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Various low-density lipoprotein receptors (LPRs) have been identified as entry factors for alphaviruses, and structures of the corresponding virion-receptor complexes have been determined. Here, we analyze the similarities and differences in the receptor binding modes of multiple alphaviruses to understand their ability to infect a wide range of hosts. We further discuss the challenges associated with the development of broad-spectrum treatment strategies against a diverse range of alphaviruses. Recently, studies have identified different low-density lipoprotein receptors as functional entry receptors for alphaviruses. In this Perspective, the authors compare the known alphavirus-receptor structures and discuss why it might be challenging to generate a broad-spectrum entry inhibitor.

Alphaviruses including chikungunya virus (CHIKV) are mosquito-borne positive-strand RNA viruses that can cause various diseases in humans. Although compounds that inhibit CHIKV and other alphaviruses have been identified in vitro , there are no licensed antivirals against CHIKV. Here, we investigated a ribonucleoside analog, 4′-fluorouridine (4′-FlU), and demonstrated that it inhibited infectious virus production by several alphaviruses in vitro and reduced virus burden in mouse models of CHIKV and Mayaro virus infection. Our studies also indicated that 4′-FlU treatment reduced CHIKV-induced footpad swelling and reduced the production of pro-inflammatory cytokines. Inhibition in the mouse model correlated with effective oral delivery of 4′-FlU and accumulation of both 4′-FlU and its bioactive form in relevant tissues. In summary, 4′-FlU exhibits potential as a novel anti-alphavirus agent targeting the replication of viral RNA.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype among breast tumors and remains a challenge even for the most current therapeutic regimes. Here, we demonstrate that oncolytic alphavirus M1 effectively kills both TNBC and non-TNBC. ER-stress and apoptosis pathways are responsible for the cell death in non-TNBC as reported in other cancer types, yet the cell death in TNBC does not depend on these pathways. Transcriptomic analysis reveals that the M1 virus activates necroptosis in TNBC, which can be pharmacologically blocked by necroptosis inhibitors. By screening a library of clinically available compounds commonly used for breast cancer treatment, we find that Doxorubicin enhances the oncolytic effect of the M1 virus by up to 100-fold specifically in TNBC in vitro, and significantly stalls the tumor growth of TNBC in vivo, through promoting intratumoral virus replication and further triggering apoptosis in addition to necroptosis. These findings reveal a novel antitumor mechanism and a new combination regimen of the M1 oncolytic virus in TNBC, and highlight a need to bridge molecular diagnosis with virotherapy.

Arthritogenic alphaviruses are a group of medically important arboviruses that cause inflammatory musculoskeletal disease in humans with debilitating symptoms, such as arthralgia, arthritis, and myalgia. The arthritogenic, or Old World, alphaviruses are capable of causing explosive outbreaks, with some viruses of major global concern. At present, there are no specific therapeutics or commercially available vaccines available to prevent alphaviral disease. Infected patients are typically treated with analgesics and non-steroidal anti-inflammatory drugs to provide often inadequate symptomatic relief. Studies to determine the mechanisms of arthritogenic alphaviral disease have highlighted the role of the host immune system in disease pathogenesis. This review discusses the current knowledge of the innate immune response to acute alphavirus infection and alphavirus-induced immunopathology. Therapeutic strategies to treat arthritogenic alphavirus disease by targeting the host immune response are also examined.

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 β-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.

The alphavirus chikungunya virus (CHIKV) is an example of an emerging human pathogen with increased and rapid global spread. Although an acute CHIKV infection is rarely fatal, many patients suffer from debilitating chronic arthralgia for years. Antivirals against chikungunya and other alphaviruses have been identified in vitro , but to date none have been shown to be efficacious and have been licensed for human use. Here, we investigated a small molecule, berberine chloride (BBC), and showed that it inhibited infectious virus production by several alphaviruses including CHIKV. BBC acted on a late step in the alphavirus exit pathway, namely the formation of the nucleocapsid containing the infectious viral RNA. Better understanding of nucleocapsid formation and its inhibition by BBC will provide important information on the mechanisms of infectious alphavirus production and may enable their future targeting in antiviral strategies. Alphaviruses are enveloped positive-sense RNA viruses that can cause serious human illnesses such as polyarthritis and encephalitis. Despite their widespread distribution and medical importance, there are no licensed vaccines or antivirals to combat alphavirus infections. Berberine chloride (BBC) is a pan-alphavirus inhibitor that was previously identified in a replicon-based small-molecule screen. This work showed that BBC inhibits alphavirus replication but also suggested that BBC might have additional effects later in the viral life cycle. Here, we show that BBC has late effects that target the virus nucleocapsid (NC) core. Infected cells treated with BBC late in infection were unable to form stable cytoplasmic NCs or assembly intermediates, as assayed by gradient sedimentation. In vitro studies with recombinant capsid protein (Cp) and purified genomic RNA (gRNA) showed that BBC perturbs core-like particle formation and potentially traps the assembly process in intermediate states. Particles produced from BBC-treated cells were less infectious, despite efficient particle production and only minor decreases in genome packaging. In addition, BBC treatment of free virus particles strongly decreased alphavirus infectivity. In contrast, the infectivity of the negative-sense RNA virus vesicular stomatitis virus was resistant to BBC treatment of infected cells or free virus. Together, our data indicate that BBC alters alphavirus Cp-gRNA interactions and oligomerization and suggest that this may cause defects in NC assembly and in disassembly during subsequent virus entry. Thus, BBC may be considered a novel alphavirus NC assembly inhibitor. IMPORTANCE The alphavirus chikungunya virus (CHIKV) is an example of an emerging human pathogen with increased and rapid global spread. Although an acute CHIKV infection is rarely fatal, many patients suffer from debilitating chronic arthralgia for years. Antivirals against chikungunya and other alphaviruses have been identified in vitro , but to date none have been shown to be efficacious and have been licensed for human use. Here, we investigated a small molecule, berberine chloride (BBC), and showed that it inhibited infectious virus production by several alphaviruses including CHIKV. BBC acted on a late step in the alphavirus exit pathway, namely the formation of the nucleocapsid containing the infectious viral RNA. Better understanding of nucleocapsid formation and its inhibition by BBC will provide important information on the mechanisms of infectious alphavirus production and may enable their future targeting in antiviral strategies.

