The impact of radiomics libraries and gray level discretization on the discovery of immunotherapy biomarkers in NSCLC patients.

OBJECTIVE: The influence of radiomics pipeline and the grey-level discretization on the discovery of immunotherapy biomarkers is still a poorly understood topic. This study is aimed at identifying robust features by comparing two radiomics libraries and their association with clinical outcomes in non-small cell lung cancer (NSCLC) patients treated with immune checkpoint inhibitors (ICIs). METHODS: A retrospective cohort of 164 NSCLC patients administered with ICIs was used in this study. Radiomic features were extracted from the pre-treatment CT scans. Univariate models were used to assess the association of radiomics features with progression free survival (PFS), PD-L1 and CD8 cell counts. We also examined the impact of gray-level discretization on feature robustness by evaluating the association of features with clinical endpoints. RESULTS: We extracted 1224, 441 radiomic features using Pyradiomics and RaCat, respectively, out of which 75 were common between them. We showed that the directionality of association between features and clinical endpoints is specific to the radiomic library used. Overall, more Pyradiomics and RaCat features were statistically associated with PFS, and PD-L1, respectively. We found intensity-based features to be more agnostic to the gray-level discretization parameters. Among features that showed significant correlation with PFS with varying gray-level discretization parameters, 45% were intensity-based, compared to PD-L1, and CD8. CONCLUSIONS: This study highlights the heterogeneity of radiomics libraries and the gray level discretization parameters that will impact the feature selection and predictive model development. Importantly, our work highlights the significance of selecting features that are agnostic to radiomics libraries for clinical translation. ADVANCES IN KNOWLEDGE: Our study emphasizes the need to select stable CT-derived handcrafted features to build immunotherapy biomarkers, which is a necessary precursor for multi-institutional validation of imaging biomarkers.

The NS1 protein of contemporary West African Zika virus potentiates viral replication and reduces innate immune activation.

Mosquito-borne Zika virus (ZIKV) from sub-Saharan Africa has recently gained attention due to its epidemic potential and its capacity to be highly teratogenic. To improve our knowledge on currently circulating strains of African ZIKV, we conducted protein sequence alignment and identified contemporary West Africa NS1 (NS1CWA) protein as a highly conserved viral protein. Comparison of NS1CWA with the NS1 of the historical African ZIKV strain MR766 (NS1MR766), revealed seven amino acid substitutions. The effects of NS1 mutations on protein expression, virus replication, and innate immune activation were assessed in human cells using recombinant NS1 proteins and a chimeric viral clone MR766 with NS1CWA replacing NS1MR766. Our data indicated higher secretion efficiency of NS1CWA compared to NS1MR766 associated with a change in subcellular distribution. A chimeric MR766 virus with NS1CWA instead of authentic protein displayed a greater viral replication efficiency, leading to more pronounced cell death compared to parental virus. Enhanced viral growth was associated with reduced activation of innate immunity. Our data raise questions of the importance of NS1 protein in the pathogenicity of contemporary ZIKV from sub-Saharan Africa and point to differences within viral strains of African lineage.

Compensatory mutations potentiate constructive neutral evolution by gene duplication.

The functions of proteins generally depend on their assembly into complexes. During evolution, some complexes have transitioned from homomers encoded by a single gene to heteromers encoded by duplicate genes. This transition could occur without adaptive evolution through intermolecular compensatory mutations. Here, we experimentally duplicated and evolved a homodimeric enzyme to determine whether and how this could happen. We identified hundreds of deleterious mutations that inactivate individual homodimers but produce functional enzymes when coexpressed as duplicated proteins that heterodimerize. The structure of one such heteromer reveals how both losses of function are buffered through the introduction of asymmetry in the complex that allows them to subfunctionalize. Constructive neutral evolution can thus occur by gene duplication followed by only one deleterious mutation per duplicate.

Replication properties of a contemporary Zika virus from West Africa.

