José González-Santamaría

Viruses pose a significant threat to global public health, yet therapeutic options remain limited. Zotatifin, a synthetic rocaglate targeting eukaryotic initiation factor 4A (eIF4A), inhibits viral protein synthesis and potentially triggers interferon responses; however, its broad-spectrum antiviral potential remains unclear. We evaluated zotatifin’s cytotoxicity using MTT assays and assessed its antiviral activity through plaque-forming assays, Western blot analysis, immunofluorescence, and flow cytometry across multiple cell lines and virus strains. Zotatifin demonstrated superior tolerability compared to rocaglamide A and CR-1-31-B. The compound exhibited potent, dose-dependent inhibition of Mayaro virus, achieving a >4-log ₁₀ reduction in viral titers at 50 nM across all cell lines or virus strains tested. Zotatifin also effectively inhibited multiple arboviruses (Chikungunya, Una, and Zika viruses), influenza A virus, vesicular stomatitis virus, and vaccinia virus. Mechanistically, Zotatifin down-regulated viral protein synthesis for all viruses tested, and RT-qPCR analysis revealed activation of the type I interferon pathway in treated cells. Collectively, these results demonstrate that zotatifin exhibits broad-spectrum antiviral activity against five virus families, through different molecular mechanisms, supporting its potential therapeutic use as a pan-antiviral drug in humans.

Viruses pose a critical global health threat, yet therapeutic options remain limited. Finding drugs with broad-spectrum antiviral activity is essential to confront this threat. Here, we investigated whether plitidepsin, a marine-derived anticancer drug targeting the host eukaryotic elongation factor 1A (eEF1A), has such broad-spectrum activity. Using in vitro infection models and complementary assays (MTT, plaque-forming assays, RT-qPCR, Western blot, flow cytometry), we demonstrated that plitidepsin exhibits potent dose-dependent antiviral activity against Mayaro virus (MAYV) and Chikungunya virus (CHIKV). The compound achieved 4-6 log ₁₀ reduction in viral titers at nanomolar concentrations across multiple cell lines and viral strains. Plitidepsin protected human dermal fibroblasts from viral cytopathic effects and disrupted both entry and post-entry replication stages by suppressing viral protein expression (E1, nsP1) and RNA synthesis. The compound also demonstrated antiviral activity against other medically important arboviruses, including Una, Punta Toro, Zika, and Oropouche viruses, as well as RNA and DNA viruses such as influenza A virus, vesicular stomatitis virus, and human cytomegalovirus. These findings establish plitidepsin as a potent host-directed antiviral agent with reduced likelihood of resistance development and therapeutic potential against multiple viral families.

Viruses pose a significant threat to global public health, yet therapeutic options are limited. Zotatifin, a synthetic rocaglate, targets eukaryotic initiation factor 4A (eIF4A), thereby inhibiting viral protein synthesis and triggering an interferon response. Whether zotatifin exhibits broad-spectrum antiviral activity is unknown. Here we evaluated zotatifin’s cytotoxicity using the MTT method and its antiviral activity using plaque-forming assays, Western blot analysis, immunofluorescence, and flow cytometry across multiple cell lines and virus strains. Zotatifin demonstrated better tolerability than rocaglates rocaglamide A and CR-1-31-B. The compound exhibited potent, dose-dependent inhibition of the Mayaro virus, achieving greater than a 4-log reduction in viral titers at a 50 nM dose, regardless of the cell line or virus strain tested. In addition, zotatifin was effective to inhibit multiple arboviruses (Chikungunya, Una, and Zika), influenza A virus, vesicular stomatitis virus, and vaccinia virus. Zotatifin down-regulated viral protein synthesis for all viruses tested and analysis of gene expression using RT-qPCR revealed activation of the type I interferon pathway in zotatifin-treated cells. Taken together, these results suggest that zotatifin exhibits broad-spectrum antiviral activity against five virus families, likely via several molecular mechanisms. This work supports the potential therapeutic use of zotatifin as a pan-antiviral drug in humans.

The Mayaro virus (MAYV), Togaviridae family, genus Alphavirus, has caused several sporadic outbreaks, affecting countries in the Americas. Currently, there are no licensed drugs against MAYV, requiring the search for effective antiviral compounds. Thus, this study aimed to evaluate the antiviral potential of polyphenol (-)-epigallocatechin-3-gallate (EGCG) against MAYV infection, in vitro. Antiviral assays against MAYV were performed in BHK-21 and Vero E6 cells. In addition, molecular docking was performed with EGCG and the MAYV non-structural and structural proteins. EGCG showed a significant protective effect against MAYV infection in both cell lines. The virucidal assay showed an effect on extracellular viral particles at the entry stage into BHK-21 cells. Finally, it also showed significant inhibition in the post-entry stages of the MAYV replication cycle, acting on the replication of the genetic material and late stages, such as assembly and release. In addition, the MAYV proteins E1 and nsP1 were significantly inhibited by the EGCG treatment in BHK-21 cells. Molecular docking analysis also showed that EGCG could interact with MAYV Capsid and Envelope proteins (E1 and E2). Therefore, this study shows the potential of EGCG as a promising antiviral against MAYV, as it acts on different stages of the MAYV replication cycle.

