Additional file 1: of Find the right sample: A study on the versatility of saliva and urine samples for the diagnosis of emerging viruses

Figure S1. Selection scheme flowchart. Abbreviation: CHIV = Chikungunya Virus, VHF = Viral Hemorrhagic Fever, WN = West Nile, YF = Yellow fever. Table S1. Analysis of different sampling methods for diagnostic of CHIK virus infection. Table S2. Analysis of different sampling methods for diagnostic of Dengue virus infection. Table S3. Analysis of different sampling methods for diagnostic of Ebola/VHF virus infections. Table S4. Analysis of different sampling methods for diagnostic of West Nile virus infection. Table S5. Analysis of different sampling methods for diagnostic of Zika virus infection. Table S6. Analysis of different sampling methods for diagnostic of Yellow Fever virus infection. Table S7. Analysis of different sampling methods for diagnostic of severe acute respiratory syndrome (SARS) virus infection. Table S8. Analysis of different sampling methods for diagnostic of Middle East respiratory syndrome coronavirus (MERS-CoV) infection. (DOCX 162 kb

International External Quality Assessment Study for Molecular Detection of Lassa Virus

<div><p>Lassa virus (LASV) is a causative agent of hemorrhagic fever in West Africa. In recent years, it has been imported several times to Europe and North America. The method of choice for early detection of LASV in blood is RT-PCR. Therefore, the European Network for Diagnostics of ‘Imported’ Viral Diseases (ENIVD) performed an external quality assessment (EQA) study for molecular detection of LASV. A proficiency panel of 13 samples containing various concentrations of inactivated LASV strains Josiah, Lib-1580/121, CSF, or AV was prepared. Samples containing the LASV-related lymphocytic choriomeningitis virus (LCMV) and negative sera were included as specificity controls. Twenty-four laboratories from 17 countries (13 European, one African, one Asian, two American countries) participated in the study. Thirteen laboratories (54%) reported correct results, 4 (17%) laboratories reported 1 to 2 false-negative results, and 7 (29%) laboratories reported 3 to 5 false-negative results. This EQA study indicates that most participating laboratories have a good or acceptable performance in molecular detection of LASV. However, several laboratories need to review and improve their diagnostic procedures.</p></div

Summary of the published protocols used by the participating laboratories.

<p>Summary of the published protocols used by the participating laboratories.</p

A: Expression and purification of DENV-2 Ewt and Equad from Drosophila S2 culture supernatants; supernatant before induction (b.ind.), after 7 days of expression culture (7 d expr.), concentrated via tangential flow (Conc.) and the two step purification with immobilized imidazole affinity (IMAC) and size exclusion chromatography (SEC) were separated on a 10% SDS-PAGE gel under reducing conditions. B: 6 μg of purified DENV1-4 (D1-D4) Equad and DENV-2 Ewt proteins were analyzed with SDS-PAGE. Proteins were stained with Coomassie blue. Size of molecular weight markers in kilo Daltons is indicated on the left.

<p>A: Expression and purification of DENV-2 Ewt and Equad from Drosophila S2 culture supernatants; supernatant before induction (b.ind.), after 7 days of expression culture (7 d expr.), concentrated via tangential flow (Conc.) and the two step purification with immobilized imidazole affinity (IMAC) and size exclusion chromatography (SEC) were separated on a 10% SDS-PAGE gel under reducing conditions. B: 6 μg of purified DENV1-4 (D1-D4) Equad and DENV-2 Ewt proteins were analyzed with SDS-PAGE. Proteins were stained with Coomassie blue. Size of molecular weight markers in kilo Daltons is indicated on the left.</p

Summary of the EQA study for molecular detection of LASV.

<p>^a with stabilizer;</p><p>^b not included in score;</p><p>+, virus correctly detected;—negative result; +/–, indeterminate result</p><p>Summary of the EQA study for molecular detection of LASV.</p

Cytoskeletal integrity is important for TBEV infection in Caco-2 cells.

<p>(<b>A</b>) Actin re-arrangements following TBEV infection. Cells infected with TBEV strain K23 were fixed at 24 h. Samples were stained for actin microfilament and the apical cell-domain (perijunctional cytoskeleton) was visualized by fluorescence microscopy with Acti-stain 488 phalloidin. Non-infected cells were used as controls. White arrows indicate representative areas of more condensed actin filaments in the right image. (<b>B</b>) Inhibition of TBEV cell entry by blocking microfilaments. Caco-2 cells were treated with cytochalasin D (Cyt D), nocodazole (Noc) or LY294002 (LY) for 30 min. DMSO treated Caco-2 cells were used as control. After treatment with inhibitors, cells were incubated with TBEV strain K23 for 1 h. Virus entry was monitored by RT-qPCR, n = 3; **<i>P</i><0.01 to control in Student's t test.</p

Tick-Borne Encephalitis Virus Replication, Intracellular Trafficking, and Pathogenicity in Human Intestinal Caco-2 Cell Monolayers

<div><p>Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent (LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route.</p></div

Antigen titration using the indicated amounts of DENV-1 (A), -2 (B), -3 (C) and -4 (D) Equad and DENV-2 Ewt (E) proteins with three different DENV, WNV and negative (NEG) human sera.

<p>Measurements were performed in duplicates in at least two independent experiments.</p

Comparison of different antigens for the detection of DENV IgG.

<p>Sera positive for IgG against DENV, WNV, TBEV or negative control sera were analyzed with the Panbio Indirect IgG ELISA (black), the DENV-2 Ewt protein (light gray, lined) or the DENV1-4 Equad mix (dark grey, lined). The absolute absorbance is indicated. Cut-Off values for the Panbio test were obtained by calculation of the internal standard of the manufacturer; these are indicated at the right and only refer to this test (horizontal bars: DENV-positive results with an OD-value higher than 1.1*cut-off, equivocal results having an OD-value between 1.1*cut-off and 0.9*cut-off, negative results with an OD-value lower than 0.9*cut-off).</p

IgM-ELISA on 300 ng of DENV-2 Ewt (A) and DENV 1–4 Equad mixture (B) with different DENV, WNV and negative (NEG) sera (n = number of individuals).

<p>Bottom and top of the boxes are the first and third quartiles. The median signal is depicted as a line inside the box. Whiskers represent the 9^th and the 91^st percentile. Measurements were performed in duplicates in at least two independent experiments.</p