Zika virus (ZIKV) has become a global health problem over the past decade due to the extension of the geographic distribution of the Asian/American genotype. Recent epidemics of Asian/American ZIKV have been associated with developmental disorders in humans. There is mounting evidence that African ZIKV may be associated with increased fetal pathogenicity necessitating to pay a greater attention towards currently circulating viral strains in sub-Saharan Africa. Here, we generated an infectious molecular clone GUINEA-18 of a recently transmitted human ZIKV isolate from West Africa, ZIKV-15555. The available infectious molecular clone MR766MC of historical African ZIKV strain MR766-NIID was used for a molecular clone-based comparative study. Viral clones GUINEA-18 and MR766MC were compared for their ability to replicate in VeroE6, A549 and HCM3 cell lines. There was a lower replication rate for GUINEA-18 associated with weaker cytotoxicity and reduced innate immune system activation compared with MR766MC. Analysis of chimeric viruses between viral clones stressed the importance of NS1 to NS4B proteins, with a particular focus of NS4B on GUINEA-18 replicative properties. ZIKV has developed strategies to prevent cytoplasmic stress granule formation which occurs in response to virus infection. GUINEA-18 was greatly efficient in inhibiting stress granule assembly in A549 cells subjected to a physiological stressor, with NS1 to NS4B proteins also being critical in this process. The impact of these GUINEA-18 proteins on viral replicative abilities and host-cell responses to viral infection raises the question of the role of nonstructural proteins in the pathogenicity of currently circulating ZIKV in sub-Saharan Africa.

Implementing Multifactorial Risk Assessment with Polygenic Risk Scores for Personalized Breast Cancer Screening in the Population Setting: Challenges and Opportunities.

Risk-stratified breast screening has been proposed as a strategy to overcome the limitations of age-based screening. A prospective cohort study was undertaken within the PERSPECTIVE I&I project, which will generate the first Canadian evidence on multifactorial breast cancer risk assessment in the population setting to inform the implementation of risk-stratified screening. Recruited females aged 40-69 unaffected by breast cancer, with a previous mammogram, underwent multifactorial breast cancer risk assessment. The adoption of multifactorial risk assessment, the effectiveness of methods for collecting risk factor information and the costs of risk assessment were examined. Associations between participant characteristics and study sites, as well as data collection methods, were assessed using logistic regression; all p-values are two-sided. Of the 4246 participants recruited, 88.4% completed a risk assessment, with 79.8%, 15.7% and 4.4% estimated at average, higher than average and high risk, respectively. The total per-participant cost for risk assessment was CAD 315. Participants who chose to provide risk factor information on paper/telephone (27.2%) vs. online were more likely to be older (p = 0.021), not born in Canada (p = 0.043), visible minorities (p = 0.01) and have a lower attained education (p < 0.0001) and perceived fair/poor health (p < 0.001). The 34.4% of participants requiring risk factor verification for missing/unusual values were more likely to be visible minorities (p = 0.009) and have a lower attained education (p ≤ 0.006). This study demonstrates the feasibility of risk assessment for risk-stratified screening at the population level. Implementation should incorporate an equity lens to ensure cancer-screening disparities are not widened.

Differential outcomes of re-stratified high-risk prostate cancer patients treated with external beam radiation therapy plus high-dose-rate brachytherapy boost.

Purpose: We report outcomes of high-risk prostate cancer (PCa) patients, initially classified according to a 3-tier NCCN classification system, treated with external beam radiation therapy (EBRT) and high-dose-rate brachytherapy boost (HDR-BT). Patients were analyzed based on a re-stratification of their risk grouping using CAPRA score and a newer 5-tier NCCN classification. Material and methods: 471 high-risk PCa patients treated with EBRT, HDR-BT, and androgen deprivation therapy (ADT) between 1999 and 2018 were included. Competing risk survival analyses to compare individuals with CAPRA scores < 6 vs. ≥ 6 for biochemical relapse (BCR) and metastasis incidence were conducted. Also, overall survival (OS) for both groups using Kaplan-Meier analysis was assessed. The same analyses were repeated using a 5-tier NCCN stratification comparing those classified as high-risk vs. very high-risk patients. Results: The median age was 71 years, and the median follow-up period was 72 months. The whole cohort received an EQD2 of 74 Gy or greater, with a median EQD2 of 106.89 Gy. Both a CAPRA score ≥ 6 and belonging to the NCCN very high-risk group were associated with BCR, with subdistribution hazard ratios (sHRs) of 3.04 (p = 0.015) and 2.53 (p = 0.013), respectively. For metastasis incidence, both the CAPRA and NCCN groups had similar sHRs of 2.60 (p = 0.094) and 2.71 (p = 0.037), respectively. For 10-year OS, patients with CAPRA score ≥ 6 and belonging to the NCCN very high-risk group presented similar HRs of 2.11 (p = 0.005) and 2.10 (p = 0.002). Conclusions: We showed that high-risk PCa patients classified according to the 3-tier NCCN system benefit from further stratification using the CAPRA score or the 5-tier NCCN stratification method. Patients with a CAPRA score ≥ 6 or classified as very high-risk demonstrate a higher hazard of BCR, metastasis, and death. These patients might benefit from further intensification of their investigations and treatment, based on ongoing research.

Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate.

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

Compensatory mutations potentiate constructive neutral evolution by gene duplication.

Protein functions generally depend on their assembly into complexes. During evolution, some complexes have transitioned from homomers encoded by a single gene to heteromers encoded by duplicate genes. This transition could occur without adaptive evolution through intermolecular compensatory mutations. Here, we experimentally duplicate and evolve an homodimeric enzyme to examine if and how this could happen. We identify hundreds of deleterious mutations that inactivate individual homodimers but produce functional enzymes when co-expressed as duplicated proteins that heterodimerize. The structure of one such heteromer reveals how both losses of function are buffered through the introduction of asymmetry in the complex that allows them to subfunctionalize. Constructive neutral evolution can thus occur by gene duplication followed by only one deleterious mutation per duplicate.

The Cross-Scale Association between Pathomics and Radiomics Features in Immunotherapy-Treated NSCLC Patients: A Preliminary Study.

BACKGROUND: Recent advances in cancer biomarker development have led to a surge of distinct data modalities, such as medical imaging and histopathology. To develop predictive immunotherapy biomarkers, these modalities are leveraged independently, despite their orthogonality. This study aims to explore the cross-scale association between radiological scans and digitalized pathology images for immunotherapy-treated non-small cell lung cancer (NSCLC) patients. METHODS: This study involves 36 NSCLC patients who were treated with immunotherapy and for whom both radiology and pathology images were available. A total of 851 and 260 features were extracted from CT scans and cell density maps of histology images at different resolutions. We investigated the radiopathomics relationship and their association with clinical and biological endpoints. We used the Kolmogorov-Smirnov (KS) method to test the differences between the distributions of correlation coefficients with the two imaging modality features. Unsupervised clustering was done to identify which imaging modality captures poor and good survival patients. RESULTS: Our results demonstrated a significant correlation between cell density pathomics and radiomics features. Furthermore, we also found a varying distribution of correlation values between imaging-derived features and clinical endpoints. The KS test revealed that the two imaging feature distributions were different for PFS and CD8 counts, while similar for OS. In addition, clustering analysis resulted in significant differences in the two clusters generated from the radiomics and pathomics features with respect to patient survival and CD8 counts. CONCLUSION: The results of this study suggest a cross-scale association between CT scans and pathology H&E slides among ICI-treated patients. These relationships can be further explored to develop multimodal immunotherapy biomarkers to advance personalized lung cancer care.

A Monte Carlo dose recalculation pipeline for durable datasets: an I-125 LDR prostate brachytherapy use case.

