Dengue virus NS1 cytokine-independent vascular leak is dependent on endothelial glycocalyx components

<div><p>Dengue virus (DENV) is the most prevalent, medically important mosquito-borne virus. Disease ranges from uncomplicated dengue to life-threatening disease, characterized by endothelial dysfunction and vascular leakage. Previously, we demonstrated that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability in a systemic mouse model and human pulmonary endothelial cells, where NS1 disrupts the endothelial glycocalyx-like layer. NS1 also triggers release of inflammatory cytokines from PBMCs via TLR4. Here, we examined the relative contributions of inflammatory mediators and endothelial cell-intrinsic pathways. <i>In vivo</i>, we demonstrated that DENV NS1 but not the closely-related West Nile virus NS1 triggers localized vascular leak in the dorsal dermis of wild-type C57BL/6 mice. <i>In vitro</i>, we showed that human dermal endothelial cells exposed to DENV NS1 do not produce inflammatory cytokines (TNF-α, IL-6, IL-8) and that blocking these cytokines does not affect DENV NS1-induced endothelial hyperpermeability. Further, we demonstrated that DENV NS1 induces vascular leak in TLR4- or TNF-α receptor-deficient mice at similar levels to wild-type animals. Finally, we blocked DENV NS1-induced vascular leak <i>in vivo</i> using inhibitors targeting molecules involved in glycocalyx disruption. Taken together, these data indicate that DENV NS1-induced endothelial cell-intrinsic vascular leak is independent of inflammatory cytokines but dependent on endothelial glycocalyx components.</p></div

HMEC-1 do not produce the inflammatory cytokines IL-6, TNF-α, or IL-8 in response to DENV2 NS1 stimulation <i>in vitro</i>.

<p><b>(A-C)</b> HMEC-1 were stimulated with LPS (10 or 100 ng/ml; red squares and orange triangles, respectively) or DENV2 NS1 (5 or 10 μg/ml; dark blue triangles and light blue diamonds, respectively), and supernatant was collected at 0, 1, 3, 6, 12, and 24 hours post-treatment. Untreated HMEC-1 monolayers were used as a control (black circles). ELISAs for <b>(A)</b> IL-6, <b>(B)</b> TNF-α, and <b>(C)</b> IL-8 were performed on all samples. All data shown represent the mean +/- SEM and were collected from two independent experiments. A repeated measure two-way ANOVA with multiple comparisons to the untreated group using Dunnett’s multiple comparison test was used to determine significance of treatment with LPS (10 and 100 ng/ml) or DENV2 NS1 (5 and 10 μg/ml). *<i>P</i> < 0.05, **<i>P</i> < 0.01, ****<i>P</i> < 0.0001.</p

Inflammatory cytokines TNF-α and IL-6 are not involved in DENV2 NS1-induced endothelial hyperpermeability <i>in vitro</i>.

<p><b>(A-B)</b> Trans-endothelial electrical resistance (TEER) of HMEC-1 monolayers incubated with 5 μg/ml DENV2 NS1 (blue squares), 10 ng/ml recombinant cytokine (<b>(A)</b> IL-6, <b>(B)</b> TNF-α; purple diamonds), 100 ng/ml anti-cytokine mAbs (<b>(A)</b> IL-6, <b>(B)</b> TNF-α; orange triangles), recombinant cytokine + specific mAb (<b>(A)</b> IL-6, <b>(B)</b> TNF-α; green diamonds), or DENV2 NS1 + specific mAb (<b>(A)</b> IL-6, <b>(B)</b> TNF-α; red circles). The background signal was subtracted (using TEER values from a blank Transwell), and data were normalized to untreated HMEC-1. All data shown represent the mean +/- SEM and were collected from two independent experiments. Data represent two replicate Transwells per condition. A repeated measure two-way ANOVA was used to determine the significance of anti-cytokine mAbs on DENV2 NS1-induced hyperpermeability in HMEC-1. ns = not significant.</p

DENV2 NS1 induces degradation of sialic acid, activation of cathepsin L, and shedding of heparan sulfate in HMEC-1 <i>in vitro</i>.

