Ethics statement and biosafety
This study was approved by the Local Research Ethics Committee of the Ankara Numune Education and Research Hospital, Turkey (Protocol # 17-1338) and Regional Ethics Committee, Stockholm (Dnr. 2017-/1712-31/2). All patients and/or their relatives were informed about the purpose of the study and signed a consent form before collection.
Study design, patients, and sample collection:
We enrolled 18 adult patients (≥18 years) diagnosed with CCHF who were followed up by the clinical service of Infectious Diseases and Clinical Microbiology of Sivas Cumhuriyet University Hospital, Sivas, Turkey. The CCHF patients were divided into three groups using the SGS scores of 1, 2 and 3 40. Blood samples were collected on the admission day (acute stage) and from the survivors one year after their recovery (Supplementary Table 1) following confirmed positive real-time RT-PCR test (Altona Diagnostics®, Hamburg, Germany) and/or serology by IgM indirect immunofluorescence antibody (IFA) assay (Euroimmun®, Luebeck, Germany). Peripheral blood mononuclear cells (PBMCs) and plasma were sent to the Public Health Agency of Sweden, Stockholm, Sweden. Serum cytokine profiling targeting 22 cytokines/chemokines was performed by Public Health England using methods as previously described 41 using a 22 -plex customised luminex kit (Merck Millipore, Darmstadt, Germany) was designed to quantitate: Eotaxin, Granulocyte-Colony Stimulating Factor (G-CSF), Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), Interferon-alpha 2 (IFN-a2), Interferon-gamma (IFN-γ), Interleukin (IL)-10, IL-12, IL-15, IL-17a, IL-1a, IL-9, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IFN-γ-Inducible Protein 10 (IP-10), Monocyte Chemotactic Protein 1 (MCP-1), Macrophage Inhibitory Protein (MIP)-1a, MIP-1b and Tumour Necrosis Factor alpha (TNF-a). Two quality control samples and a standard included with the kit were prepared as per the manufacturer’s instructions. A Luminex MAGPIX instrument using Exponent software (Invitrogen, Paisley, UK) was used to obtain results in the form of MFI (median fluorescence intensity), which was converted to concentration (pg/ml) using the standard curve prepared. Plate data was accepted based on negative background wells and QC data within the expected ranges.
RNA extraction and sequencing (Illumina RNAseq) and transcriptomics analysis:
Total RNA was extracted from Trizol-treated PBMC using the Direct-zol RNA Miniprep (Zymo Research, CA, USA) according to the manufacturer's protocol. RNA-Seq was performed at the National Genomics Infrastructure, Science for Life Laboratory, Stockholm, Sweden, as described by us previously42. The transcriptomics data analysis was performed as described by us recently14. The distribution of all samples was then visualized after reducing the dimension of the data by applying the Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) technique using R package umap v0.2.6.043. The reduced dimensions of the data were plotted in 2D space using R package ggplot2 v3.3.244. Differential gene expression analysis was performed using raw read counts of remaining samples using the R/Bioconductor package DESeq2 v1.26.045. Genes with adjusted p-values of less than 0.05 were considered as significantly regulated. Functional analysis of the significantly regulated genes was performed using the enrichr module of the python package GSEAPY v 0.9.1646, 47. Gene ontology (GO) enrichment analysis was performed using the Enrichr web resource 47, and the GO biological process 2018 category was selected. Redundant GO terms were removed using the online tool REVIGO15. Heatmaps were generated using the R/Bioconductor package ComplexHeatmap v2.2.048. Bubble plots, MA plots, Volcanoplots, violin plots and bar plots were created using R package ggplot2 v3.3.2. Network visualization was performed using Cytoscape v3.6.149. Venn diagrams were constructed using the online tool InteractiVenn50. All the codes are available in GitHub (https://neogilab.github.io/CCHF-Turkey/). Raw RNAseq data is available in Sequence Read Archive (SRA) with temporary id SUB8608640.
Cells and viruses:
The CCHFV strain IbAr10200 (originally isolated from Hyalomma excavatum ticks from Sokoto, Nigeria, in 1966) was used in this study. The SW13- ATCC®-CCL-105TM and human hepatocyte-derived cellular carcinoma cell line Huh7 was obtained from Marburg Virology Laboratory, (Philipps-Universität Marburg, Marburg, Germany) and matched the STR reference profile of Huh751. CCHFV was propagated and titered in SW13 cells. The virus was serially diluted 10-fold and 100ul of each dilution was added to SW13 cells in a 96 well plate. At 48 h post-infection (hpi), cells were fixed in ice-cold acetone-methanol (1:1) and stained using a rabbit polyclonal anti-CCHFV nucleocapsid antibody followed by a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody (Thermo Fisher Scientific, US) and DAPI (Roche, US). The number of fluorescent foci in each well was counted using a fluorescence microscope and the titer was determined. Human 293 FLP-IN TRex cells expressing CAT or ISG20 under the control of a Tet-on promoter were kindly provided by Ju-Tao Guo (Drexel University College of Medicine, Pennsylvania, USA).52 The cells were propagated in DMEM supplemented with 5% Tet-negative FCS (PAA Lab, US).
