Image_1_Position Paper on Road Map for RNA Virus Research in India.PDF

<p>The Indian subcontinent with its population density, climatic conditions, means of subsistence, socioeconomic factors as well as travel and tourism presents a fertile ground for thriving of RNA viruses. Despite being pathogens of huge significance, there is very little focus on research into the biology and pathogenesis of RNA viruses in India. Studies on epidemiology and disease burden, risk factors, the immune response to RNA viruses, circulating virus strains and virus evolution, animal models of disease, antivirals and vaccines are strikingly absent. Emerging RNA viruses such as Zika virus, Nipah virus and Crimean-Congo haemorrhagic fever virus are a matter of grave concern to India. Here we summarize the outcome of the India|EMBO symposium on “RNA viruses: immunology, pathogenesis and translational opportunities” organized at Faridabad, National Capital Region, India, on March 28–30, 2018. The meeting focused on RNA viruses (non-HIV), and both national and international experts on RNA viruses covered topics ranging from epidemiology, immune response, virus evolution and vaccine trials concerning RNA viruses. The aim of the symposium was to create a road map for RNA virus research in India. Both concrete and tentative ideas pointing towards short-term and long-term goals were presented with recommendations for follow-up at government level.</p

Production of soluble factors associated with protection against dengue by monocyte subsets.

<p>Isolated monocyte subsets were either exposed to dengue virus (DENV2, NGC) at a MOI of 10 or medium without virus. Supernatants were harvested over the course of 6 days. (A) Levels of IFN-α were determined by a multi-subtype specific ELISA kit. (B and C) Levels of CXCL10 and TRAIL were determined using multiplex bead arrays. Results are mean ± SE for 6 different donors. There were no significant differences were found between infected CD16⁻ and CD16⁺ monocytes. (D) Supernatants from CD16⁻ and CD16⁺ monocytes exposed to dengue virus or medium without virus were harvested at day 6. These supernatants were passed through 100 kDa centrifuge filters to remove dengue virus. K562 cells were pretreated for 24 hours with either culture medium, supernatants of CD16⁻ or CD16⁺ monocytes with or without virus exposure. Pre-treated K562 cells were washed and infected with dengue virus at a MOI of 2. After 2 days, the extent of infection was determined by intracellular labeling of K562 cells with anti-NS1 antibody. The percentage of NS1⁺ K562 cells after 2 days is shown. Data are representative of 2 experiments using different donors. **, <i>p</i><0.005 between respective monocyte subset with and without virus.</p

Production of inflammatory cytokines by monocyte subsets.

<p>Monocyte subsets were exposed to dengue virus or medium without virus. Supernatants were harvested over the course of 6 days. Levels of (A) IL-1β (B) TNF-α (C) IL-6 (D) CCL2 (E) CCL3 and (F) CCL4 were measured using multiplex bead arrays. Results are mean ± SE of 5 different donors. <b>*</b><i>p</i><0.05, **, <i>p</i><0.005 between CD16⁺ and CD16⁻ monocytes with virus.</p

IL-4 treatment enhances the susceptibility of the CD16⁺ monocyte subset to a greater extent.

<p>Isolated CD16⁻ or CD16⁺ monocyte subsets were pretreated with 25 ng/ml of IL-4 for two days. Cells were subsequently washed and harvested before exposure to dengue virus (DENV2, NGC) at a MOI of 10 or medium without virus. Percentages of CD16⁻ and CD16⁺ monocytes that are (A) NS1⁺ or (B) E-protein⁺ over the course of 6 days after virus exposure. Results are mean ± SE of 5 different donors. (C) Plaque assays with BHK-21 cells were performed with supernatants taken from virus exposed IL-4 treated CD16⁻ or CD16⁺ monocytes over the course of 6 days. Results are mean ± SE from 4 different donors. <b>*</b><i>p</i><0.05, between IL-4 treated CD16⁺ and IL-4 treated CD16⁻ monocytes with virus.</p

Susceptibility of monocyte subsets to dengue virus infection.

<p>(A) Flow cytometric profile of CD16⁻ and CD16⁺ monocytes after isolation. (B) Isolated CD16⁻ or CD16⁺ monocyte subsets were either exposed to dengue virus (DENV2, NGC) at a MOI of 10 or medium without virus. After 2 days, monocytes were fixed, permeabilized and labeled with anti-E-protein and anti-NS1 specific antibodies. Quadrants for virus exposed monocytes (right panel) were set based on monocytes without virus exposure (left panel). Percentage positive cells in each quadrant are shown. Representative data for 6 different donors. (C and D) Percentages of CD16⁻ and CD16⁺ monocytes that are NS1⁺ or E-protein⁺ over the course of 6 days after virus exposure. Results are mean ± SE of 6 different donors. (E) Plaque assays with BHK-21 cells were performed with supernatants taken from virus exposed CD16⁻ or CD16⁺ monocytes over the course of 6 days. Results are mean ± SE from 5 different donors.</p

Infection characteristics of DENV in DC subsets from human skin.