Monte Carlo (MC) dose datasets are valuable for large-scale dosimetric studies. This work aims to build and validate a DICOM-compliant automated MC dose recalculation pipeline with an application to the production of I-125 low dose-rate prostate brachytherapy MC datasets. Built as a self-contained application, the recalculation pipeline ingested clinical DICOM-RT studies, reproduced the treatment into the Monte Carlo simulation, and outputted a traceable and durable dose distribution in the DICOM dose format. MC simulations with TG43-equivalent conditions using both TOPAS andegs_brachyMC codes were compared to TG43 calculations to validate the pipeline. The consistency of the pipeline when generating TG186 simulations was measured by comparing simulations made with both MC codes. Finally,egs_brachysimulations were run on a 240-patient cohort to simulate a large-scale application of the pipeline. Compared to line source TG43 calculations, simulations with both MC codes had more than 90% of voxels with a global difference under ±1%. Differences of 2.1% and less were seen in dosimetric indices when comparing TG186 simulations from both MC codes. The large-scale comparison ofegs_brachysimulations with treatment planning system dose calculation seen the same dose overestimation of TG43 calculations showed in previous studies. The MC dose recalculation pipeline built and validated against TG43 calculations in this work efficiently produced durable MC dose datasets. Since the dataset could reproduce previous dosimetric studies within 15 h at a rate of 20 cases per 25 min, the pipeline is a promising tool for future large-scale dosimetric studies.

Dengue virus M and E proteins belonging to genotype II (Cosmopolitan) of serotype 2 are influenced by the nature of M residue 36.

Mosquito-borne dengue disease is caused by the dengue virus serotype-1 to serotype-4. The contemporary dengue outbreaks in the southwestern Indian ocean coincided with the widespread of dengue virus serotype 2 genotype II (Cosmopolitan), including epidemic viral strains DES-14 and RUN-18 isolated in Dar es Salaam (Tanzania) in 2014 and La Reunion Island (France) in 2018, respectively. Heterodimeric interaction between prM (intracellular precursor of surface structural M protein) and envelope E proteins is required during the initial stage of dengue virus assembly. Amino acid 127 of DES-14 prM protein (equivalent to M36) has been identified as an infrequent valine whereas RUN-18 has a common isoleucine. In the present study, we examined the effect of M-I36V mutation on the expression of a recombinant RUN-18 E protein co-expressed with prM in human epithelial A549 cells. The M ectodomain of dengue virus serotype 2 embeds a pro-apoptotic peptide referred as D2AMP. The impact of M-I36V mutation on the death-promoting capability of D2AMP was assessed in A549 cells. We showed that valine at position M36 affects expression of recombinant RUN-18 E protein and potentiates apoptosis-inducing activity of D2AMP. We propose that the nature of M residue 36 influences the virological characteristics of dengue 2 M and E proteins belonging to genotype II that contributes to global dengue burden.

Risk of Bias Mitigation for Vulnerable and Diverse Groups in Community-Based Primary Health Care Artificial Intelligence Models: Protocol for a Rapid Review.

BACKGROUND: The current literature identifies several potential benefits of artificial intelligence models for populations' health and health care systems' efficiency. However, there is a lack of understanding on how the risk of bias is considered in the development of primary health care and community health service artificial intelligence algorithms and to what extent they perpetuate or introduce potential biases toward groups that could be considered vulnerable in terms of their characteristics. To the best of our knowledge, no reviews are currently available to identify relevant methods to assess the risk of bias in these algorithms. The primary research question of this review is which strategies can assess the risk of bias in primary health care algorithms toward vulnerable or diverse groups? OBJECTIVE: This review aims to identify relevant methods to assess the risk of bias toward vulnerable or diverse groups in the development or deployment of algorithms in community-based primary health care and mitigation interventions deployed to promote and increase equity, diversity, and inclusion. This review looks at what attempts to mitigate bias have been documented and which vulnerable or diverse groups have been considered. METHODS: A rapid systematic review of the scientific literature will be conducted. In November 2022, an information specialist developed a specific search strategy based on the main concepts of our primary review question in 4 relevant databases in the last 5 years. We completed the search strategy in December 2022, and 1022 sources were identified. Since February 2023, two reviewers independently screened the titles and abstracts on the Covidence systematic review software. Conflicts are solved through consensus and discussion with a senior researcher. We include all studies on methods developed or tested to assess the risk of bias in algorithms that are relevant in community-based primary health care. RESULTS: In early May 2023, almost 47% (479/1022) of the titles and abstracts have been screened. We completed this first stage in May 2023. In June and July 2023, two reviewers will independently apply the same criteria to full texts, and all exclusion motives will be recorded. Data from selected studies will be extracted using a validated grid in August and analyzed in September 2023. Results will be presented using structured qualitative narrative summaries and submitted for publication by the end of 2023. CONCLUSIONS: The approach to identifying methods and target populations of this review is primarily qualitative. However, we will consider a meta-analysis if quantitative data and results are sufficient. This review will develop structured qualitative summaries of strategies to mitigate bias toward vulnerable populations and diverse groups in artificial intelligence models. This could be useful to researchers and other stakeholders to identify potential sources of bias in algorithms and try to reduce or eliminate them. TRIAL REGISTRATION: OSF Registries qbph8; https://osf.io/qbph8. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/46684.

