Tomatidine, a natural steroidal alkaloid shows antiviral activity towards chikungunya virus in vitro

In recent decades, chikungunya virus (CHIKV) has re-emerged, leading to outbreaks of chikungunya fever in Africa, Asia and Central and South America. The disease is characterized by a rapid onset febrile illness with (poly)arthralgia, myalgia, rashes, headaches and nausea. In 30 to 40% of the cases, CHIKV infection causes persistent (poly)arthralgia, lasting for months or even years after initial infection. Despite the drastic re-emergence and clinical impact there is no vaccine nor antiviral compound available to prevent or control CHIKV infection. Here, we evaluated the antiviral potential of tomatidine towards CHIKV infection. We demonstrate that tomatidine potently inhibits virus particle production of multiple CHIKV strains. Time-of -addition experiments in Huh7 cells revealed that tomatidine acts at a post-entry step of the virus replication cycle. Furthermore, a marked decrease in the number of CHIKV-infected cells was seen, suggesting that tomatidine predominantly acts early in infection yet after virus attachment and cell entry. Antiviral activity was still detected at 24 hours post-infection, indicating that tomatidine controls multiple rounds of CHIKV replication. Solasodine and sarsasapogenin, two structural derivatives of tomatidine, also showed strong albeit less potent antiviral activity towards CHIKV. In conclusion, this study identifies tomatidine as a novel compound to combat CHIKV infection in vitro

Complex interaction between dengue virus replication and expression of miRNA-133a

Background: Dengue virus (DENV) is the most common vector-borne viral infection worldwide with approximately 390 million cases and 25,000 reported deaths each year. MicroRNAs (miRNAs) are small non-coding RNA molecules responsible for the regulation of gene expression by repressing mRNA translation or inducing mRNA degradation. Although miRNAs possess antiviral activity against many mammalian-infecting viruses, their involvement in DENV replication is poorly understood. Methods: Here, we explored the relationship between DENV and cellular microRNAs using bioinformatics tools. We overexpressed miRNA-133a in Vero cells to test its role in DENV replication and analyzed its expression using RT-qPCR. Furthermore, the expression of polypyrimidine tract binding protein (PTB), a protein involved in DENV replication, was analyzed by western blot. In addition, we profiled miRNA-133a expression in Vero cells challenged with DENV-2, using Taqman miRNA. Results: Bioinformatic analysis revealed that the 3' untranslated region (3'UTR) of the DENV genome of all four DENV serotypes is targeted by several cellular miRNAs, including miRNA-133a. We found that overexpression of synthetic miRNA-133a suppressed DENV replication. Additionally, we observed that PTB transcription, a miRNA-133a target, is down-regulated during DENV infection. Based in our results we propose that 3' UTR of DENV downregulates endogenous expression of miRNA-133a in Vero cells during the first hours of infection. Conclusions: miRNA-133a regulates DENV replication possibly through the modulation of a host factor such as PTB. Further investigations are needed to verify whether miRNA-133a has an anti-DENV effect in vivo

Tomatidine, a novel antiviral compound towards dengue virus

Dengue is the most common arboviral disease worldwide with 96 million symptomatic cases annually. Despite its major impact on global human health and huge economic burden there is no antiviral drug available to treat the disease. The first tetravalent dengue virus vaccine was licensed in 2015 for individuals aged 9 to 45, however, most cases are reported in infants and young children. This, together with the limited efficacy of the vaccine to dengue virus (DENV) serotype 2, stresses the need to continue the search for compounds with anti-viral activity to DENV. In this report, we describe tomatidine as a novel compound with potent antiviral properties towards all DENV serotypes and the related Zika virus. The strongest effect was observed for DENV-2 with an EC50 and EC90 value of 0.82 and 1.61 mu M, respectively, following infection of Huh7 cells at multiplicity of infection of 1. The selectivity index is 97.7. Time-of-drug-addition experiments revealed that tomatidine inhibits virus particle production when added pre, during and up to 12 h post-infection. Subsequent experiments show that tomatidine predominantly acts at a step after virus-cell binding and membrane fusion but prior to the secretion of progeny virions. Tomatidine was found to control the expression of the cellular protein activating transcription factor 4 (ATF4), yet, this protein is not solely responsible for the observed antiviral effect. Here, we propose tomatidine as a candidate for the treatment of dengue given its potent antiviral activity

Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses

The dogma is that the human immune system protects us against pathogens. Yet, several viruses, like dengue virus, antagonize the hosts' antibodies to enhance their viral load and disease severity; a phenomenon called antibody-dependent enhancement of infection. This study offers novel insights in the molecular mechanism of antibody-mediated enhancement (ADE) of dengue virus infection in primary human macrophages. No differences were observed in the number of bound and internalized DENV particles following infection in the absence and presence of enhancing concentrations of antibodies. Yet, we did find an increase in membrane fusion activity during ADE of DENV infection. The higher fusion activity is coupled to a low antiviral response early in infection and subsequently a higher infection efficiency. Apparently, subtle enhancements early in the viral life cycle cascades into strong effects on infection, virus production and immune response. Importantly, and in contrast to other studies, the antibody-opsonized virus particles do not trigger immune suppression and remain sensitive to interferon. Additionally, this study gives insight in how human macrophages interact and respond to viral infections and the tight regulation thereof under various conditions of infection

Overexpression of miR-3614-5p regulates protein expression in Huh7 cells.

<p>Huh7 cells were transfected with a mimic NC and with the mimic of miR-3614-5p. At 24 hpt changes in protein expression were analyzed by LC/MS. (A) Clustered heat map displaying differentially expressed proteins between the groups (two independent experiments per group). Underlined proteins were also predicted by bioinformatics tools. (B) Gene set enrichment analysis of the proteins regulated by the miR-3614-5p. (C) Enriched KEGG pathways upregulated by the miR-3614-5p (PR: positive regulation, NR: negative regulation).</p

Differentially expressed miRNAs in MDMs challenged with DENV.

<p>(A) MDMs obtained from three different blood donors (D28, D29 and D30) were treated as follows: 1) mock-infected and non-sorted, 2) treated with UVi-DENV, 3) challenged with GFP-DENV (DENV-challenge), 4) mock-infected and passed through the FACS sorter, 5) challenged with GFP-DENV and sorted for GFP positive cells (DENV-plus), 6) challenged with GFP-DENV and sorted for GFP negative cells (DENV-neg). (B) Hierarchical unsupervised Pearson correlation of miRNAs showing differentially expressed miRNAs across the samples (ANOVA, p<0.05). (C) Differentially expressed miRNAs across the samples after the Benjamini and Hochberg correction for multiple testing (p<0.05).</p

Effect of miR-181a, miR-4301, miR-3960 and miR-4508 on DENV infection of Huh7 cells.

<p>Huh7 cells were transfected with a mimic negative control (NC) and with the indicated miRNA mimics. At 24 hpt, cells were infected at MOIs 1, 5 and 10. The percentage of E-positive cells (grey bars) and infectious virus particle production (black bars) were determined at 24 hpi. The percentages of infection of cells transfected with the NC and infected at MOIs 1, 5 and 10 were 6.57±2.07; 13.9±3.48 and 18.53±2.64 respectively; while the viral titers were 1.2x10⁵±5x10⁴; 1.08x10⁶±2x10⁵ and 7.06 x10⁶±6x10⁴ PFU/ml respectively. Data is presented as the percentage relative to the NC and shows mean ± SEM from three independent experiments. Statistical differences were assessed with Student’s t-test.</p

Dengue tropism for macrophages and dendritic cells:the host cell effect

Dengue virus infects immune cells, including monocytes, macrophages and dendritic cells (DC). We compared virus infectivity in macrophages and DC, and found that the virus origin determined the cell tropism of progeny virus. The highest efficiency of re-infection was seen for macrophage-derived dengue virus. Furthermore, in the presence of enhancing antibodies, macrophage-derived virus gave greater enhancement of infection compared with immature DC derived virus. Taken together, our results highlight the importance of macrophages in dengue infection.</p

DENV infection induces ADAR1 expression in MEFs.

<p>MEFs were infected with DENV at the indicated MOIs. The methodology is as described in the legend to <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005981#pntd.0005981.g006" target="_blank">Fig 6</a>. The expression of ADAR1 was normalized to that of GAPDH and it is expressed as the percentage of the mock-infected cells. Data shows mean ± SEM from three independent experiments.</p

ADAR KO MEFs are less permissive to DENV than wild-type MEFs.

<p>Wild-type (WT) MEFs, p53 KO MEFs (p53^-/-) and p53/ADAR double KO MEFs (ADAR^-/-) were infected with DENV at MOIs 1 and 5. At the indicated time points, the percentage of infection (upper panels) was determined by flow cytometry and the infectious virus particle production (lower panels) by plaque assay. Data shows mean ± SEM from three independent experiments.</p