<p><b>(A-D)</b> HMEC-1 monolayers treated with 5 μg/ml of DENV2 NS1 (middle column) or 5 μg/ml of DENV2 NS1 and an inhibitor cocktail (Zanamivir, 100 μM; Cathepsin L Inhibitor, 10 μM; OGT 2115, 1.0 μM; right column). Untreated monolayers were used as a control (left column). Six hours post-treatment, cells were stained for <b>(B)</b> sialic acid (WGA-A647, red; top row images), <b>(C)</b> cathepsin L activity (Magic Red Cathepsin L detection kit, red; middle row images), or <b>(D)</b> heparan sulfate (Heparan Sulfate mAb clone F58-10E4, green; bottom row images) and imaged on a Zeiss LSM 710 Axio Observer inverted fluorescence microscope equipped with a 34-channel spectral detector at 20x magnification. <b>(A)</b> Images were acquired using the Zen 2010 software (Zeiss). Nuclei were stained with <i>Hoechst</i> (blue). Images shown at 20X; scale bar, 10 μM. Representative images shown. <b>(B-D)</b> Quantification of MFI in <b>Fig 6A</b>.</p

Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism.

Flaviviruses cause systemic or neurotropic-encephalitic pathology in humans. The flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein involved in viral replication, immune evasion, and vascular leakage during dengue virus infection. However, the contribution of secreted NS1 from related flaviviruses to viral pathogenesis remains unknown. Here, we demonstrate that NS1 from dengue, Zika, West Nile, Japanese encephalitis, and yellow fever viruses selectively binds to and alters permeability of human endothelial cells from lung, dermis, umbilical vein, brain, and liver in&nbsp;vitro and causes tissue-specific vascular leakage in mice, reflecting the pathophysiology of each flavivirus. Mechanistically, each flavivirus NS1 leads to differential disruption of endothelial glycocalyx components, resulting in endothelial hyperpermeability. Our findings reveal the capacity of a secreted viral protein to modulate endothelial barrier function in a tissue-specific manner both in&nbsp;vitro and in&nbsp;vivo, potentially influencing virus dissemination and pathogenesis and providing targets for antiviral therapies and vaccine development

Inhibition of sialidases, cathepsin L, and heparanase prevents DENV2 NS1-induced endothelial hyperpermeability <i>in vivo</i>.

<p>Hair was removed from the dorsal dermis of wild-type B6 mice, and mice were allowed to recover for 3 days. On the day of the assay, mice received an intraperitoneal dose of inhibitor cocktail (Zanamivir, Cathepsin L Inhibitor, and OGT 2115; 1 mg/ml of each inhibitor) 6 hours pre-assay and then immediately preceding the start of the assay (n = 4; closed symbols). Control mice received injections of DMSO, PBS, and water as a vehicle control (n = 3; open symbols). Retro-orbital injections of Alexa Fluor 680-conjugated dextran were then administered, followed by intradermal injections of PBS (black circles), 200 ng VEGF (purple squares), 15 μg DENV2 NS1 (blue triangles), and 7.5 μg DENV2 NS1 (green triangles). The dermis from each mouse was collected and processed two hours post-injection. Data represent the fold change of mean fluorescent intensity from VEGF and DENV2 NS1 injections to PBS injections. Data represent mean +/- SEM and were collected from 2 independent experiments. Unpaired, parametric, two-tailed t-tests were used to determine significance between inhibitor-treated and mock-treated groups. ns = not significant, *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

A 2-Gene Host Signature for Improved Accuracy of COVID-19 Diagnosis Agnostic to Viral Variants.