Stability of Dengue 2 Nonstructural Glycoprotein 1 (NS1) Is Affected by the Nature of Basic Residue at Position NS1-324.

Dengue is the most prevalent mosquito-borne viral disease. It is caused by the infection of any of the four dengue virus (DENV) serotypes DENV-1 to DENV-4. The DENV non-structural glycoprotein 1 (NS1) plays an important role in virus replication and the immunopathogenesis of virus infection. The NS1 protein has been identified as both a cell-associated homodimer and a soluble secreted lipoprotein nanoparticle. The nature of the residues at positions NS1-272 and NS1-324 in the ß-ladder domain may have an effect on the biological behaviors of DENV-2 NS1 protein in human hepatoma Huh7 cells. The stability of the NS1 protein from the Reunion 2018 DENV-2 strain was affected by the presence of lysine residues at positions 272 and 324. In the present study, we evaluated the impact of mutations into lysine at positions 272 and 324 on recombinant NS1 protein from the DES-14 DENV-2 strain bearing arginine residue on these two positions. The DES-14 NS1 protein mutant bearing a lysine at position 324 was deficient in protein stability and secretion compared to wild-type protein. The defect in the DES-14 NS1 protein mutant was associated to oxidative stress and pro-inflammatory cytokine activation in Huh7 cells. The ubiquitin-proteasome proteolytic pathway might play a key role in the stability of DENV-2 protein bearing a lysine residue at position 324.

Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection.

Extracellular vesicles (EVs), produced during viral infections, are of emerging interest in understanding infectious processes and host-pathogen interactions. EVs and exosomes in particular have the natural ability to transport nucleic acids, proteins, and other components of cellular or viral origin. Thus, they participate in intercellular communication, immune responses, and infectious and pathophysiological processes. Some viruses are known to hijack the cell production and content of EVs for their benefit. Here, we investigate whether two pathogenic flaviviruses i.e., Zika Virus (ZIKV) and Dengue virus (DENV2) could have an impact on the features of EVs. The analysis of EVs produced by infected cells allowed us to identify that the non-structural protein 1 (NS1), described as a viral toxin, is associated with exosomes. This observation could be confirmed under conditions of overexpression of recombinant NS1 from each flavivirus. Using different isolation methods (i.e., exosome isolation kit, size exclusion chromatography, Polyethylene Glycol enrichment, and ELISA capture), we showed that NS1 was present as a dimer at the surface of excreted exosomes, and that this association could occur in the extracellular compartment. This finding could be of major importance in a physiological context. Indeed, this capacity of NS1 to address EVs and its implication in the pathophysiology during Dengue or Zika diseases should be explored. Furthermore, exosomes that have demonstrated a natural capacity to vectorize NS1 could serve as useful tools for vaccine development.

Epistasis between promoter activity and coding mutations shapes gene evolvability.

The evolution of protein-coding genes proceeds as mutations act on two main dimensions: regulation of transcription level and the coding sequence. The extent and impact of the connection between these two dimensions are largely unknown because they have generally been studied independently. By measuring the fitness effects of all possible mutations on a protein complex at various levels of promoter activity, we show that promoter activity at the optimal level for the wild-type protein masks the effects of both deleterious and beneficial coding mutations. Mutations that are deleterious at low activity but masked at optimal activity are slightly destabilizing for individual subunits and binding interfaces. Coding mutations that increase protein abundance are beneficial at low expression but could potentially incur a cost at high promoter activity. We thereby demonstrate that promoter activity in interaction with protein properties can dictate which coding mutations are beneficial, neutral, or deleterious.