Mayaro (MAYV) and Una (UNAV) are emerging alphaviruses circulating in the Americas. Earlier reports have revealed that MAYV infects different human cell lines, including synovial and dermal fibroblasts, chondrocytes, osteoblasts, astrocytes and pericytes, as well as neural progenitor cells. In this study we evaluated the susceptibility of immortalized human microglia HMC3 cells and brain microvascular endothelial HBEC-5i cells to MAYV and UNAV infection. Cytopathic effects, cell viability, viral progeny yields, and the presence of E1 and nsP1 proteins in HMC3 and HBEC-5i cells infected with several MAYV or UNAV strains were assessed using an inverted microscope, MTT assay, plaque-forming assays, and immunofluorescence or Western blot, respectively. Finally, the expression of immune response genes was analyzed using RT-qPCR. MAYV and UNAV demonstrated strong cytopathic effects and significantly reduced cell viability in HMC3 cells. Moreover, the HMC3 cells were efficiently infected regardless of the virus strain tested, and E1 and nsP1 viral proteins were detected. In contrast, only MAYV appeared to infect HBEC-5i cells, and minimal effects on cell morphology or viability were observed. Furthermore, the MAYV titer and viral protein levels were substantially lower in the infected HBEC-5i cells when compared to those of the infected microglia cells. Finally, unlike UNAV, MAYV elicited a strong expression of specific interferon-stimulated genes in microglia cells, along with pro-inflammatory cytokines implicated in the immune response. Collectively, these findings demonstrate that MAYV and UNAV are capable of infecting relevant human brain cells.

Mayaro (MAYV) and Una (UNAV) are emerging alphaviruses circulating in the Americas. Earlier reports have revealed that MAYV infects different human cell lines, including synovial and dermal fibroblasts, chondrocytes, osteoblasts, astrocytes and pericytes, as well as neural progenitor cells. In this study we evaluated the susceptibility of immortalized human microglia HMC3 cells and brain microvascular endothelial HBEC-5i cells to MAYV and UNAV infection. Cytopathic effects, cell viability, viral progeny yields, and the presence of E1 and nsP1 proteins in HMC3 and HBEC-5i cells infected with several MAYV or UNAV strains were assessed using an inverted microscope, MTT assay, plaque-forming assays, and immunofluorescence or Western blot, respectively. Finally, the expression of immune response genes was analyzed using qRT-PCR. MAYV and UNAV demonstrated strong cytopathic effects and significantly reduced cell viability in HMC3 cells. Moreover, the HMC3 cells were efficiently infected regardless of the virus strains tested, and E1 and nsP1 viral proteins were detected. In contrast, only MAYV appeared to infect HBEC-5i cells, and minimal effects on cell morphology or viability were observed. Furthermore, the MAYV titer and viral protein levels were substantially lower in the infected HBEC-5i cells when compared to those of the infected microglia cells. Finally, unlike UNAV, MAYV elicited a strong expression of specific interferon-stimulated genes in microglia cells, along with pro-inflammatory cytokines implicated in the immune response. Collectively, these findings demonstrate that MAYV and UNAV are capable of infecting relevant human brain cells.

Mayaro virus (MAYV) is an emerging arbovirus with an increasing circulation across the Americas. In the present study, we evaluated the potential antiviral activity of the following natural compounds against MAYV and other arboviruses: Sanguinarine, (R)-Shikonin, Fisetin, Honokiol, Tanshinone IIA, and α-Mangostin. Sanguinarine and Shikonin showed significant cytotoxicity, whereas Fisetin, Honokiol, Tanshinone IIA, and α-Mangostin were well tolerated in all the cell lines tested. Honokiol and α-Mangostin treatment protected Vero-E6 cells against MAYV-induced damage and resulted in a dose-dependent reduction in viral progeny yields for each of the MAYV strains and human cell lines assessed. These compounds also reduced MAYV viral RNA replication in HeLa cells. In addition, Honokiol and α-Mangostin disrupted MAYV infection at different stages of the virus life cycle. Moreover, Honokiol and α-Mangostin decreased Una, Chikungunya, and Zika viral titers and downmodulated the expression of E1 and nsP1 viral proteins from MAYV, Una, and Chikungunya. Finally, in Honokiol- and α-Mangostin-treated HeLa cells, we observed an upregulation in the expression of type I interferon and specific interferon-stimulated genes, including IFNα, IFNβ, MxA, ISG15, OAS2, MDA-5, TNFα, and IL-1β, which may promote an antiviral cellular state. Our results indicate that Honokiol and α-Mangostin present potential broad-spectrum activity against different arboviruses through different mechanisms.

Mayaro virus (MAYV) is an emerging arbovirus with increasing circulation across the Americas. In the present study, we evaluated the potential antiviral activity of the following natural compounds against MAYV and other arboviruses: Sanguinarine, (R)-Shikonin, Fisetin, Honokiol, Tanshinone IIA and α-Mangostin. Sanguinarine and Shikonin showed significant cytotoxicity, whereas Fisetin, Honokiol, Tanshinone IIA and α-Mangostin were well-tolerated in all the cell lines tested. Honokiol and α-Mangostin treatment protected Vero-E6 cells against MAYV-induced damage and resulted in a dose-dependent reduction in viral progeny yields for each of the MAYV strains and human cell lines assessed. Also, Honokiol and α-Mangostin disrupted MAYV infection at different stages of the virus life cycle. Moreover, these compounds decreased Una, Chikungunya and Zika viral titers and downmodulated the expression of E1 and nsP1 viral proteins from MAYV, Una and Chikungunya. Finally, in Honokiol- and α-Mangostin-treated cells, we observed an upregulation in the expression of type I interferon and specific interferon-stimulated genes, including IFNα, IFN β, MxA, ISG15, OAS2, MDA-5, TNFα and IL-1β, which may promote an antiviral cellular state. Our results indicate that Honokiol and α-Mangostin present potential broad-spectrum activity against different arboviruses through a possible modulation of the interferon pathway.

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.