Rescue of recombinant MARV from R06E cells.

<p>R06E cells were transfected with all the plasmids sufficient for replication and transcription, a T7 polymerase construct and a T7-driven full-length cDNA construct of MARV (clone #16). (A) CPE formation was monitored at day 7 post transfection (p.t.). (B) Supernatant of transfected cells collected on day 7 was used to infect fresh VeroE6 cells (passage 1, p1). CPE formation was monitored 8 days post infection. (C) Supernatant of p1 was collected on day 8 p.i. and viral RNA was extracted. Glycoprotein gene-specific RT-PCR and subsequent restriction of the DNA with <i>KpnI</i>, a restriction site present only in the wild type genome, revealed the rescue of recombinant virus. (D) On day 8 p.i. supernatant and cells were lysed and subjected to Western blot analysis using monoclonal antibodies to detect the MARV proteins NP and VP40. * unknown cellular protein.</p

Additional file 3: Table S3. of Host-feeding patterns of mosquito species in Germany

Chi-square tests on the differences in the frequencies of detected mammalian or avian hosts among all pairs of used trapping methods with adjusted P-values for multiple comparisons. (DOCX 13 kb

MARV-specific <i>in vitro</i> assays.

<p>(A) Minigenome assay. Different cell lines were transfected with all of the plasmids necessary for replication and transcription of a MARV (3M–5M) minigenome. Relative light units (RLU) represent the efficiency of replication and transcription of the minigenome (upper panel). This experiment was performed in triplicate and standard deviations are shown. Asterisks indicate statistically significant differences (*** P-value≤0.002) RLUs shown in the upper panel were normalized to the transfection efficiency of the cells, as analyzed by a GFP-reporter construct. (lower panel) (B) iVLP assay with pretransfected indicator cells. HEK293 or R06E cells were transfected with all of the plasmids necessary to produce MARV iVLPs (producer cells, pc). Supernatants were collected 72 h p.t. and used to infect new indicator cells (HUH7 or R06E cells) pretransfected with all of the plasmids necessary for replication and transcription (indicator cells, ic). Three days p.i. the luciferase activity in the ic was determined, reflecting iVLP formation, budding, iVLP entry, minigenome delivery and secondary transcription. (C) iVLP assay with naïve indicator cells. R06E cells were transfected with all of the plasmids necessary to produce iVLPs (pc). Supernatant was collected 72 h p.t. and used to infect R06E cells that were not pretransfected. Luciferase activity in the indicator cells was determined 48 or 72 h p.i., reflecting iVLP formation, budding, iVLP entry, minigenome delivery and primary transcription of minigenomes. This experiment was performed in triplicate and standard deviations are shown.</p

Expression of viral proteins in different cell lines.

<p>(A) 4×10⁵ HUH7, VeroE6 or R06E cells were infected with 0.1 TCID₅₀/cell MARV or ZEBOV. Cell lysates were prepared at 48 and 72 h p.i., subjected to Western blot analysis to detect cellular tubulin and VP40 of MARV and ZEBOV using mouse monoclonal antibodies. The total amount of cellular proteins in the samples was quantified by separating cell lysates on SDS PAGE, which were then stained with Coomassie Blue. Using the Odyssey Infrared Imaging Application Software, the protein signals were quantified. VP40 levels normalized to total cell protein are shown in (B).</p

Additional file 2: Table S2. of Host-feeding patterns of mosquito species in Germany

Kruskal-Wallis tests on the differences of the percentages of detected birds, non-human mammals and humans between the three land use classes (natural, rural and urban) for the three most frequent mosquito species (Fig. 3). (DOCX 14 kb

Detection of Puumala Hantavirus Antigen in Human Intestine during Acute Hantavirus Infection