Use of Envelope Domain III Protein for the Detection of IgG Type Antibodies Specific to Zika Virus by Indirect ELISA.

Zika virus (ZIKV) diagnostics are crucial for proper antenatal and postnatal care and also for surveillance and serosurvey studies. Since the viremia during ZIKV infection is fleeting, serological testing is highly valuable to inform diagnosis. However, current serology tests using whole virus antigens frequently suffer from cross reactivity issues, delays, and technical complexity, especially in low and middle income countries (LMICs) and endemic countries. Here, we describe an indirect ELISA to detect specific IgG antibodies using the ZIKV envelope domain III (EDIII) protein expressed in Drosophila S2 cells as an immunogen. Using a total of 367 clinical samples, we showed that the EDIII-ELISA was able to detect IgG antibodies against ZIKV with high sensitivity of 100.0% and specificity of 94.7% when compared to plaque reduction neutralization tests (PRNTs) as the gold standard and using 0.208 as the cut-off OD value. These results show the usefulness of the recombinant envelope domain III as an alternative to standard whole virus proteins for ZIKV diagnostics as it improves the sensitivity and specificity of IgG ELISA assay when used as an immunogen. This method should, therefore, be extended to serological diagnostic techniques for other members of the flavivirus genus and for use in IgM diagnostic testing.

Rhizophora mucronata Lam., a halophyte from Mauritius Island, inhibits the entry of Zika virus in human cells (A549)- an in vitro and in silico analysis.

The recent appearance of Zika virus (ZIKV) in Brazil should serve as a wake-up call to international authorities, as it poses a threat to global public health. In the present study, we investigated whether a mangrove plant, Rhizophora mucronata Lam. (R. mucronata) collected in Mauritius, possesses anti-ZIKV activity at the non-cytotoxic doses. ZIKVMC-MR766NIID (ZIKVGFP) was used for assessing anti ZIKV activity. In silico docking (Autodock 4) and molecular simulation were performed on collected data. Using a recombinant ZIKV expressing reporter green fluorescent protein(GFP) protein, we discovered that fruit and root methanolic, decocted fruit and root extracts were effective inhibitors of ZIKV infection in human epithelial A549 cells at negligible cytotoxicity. The mechanisms by which such extracts prevented ZIKV infection are linked to the inability of the virus to attach to the host cell surface. The outcomes of this study were supported by the docking calculations in which some of the dominant compounds have shown high binding affinity against ZIKV. The scientific data gathered in this study might pave the way for the future development of possible R. mucronata inhibitors to combat ZIKV.fCommunicated by Ramaswamy H. Sarma.

Antiviral Effect of Stenocline ericoides DC. and Stenocline inuloides DC., Two Flavonoid-Rich Endemic Plants from Madagascar, against Dengue and Zika Viruses.

Dengue and Zika viruses are identified as the most medically important arthropod-borne viral pathogens. Over the past 20 years, the global dengue incidence has dramatically increased with epidemics of severe dengue where the case fatality rate can reach up to 20% in untreated patients. The association between Zika virus infection and severe congenital anomalies was first reported in 2015. Today no specific antiviral therapies are available for dengue and Zika virus infections, accentuating the need of adapted antiviral strategies based on medicinal plant drug discovery. Plants are a potential source of antiviral phytocompounds which act primarily by blocking virus entry in the host-cell. In the present study, we evaluated whether crude extracts from Stenocline ericoides DC. and Stenocline inuloides DC., two endemic plants from Madagascar, may have antiviral effects against dengue and Zika viruses. We showed that S. ericoides has virucidal action whereas S. inuloides inhibits the early steps of virus infection with a non-cytotoxic effect in human cells. The administration of S. ericoides and S. inuloides extracts in zebrafish had no effect on the behavior of animals at the active doses against dengue and Zika viruses, suggesting the absence of adverse effects at these doses. LC-HRMS2 and molecular networking analyses revealed the richness of these two plants in polyphenols and flavonoid with the presence of clusters of phytocompounds specific to each Stenocline species. Consequently, S. ericoides and S. inuloides represent potential sources for natural and safe antiviral phytocompounds against flaviviruses of medical concern.