<p>(A) Cells from collagenase-treated healthy human skin were exposed to DENV-2 at MOI 2 for the indicated times. Presence of DENV E protein in DC subsets was established by flow cytometry. (B) Amount of live virus in supernatants of cells from (A) was quantified by plaque-forming assay and used to calculate titer in plaque-forming units per ml (pfu/ml), n = 3 donors, mean ± SEM from three independent experiments. (C) Sorted DC subsets were infected with DENV-2 at MOI 2 and viral RNA was measured in the supernatant by quantitative real time PCR at 0, 24 and 48 hpi. n = 3, mean ± SEM from three independent experiments. (D) Analysis of skin DC subsets and macrophages by flow cytometry in whole human skin. Each dot represents one donor, mean ± SEM. (E) Results from (C) and (D) were used to calculate the relative contribution of each DC subset to the total viral load at 24 and 48 hpi. (F) Infected DC subsets from skin were stained with Annexin V and labeled for DENV E protein after 24 h of exposure to virus to determine the extent of apoptosis (one representative donor of three), mean % per quadrant ± SEM.</p

<i>In vivo</i> infection of resident and migratory DCs in the skin-draining lymph node of IFNAR^−/− mice and their migratory behavior.

<p>(A) Gating strategy to identify lymph node (LN)-resident and migratory DCs in the skin-draining LN of IFNAR^−/− mice. Resident DC subsets are either CD8⁺ (1), CD11b⁻ (2) or CD11b⁺ (3), while migratory DC subsets incorporate those found in the skin: CD103⁺ (4), CD11b⁻ (5), CD11b⁺ (6) and Langerhans Cells (LCs, 7). (B) Mice were inoculated i.d. with 1×10⁶ pfu of DENV-2 D2Y98P/ear and skin-draining lymph nodes (LN) were harvested 2 and 4 days post infection (dpi). Presence of DENV E protein was established by flow cytometry in both LN-resident and migratory DC subsets. (C) Summary of data from (B), 4 to 5 mice (average of two LN/mouse) per group pooled from two independent experiments, mean ± SEM, analyzed with one-way ANOVA followed by Tukey's multiple comparison test, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; non-significant differences are not indicated. (D) Quantification of DCs isolated from LNs at 2 and 4 dpi to address infiltration and migration of different cell subsets. Same conditions as in (C).</p

Identification of DENV susceptible cells from healthy human skin samples.

<p>(A) Flow cytometry of collagenase-treated healthy human skin incubated with medium (mock) or DENV-2 (multiplicity of infection, MOI 2) for 48 h (left). All cells (grey) were overlaid with infected cells (red) to determine expression of CD45 and HLA-DR. (B) Summary data of infected cells from (A) according to their expression of CD45 and HLA-DR of n = 7 donors, mean ± SEM from 7 independent experiments. (C) Mock, UV-inactivated and LIVE DENV-2 treated HLA-DR⁺ cells from (A) were subjected to surface molecule expression analysis to identify four different subsets of dendritic cells (DCs) (gating strategy shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004548#ppat.1004548.s001" target="_blank">Figure S1</a>): CD14⁺ dermal DCs, epidermal Langerhans cells (LCs), CD1c⁺ DCs and CD141⁺ DCs. Infection was quantified by flow cytometry according to detection of DENV E protein. (D) Summary data of (C), each dot represents one donor, mean ± SEM. (E) Cells were exposed to DENV-1, -3 and -4 (MOI 10) for 48 h and presence of DENV E protein detected by flow cytometry, n = 3 donors, mean ± SEM from three independent experiments.</p

<i>In vivo</i> infection of migratory DCs in the skin of IFNAR−/− mice.

<p>(A) Mice were inoculated intra-dermally (i.d) with 1×10⁶ pfu of DENV-2 D2Y98P/ear, and ears were harvested 2 and 4 days post infection. DENV E protein expression was detected by flow cytometry of skin-resident DC subsets and infiltrating monocytes (Gating strategy to identify DC subsets/monocytes shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004548#ppat.1004548.s002" target="_blank">Figure S2</a>) (B) Summary data of (A), 4 to 5 mice (taking the average of two ears/mouse) per group pooled from two independent experiments, mean ± SEM, analyzed with one-way ANOVA followed by Tukey's multiple comparison test, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; non-significant differences are not indicated (C) Quantification of DCs isolated from ears at 2 and 4 dpi to address infiltration and migration of different cell subsets. Same conditions as in (B).</p

T cell stimulation capacity of DENV-exposed DC subsets and their migratory response towards the chemokine CCL19.

<p>(A) Co-culture of sorted infected DCs with allogeneic, CFSE labeled CD3⁺ T cells (ratio 1∶10). Proliferation was measured after 5 days by flow cytometry, one representative result for CD14⁺ dermal DCs is shown. (B) Summary data from all three susceptible DC subsets are shown, each line represents one donor. Statistical analysis was performed using paired two-sided t-test, *p<0.05; ns, not significant, p-values are indicated for each DC subset (C) CCR7 expression on non-treated and DENV-2-exposed DCs at 24 hpi. One representative experiment of three is shown. (D) Skin cell migration was assessed using a 5 µm pore-sized membrane (see Methods Section) with either medium alone or CCL19 (20 ng/ml) in the bottom chamber. Cells were allowed to migrate for 2 h at 37°C before CellTiter Glo activity was measured (RLU, relative light units). Composite data of 4–6 donors is shown, mean ± SEM from 4 independent experiments. (E) Whole skin cells were analyzed by flow cytometry before and after migration towards CCL19. Migrated cells (in the lower well of the chemotaxis plate) were enriched in HLA-DR⁺ cells compared to input cells (left graph). Migrated HLA-DR⁺ cells were enriched in CD1c⁺ DCs and LCs, but not CD14⁺ DCs (right graph; non-infected cells are illustrated; similar results were obtained for infected cells). n = 3 donors, mean ± SEM from three individual experiments.</p