<div><p>Background</p><p>Puumala virus (PUUV) is the most important hantavirus species in Central Europe. Nephropathia epidemica (NE), caused by PUUV, is characterized by acute renal injury (AKI) with thrombocytopenia and frequently gastrointestinal symptoms.</p><p>Methods</p><p>456 patients with serologically and clinically confirmed NE were investigated at time of follow-up in a single clinic. The course of the NE was investigated using medical reports. We identified patients who had endoscopy with intestinal biopsy during acute phase of NE. Histopathological, immunohistochemical and molecular analyses of the biopsies were performed.</p><p>Results</p><p>Thirteen patients underwent colonoscopy or gastroscopy for abdominal pain, diarrhea, nausea and vomiting during acute phase of NE. Immunohistochemistry (IHC) revealed PUUV nucleocapsid antigen in 11 biopsies from 8 patients; 14 biopsies from 5 patients were negative for PUUV nucleocapsid antigen. IHC localized PUUV nucleocapsid antigen in endothelial cells of capillaries or larger vessels in the lamina propria. Rate of AKI was not higher and severity of AKI was not different in the PUUV-positive compared to the PUUV-negative group. All IHC positive biopsies were positive for PUUV RNA using RT-PCR. Phylogenetic reconstruction revealed clustering of all PUUV strains from this study with viruses previously detected from the South-West of Germany. Long-term outcome was favorable in both groups.</p><p>Conclusions</p><p>In patients with NE, PUUV nucleocapsid antigen and PUUV RNA was detected frequently in the intestine. This finding could explain frequent GI-symptoms in NE patients, thus demonstration of a more generalized PUUV infection. The RT-PCR was an effective and sensitive method to detect PUUV RNA in FFPE tissues. Therefore, it can be used as a diagnostic and phylogenetic approach also for archival materials. AKI was not more often present in patients with PUUV-positive IHC. This last finding should be investigated in larger numbers of patients with PUUV infection.</p></div

Detection of PUUV nucleocapsid antigen in the capillaries in the lamina propria of a gastric biopsy 8 days after beginning of symptoms associated with NE (fever, abdominal pain, nausea and vomiting) A Higher magnification demonstrates capillaries with positive endothelial cells in the lamina propria (same patient) B Endothelial cell with PUUV nucleocapsid antigen (same patient) C.

<p>Detection of PUUV nucleocapsid antigen in the capillaries in the lamina propria of a gastric biopsy 8 days after beginning of symptoms associated with NE (fever, abdominal pain, nausea and vomiting) A Higher magnification demonstrates capillaries with positive endothelial cells in the lamina propria (same patient) B Endothelial cell with PUUV nucleocapsid antigen (same patient) C.</p

Detection of PUUV nucleocapsid antigen in the capillaries in the lamina propria (gastric biopsy) 10 days after beginning of symptoms A and B Immunohistochemical double labeling with the monoclonal antibody A1C5 (brown) and the monoclonal antibody to CD34 reacting the endothelial cells (blue).

<p>The PUUV nucleocapsid antigen is granular C Detection of PUUV nucleocapsid antigen in interstitial cells (gastric biopsy) D.</p

Maximum-likelihood phylogenetic tree of PUUV based on partial (144 bp) sequences of the small segment showing the placement of the PUUV strains from this study into the German subtypes.

<p>Bootstrap values >70%, calculated from 10,000 replicates are shown at the tree branches. For clarity, the Central European PUUMV clade include only German strains. Previously characterized PUUMV clades: <b>ALAD</b>, Alpe-Adrian; <b>CE</b>, Central European; <b>S-SCA</b>, South Scandinavian; <b>FIN</b>, Finnish; <b>RUS</b>, Russian; <b>N-SCA</b>, North Scandinavian; <b>DAN</b>, Dannish. The tree was rooted using murine-associated hantavirus: Hantaan virus (HNTV). Scale bar indicates mean number of nucleotide substitutions per site. GenBank accession numbers and the name of the strains are shown.</p

Baseline characteristics of study population at follow-up.

<p>Baseline characteristics of study population at follow-up.</p