Background The arthropod-borne Mayaro virus (MAYV) causes “Mayaro fever,” a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. There are currently no licensed drugs against most mosquito-transmitted viruses. Punica granatum (pomegranate) fruits cultivated in Brazil have been subjected to phytochemical investigation for the identification and isolation of antiviral compounds. In the present study, we explored the antiviral activity of pomegranate extracts in Vero cells infected with Mayaro virus. Methods The ethanol extract and punicalagin of pomegranate were extracted solely from the shell and purified by chromatographic fractionation, and were chemically identified using spectroscopic techniques. The cytotoxicity of the purified compounds was measured by the dye uptake assay, while their antiviral activity was evaluated by a virus yield inhibition assay. Results Pomegranate ethanol extract (CC 50  = 588.9, IC 50  = 12.3) and a fraction containing punicalagin as major compound (CC 50  = 441.5, IC 50  = 28.2) were shown to have antiviral activity (SI 49 and 16, respectively) against Mayaro virus, an alphavirus. Immunofluorescence analysis showed the virucidal effect of pomegranate extract, and transmission electron microscopy (TEM) revealed damage in viral particles treated with this extract. Conclusions The P. granatum extract is a promising source of antiviral compounds against the alphavirus MAYV and represents an excellent candidate for future studies with other enveloped RNA viruses. Graphical abstract

The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host's primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus.

Chikungunya and Mayaro fevers are viral infectious diseases characterized by fever and arthralgia, for which there are currently no effective vaccines or treatments. The urgent need for novel antiviral agents against Chikungunya virus (CHIKV) and Mayaro virus (MAYV) has led to interest in plant-based compounds that can disrupt the viral replication cycle. Chiococca alba (L.) Hitchc., a Neotropical plant traditionally used by Yucatec Maya healers as an antipyretic and antirheumatic, may hold potential as a source of antiviral agents. This study aimed to evaluate the antiviral potential of C. alba methanolic extracts (CAH21 and CAH24) against CHIKV and MAYV through preliminary in vitro and in silico analyses. The cytotoxicity of two methanolic extracts from C. alba roots was assessed in Vero cells using the neutral red assay, and their viral activity was determined via plaque assay post-treatment. Given the observed antiviral effects, we used computational predictions to explore interactions between the multifunctional nsP2 proteases and secondary metabolites identified in C. alba extracts. The metabolites were identified using high-performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC-MS). Phytochemical analysis revealed the presence of flavonoids, coumarins, and phenolic acids in the C. alba extracts. In vitro assays demonstrated that both extracts inhibited over 70% of activity against CHIKV and MAYV at a concentration of 60 µg/mL. In silico predictions suggested that the flavonoids naringin and vitexin had the highest affinity for the nsP2 proteases of CHIKV and MAYV, indicating their potential as viral inhibitors. Our findings revealed that C. alba extract represents a promising source of novel antiviral compounds.

Mayaro virus (MAYV) is a neglected arthropod-borne virus found in the Americas. MAYV infection results in Mayaro fever, a non-lethal debilitating disease characterized by a strong inflammatory response affecting the joints and muscles. MAYV was once considered endemic to forested areas in Brazil but has managed to adapt and spread to urban regions using new vectors, such as Aedes aegypti, and has the potential to cause serious epidemics in the future. Currently, there are no vaccines or specific treatments against MAYV. In this study, the antiviral activity of a series of synthetic cyclic ketones were evaluated for the first time against MAYV. Twenty-four compounds were screened in a cell viability assay, and eight were selected for further evaluation. Effective concentration (EC50) and selectivity index (SI) were calculated and compound 9-(5-(4-chlorophenyl]furan-2-yl)-3,6-dimethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2))-dione (9) (EC50 = 21.5 µmol·L−1, SI = 15.8) was selected for mechanism of action assays. The substance was able to reduce viral activity by approximately 70% in both pre-treatment and post-treatment assays.