pathway. In addition, some viruses have acquired mechanisms to exploit specific functions of either autophagy or the key components of this process, the autophagy-related (ATG) proteins, to promote viral replication and pathogenesis. In this chapter, we describe several examples where the strategy employed by a virus to subvert autophagy has been described with molecular detail. Their stratagems positively or negatively target practically all the steps of autophagy, including the signaling pathways regulating this process. This highlights the intricate relationship between autophagy and viruses and how by commandeering autophagy, viruses have devised ways to fine-tune their replication.

TLR2 on blood monocytes senses dengue virus infection and its expression correlates with disease pathogenesis.

Vascular permeability and plasma leakage are immune-pathologies of severe dengue virus (DENV) infection, but the mechanisms underlying the exacerbated inflammation during DENV pathogenesis are unclear. Here, we demonstrate that TLR2, together with its co-receptors CD14 and TLR6, is an innate sensor of DENV particles inducing inflammatory cytokine expression and impairing vascular integrity in vitro. Blocking TLR2 prior to DENV infection in vitro abrogates NF-κB activation while CD14 and TLR6 block has a moderate effect. Moreover, TLR2 block prior to DENV infection of peripheral blood mononuclear cells prevents activation of human vascular endothelium, suggesting a potential role of the TLR2-responses in vascular integrity. TLR2 expression on CD14 + + classical monocytes isolated in an acute phase from DENV-infected pediatric patients correlates with severe disease development. Altogether, these data identify a role for TLR2 in DENV infection and provide insights into the complex interaction between the virus and innate receptors that may underlie disease pathogenesis.

Serotonergic Drugs Inhibit Chikungunya Virus Infection at Different Stages of the Cell Entry Pathway.

Chikungunya virus (CHIKV) is an important reemerging human pathogen transmitted by mosquitoes. The virus causes an acute febrile illness, chikungunya fever, which is characterized by headache, rash, and debilitating (poly)arthralgia that can reside for months to years after infection. Currently, effective antiviral therapies and vaccines are lacking. Due to the high morbidity and economic burden in the countries affected by CHIKV, there is a strong need for new strategies to inhibit CHIKV replication. The serotonergic drug 5-nonyloxytryptamine (5-NT) was previously identified as a potential host-directed inhibitor for CHIKV infection. In this study, we determined the mechanism of action by which the serotonin receptor agonist 5-NT controls CHIKV infection. Using time-of-addition and entry bypass assays, we found that 5-NT predominantly inhibits CHIKV in the early phases of the replication cycle, at a step prior to RNA translation and genome replication. Intriguingly, however, no effect was seen during virus-cell binding, internalization, membrane fusion and genomic RNA (gRNA) release into the cell cytosol. In addition, we show that the serotonin receptor antagonist methiothepin mesylate (MM) also has antiviral properties toward CHIKV and specifically interferes with the cell entry process and/or membrane fusion. Taken together, pharmacological targeting of 5-HT receptors may represent a potent way to limit viral spread and disease severity.IMPORTANCE The rapid spread of mosquito-borne viral diseases in humans puts a huge economic burden on developing countries. For many of these infections, including those caused by chikungunya virus (CHIKV), there are no specific treatment possibilities to alleviate disease symptoms. Understanding the virus-host interactions that are involved in the viral replication cycle is imperative for the rational design of therapeutic strategies. In this study, we discovered an antiviral compound, elucidated its mechanism of action, and propose serotonergic drugs as potential host-directed antivirals for CHIKV.