The continued emergence of SARS-CoV-2 variants is one of several factors that may cause false-negative viral PCR test results. Such tests are also susceptible to false-positive results due to trace contamination from high viral titer samples. Host immune response markers provide an orthogonal indication of infection that can mitigate these concerns when combined with direct viral detection. Here, we leverage nasopharyngeal swab RNA-seq data from patients with COVID-19, other viral acute respiratory illnesses, and nonviral conditions (n = 318) to develop support vector machine classifiers that rely on a parsimonious 2-gene host signature to diagnose COVID-19. We find that optimal classifiers include an interferon-stimulated gene that is strongly induced in COVID-19 compared with nonviral conditions, such as IFI6, and a second immune-response gene that is more strongly induced in other viral infections, such as GBP5. The IFI6+GBP5 classifier achieves an area under the receiver operating characteristic curve (AUC) greater than 0.9 when evaluated on an independent RNA-seq cohort (n = 553). We further provide proof-of-concept demonstration that the classifier can be implemented in a clinically relevant RT-qPCR assay. Finally, we show that its performance is robust across common SARS-CoV-2 variants and is unaffected by cross-contamination, demonstrating its utility for improved accuracy of COVID-19 diagnostics. IMPORTANCE In this work, we study upper respiratory tract gene expression to develop and validate a 2-gene host-based COVID-19 diagnostic classifier and then demonstrate its implementation in a clinically practical qPCR assay. We find that the host classifier has utility for mitigating false-negative results, for example due to SARS-CoV-2 variants harboring mutations at primer target sites, and for mitigating false-positive viral PCR results due to laboratory cross-contamination. Both types of error carry serious consequences of either unrecognized viral transmission or unnecessary isolation and contact tracing. This work is directly relevant to the ongoing COVID-19 pandemic given the continued emergence of viral variants and the continued challenges of false-positive PCR assays. It also suggests the feasibility of pan-respiratory virus host-based diagnostics that would have value in congregate settings, such as hospitals and nursing homes, where unrecognized respiratory viral transmission is of particular concern

Systems immunology of transcriptional responses to viral infection identifies conserved antiviral pathways across macaques and humans

Summary: Viral pandemics and epidemics pose a significant global threat. While macaque models of viral disease are routinely used, it remains unclear how conserved antiviral responses are between macaques and humans. Therefore, we conducted a cross-species analysis of transcriptomic data from over 6,088 blood samples from macaques and humans infected with one of 31 viruses. Our findings demonstrate that irrespective of primate or viral species, there are conserved antiviral responses that are consistent across infection phase (acute, chronic, or latent) and viral genome type (DNA or RNA viruses). Leveraging longitudinal data from experimental challenges, we identify virus-specific response kinetics such as host responses to Coronaviridae and Orthomyxoviridae infections peaking 1–3 days earlier than responses to Filoviridae and Arenaviridae viral infections. Our results underscore macaque studies as a powerful tool for understanding viral pathogenesis and immune responses that translate to humans, with implications for viral therapeutic development and pandemic preparedness

Upper airway gene expression shows a more robust adaptive immune response to SARS-CoV-2 in children.

Unlike other respiratory viruses, SARS-CoV-2 disproportionately causes severe disease in older adults whereas disease burden in children is lower. To investigate whether differences in the upper airway immune response may contribute to this disparity, we compare nasopharyngeal gene expression in 83 children (&lt;19-years-old; 38 with SARS-CoV-2, 11 with other respiratory viruses, 34 with no virus) and 154 older adults (&gt;40-years-old; 45 with SARS-CoV-2, 28 with other respiratory viruses, 81 with no virus). Expression of interferon-stimulated genes is robustly activated in both children and adults with SARS-CoV-2 infection compared to the respective non-viral groups, with only subtle distinctions. Children, however, demonstrate markedly greater upregulation of pathways related to B cell and T cell activation and proinflammatory cytokine signaling, including response to TNF and production of IFNγ, IL-2 and IL-4. Cell type deconvolution confirms greater recruitment of B cells, and to a lesser degree macrophages, to the upper airway of children. Only children exhibit a decrease in proportions of ciliated cells, among the primary targets of SARS-CoV-2, upon infection. These findings demonstrate that children elicit a more robust innate and especially adaptive immune response to SARS-CoV-2 in the upper airway that likely contributes to their protection from severe disease in the lower airway