In vitro infection assay in Huh7 cells and tandem mass tag (TMTpro) labelled reverse phase liquid chromatography mass-spectrometric (RPLC-MS/MS) analysis:
Huh7 cells were infected with the CCHFV in triplicate, as described by us previously14. Briefly, Huh7 cells were infected with CCHFV IbAr10200 at a multiplicity of infection (MOI) of 1. After 1 h of incubation (37°C, 5% CO2) the inoculum was removed, the cells were washed with PBS, and 2 ml DMEM supplemented with 5% heat-inactivated FBS was added to each well. Samples were collected in triplicate at 24 and 48 hpi along with controls. Following the protein digestion in S-Trap microcolumns (Protifi, Huntington, NY), the resulting peptides were labeled with TMTpro tags. Labeled peptides were fractionated by high pH (HpH) reversed-phase chromatography, and each fraction was analyzed on an Ultimate 3000 UHPLC (Thermo Scientific, San Jose, CA) in a 120 min linear gradient. Proteins were searched against the SwissProt human database using the search engine Mascot v2.5.1 (MatrixScience Ltd, UK) in Proteome Discoverer v2.4 (Thermo Scientific) software allowing up to two missed cleavages.
Proteomics data analysis:
The raw data were first filtered to remove missing data. Proteins detected in all samples were retained for analysis resulting in 8,501 proteins in the filtered dataset. The filtered data was then normalized by applying eight different methods using R/Bioconductor package NormalyzerDE v1.4.053. The quantile normalization was found superior to other methods and was selected for further use. Differential protein expression analysis was performed using R/Bioconductor package limma v3.42.254. Proteins with adjusted p-values of less than 0.05 were regarded as significant. KEGG pathway enrichment analysis of significantly regulated proteins was performed using the enrichr module of python package GSEAPY v 0.9.16. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD022672.
ISG20 inhibition assay:
Tet-inducible HEK-293 FLP-IN TRex cells containing a CAT or ISG20 expression cassette were seeded on in six-well dishes and treated with doxycycline at a final concentration of 2 μg/ml and incubated for 36 h. Cells were infected with CCHFV strain IbAr10200 at an MOI of 1, and supernatants were titrated at 48 hpi by enumeration of ﬂuorescent foci and calculation of progeny virus titers as described by us previously 11.
RNAscope targeting IFI27 (ISG12) and CCHF:
The RNAscope® ISH Assays (ACD Bioscience,US) targeting IFI27 (440111, ACD Bioscience, US) and CCHFV (510621, ACD Bioscience, US) were perfomed as described previously55. SW13 cells were infected with CCHFV (Ibar 10200 strain) at MOI of 0.1. After 1h, inoculum was replenished with fresh Leitbovitz medium containing 5% FBS and incubated 48h. After infection cells were fixed 30 min using ice-cold acetone. Cells were permeabilized using 0.1% Triton x-100 for 10 min at RT prior probe incubation at 40°C of IFI27 and CCHFV probes (RNAscope® ISH ACD,US) for 2h, 30min, 15 min, 30 min and 15 min, respectively. Cells were further stained using DAPI and coverslip attached using ProLong Gold Antifade reagent (P10144, Thermofisher). Images were acquired using Nikon Single Point scanning confocal with 60/1.4 oil objective.
Metabolic perturbation and virus infection.
To inhibit glycolysis, glutaminolysis and Akt/mTOR pathway, following 1hpi (moi 0.1) the cells were treated with 2-deoxy-D-glucose (2-DG, 5mM), diazo-5-oxo-L-norleucine (DON, 50mM) and MK-2206 (10mM) respectively. The cells were collected after 24 hpi and the cells were lysed in Trizol reagent. RNA was extracted using the Direct-zol RNA Miniprep kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. Viral RNA was measured by quantitative real real-time polymerase chain reaction (qRT-PCR) using TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) with primers and probe specific for the CCHFV L gene; Forward: 5-GCCAACTGTGACKGTKTTCTAYATGCT-3’, Reverse-1: 5’- CGGAAAGCCTATAAAACCTACCTTC-3’, Reverse-2: 5’-CGGAAAGCCTATAAAACCTGCCYTC-3’ and Reverse-3: 5’-CGGAAAGCCTAAAAAATCTGCCTTC-3’ and Probe FAM-CTGACAAGYTCAGCAAC –MGB. RNAse was used as endogenous control. The cycling reactions was performed using a capillary Roche LightCycler 2.0 system.
Messanger RNA (mRNA) expression of a few ISG transcripts (ISG15, IFIT1, MX1 and MX2) and human b-actin were measured by qRT-PCR. The sequences of the qPCR primers are listed in supplemental table S1. Reverse transcription of extracted RNA was performed using a high capacity reverse transcription kit (Applied Biosystems, USA) for 10 min at 25°C, followed by 37°C for 120 min and 85°C for 5 min. Quantitative RT-PCR assays were setup using the Power SYBR Green PCR Master Mix (Applied Biosystems, UK) using 250nM of primer pairs with cycling conditions: initial denaturation 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min. Melting curves were run by incubating the reaction mixtures at 95°C for 15 sec, 60°C for 20 sec, 95°C for 15 sec, ramping from 60°C to 95°C in 1°C/sec. The values were normalized to endogenous b-actin. Fold change was calculated as: Fold Change = 2-Δ(ΔCt).