Journal: PLoS ONE

Article Title: A Model System for In Vitro Studies of Bank Vole Borne Viruses

doi: 10.1371/journal.pone.0028992

Figure Lengend Snippet: Cells grown on coverslips were infected at a MOI of 0.1, fixed and stained (in green) for PUUV Kazan-E6 at 5 days post infection (dpi) (A), or for PUUV Umeå at 11 dpi (B). Nuclei were visualized with DAPI staining (blue). Cultivation media from VEFs infected with PUUV Kazan-E6 (C) or PUUV Umeå (D) were collected at the indicated time points post infection and the virus titre, determined as focus forming units (FFU)/ml, were assayed. Graphs represent the means from one of two or more different experiments.

Article Snippet: Wild type PUUV strain Kazan (PUUV-wt) , Vero E6 cell line-adapted PUUV strain Kazan (PUUV Kazan-E6) and Umeå/305/human/95 (PUUV Umeå) , TBEV strain 93–783 , CPXV strain ATCC VR 302, LV strain 145SLG , and green fluorescent protein-expressing Newcastle disease virus (NDV-GFP) were used in the present study.

Techniques: Infection, Staining, Virus

Journal: PLoS ONE

Article Title: A Model System for In Vitro Studies of Bank Vole Borne Viruses

doi: 10.1371/journal.pone.0028992

Figure Lengend Snippet: Cells grown on coverslips were infected with indicated viruses at a MOI of 0.1, fixed and stained (in green) for CPXV at 17 hours post infection (hpi) (A), LV at 8 hpi (B), and for TBEV at 24 hpi (C). Nuclei were visualized with DAPI staining (blue). To measure virus production, VEFs were infected with CPXV at a MOI of 0.1 (D), LV at a MOI of 60 (E) or TBEV at a MOI of 100 (F) and cultivation media was collected at the indicated time points post infection. Virus progeny production (virus/ml cell culture medium) was determined by titration on Vero E6. Error bars represent standard deviations of the means from one experiment.

Article Snippet: Wild type PUUV strain Kazan (PUUV-wt) , Vero E6 cell line-adapted PUUV strain Kazan (PUUV Kazan-E6) and Umeå/305/human/95 (PUUV Umeå) , TBEV strain 93–783 , CPXV strain ATCC VR 302, LV strain 145SLG , and green fluorescent protein-expressing Newcastle disease virus (NDV-GFP) were used in the present study.

Techniques: Infection, Staining, Virus, Cell Culture, Titration

Journal: The Journal of General Virology

Article Title: Remodelling of P-bodies and the cytoskeleton by Orthohantavirus puumalaense (Puumala virus)

doi: 10.1099/jgv.0.002220

Figure Lengend Snippet: Quantitative analysis of PUUV infection kinetics obtained by MuSeq-FISH over 10 days of infection. Vero E6 cells were infected with PUUV and investigated at different time points post-infection using MuSeq-FISH as shown in . ( a ) Per cell counts (abundance) of P-body, N protein, all vRNA species and all viral mRNA spots in non-infected and infected cells. ( b ) Intracellular localization analysis, assessing the distance of each spot from the cellular centre of mass for P-bodies and N protein. Bars show the mean with sem and single cell values are displayed as dots for different time points post-infection. ( c ) Analysis of per cell counts and localization analysis for individual vRNA and viral mRNA species. **** P ≤0.0001, *** P ≤0.001, ** P =0.001 to 0.01 and * P =0.01 to 0.05. Significance was analysed using one-way ANOVA and Tukey’s multiple comparison tests ( n >50).

Article Snippet: If not otherwise stated, PUUV infection (strain Sotkamo: V-2969/81 Charite University Hospital, Berlin, Germany) was conducted in African green monkey kidney epithelial cells (Vero E6, ATCC CRL-1586; American Type Culture Collection, Manassas, VA) maintained in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated FBS, 2 mM l -glutamine, 100 U ml −1 penicillin and 100 μg ml −1 streptomycin (all from PAA Laboratories GmbH, Austria) under standard cell culture conditions.

Techniques: Infection, Single Cell, Comparison

Journal: The Journal of General Virology

Article Title: Remodelling of P-bodies and the cytoskeleton by Orthohantavirus puumalaense (Puumala virus)

doi: 10.1099/jgv.0.002220

Figure Lengend Snippet: Graphical summary of cellular remodelling upon orthohantavirus infection and a putative orthohantavirus replication complex assembly regimen. Based on our experimental results, we propose a model in which PUUV infection induces a relocation of cellular actin and microtubules towards the cell centre, whereas P-bodies exhibit an outward movement and increase in overall abundance. Moreover, our data suggest that virus factories can form in infected cells through a stepwise association of N protein with P-bodies, followed by recruitment of vRNAs and eventually incorporation of viral transcripts.

Article Snippet: If not otherwise stated, PUUV infection (strain Sotkamo: V-2969/81 Charite University Hospital, Berlin, Germany) was conducted in African green monkey kidney epithelial cells (Vero E6, ATCC CRL-1586; American Type Culture Collection, Manassas, VA) maintained in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated FBS, 2 mM l -glutamine, 100 U ml −1 penicillin and 100 μg ml −1 streptomycin (all from PAA Laboratories GmbH, Austria) under standard cell culture conditions.

Techniques: Infection, Virus

Journal: Journal of Virology

Article Title: Interactions and Oligomerization of Hantavirus Glycoproteins

doi: 10.1128/jvi.00481-09

Figure Lengend Snippet: FIG. 1. Effect of low-pH treatment on PUUV Gn-Gc complex in immunoprecipitation of virion lysates. (A) Gn-specific immunoprecipitation with MAb 5A2. Autoradiography of MAb 5A2-Sepharose-bound, [35S]cysteine-labeled Gn and Gc after washing at indicated pH values. The control lane C represents background binding to protein G Sepharose at pH 8.0 without MAb. SDS-PAGE was run under reducing conditions with 2-mercaptoethanol as the reducing agent. The band labeled (Gn)n may also contain Gc. (B) Loss of PUUV Gc recognition by Gc-specific MAb 4G2. Gc in nonlabeled PUUV lysate was bound to MAb 4G2-Sepharose in buffers at the indicated pH. Proteins were separated by reducing SDS-PAGE and immunoblotted with anti-Gc serum. (C) MAb 4G2 binding to Gc prevents dissociation at low pH. Nonlabeled PUUV lysate was bound to MAb 4G2-Sepharose at pH 8.0 and washed in buffers at the indicated pH. The material remaining bound was separated by reducing SDS-PAGE and immunoblotted with anti-Gc serum. C1 and C2 represent controls; C1 is a virus preparation used in immunoprecipitation, and C2 is MAb 4G2-Sepharose without addition of virus. IgG() (50 kDa) and IgG( / ) (25 kDa; migrating at front) chains are indicated. (D) The low-pH-induced dissociation of Gn-Gc complex is reversible. Lysates of metabolically ([35S]cysteine) labeled PUUV treated at pH 5.8 or kept at pH 8.0, as indicated, were immunoprecipitated with Gc-specific MAbs 4G2 and 1C9 at pH 8.0. Autoradiography of SDS-PAGE-separated proteins and the control (lane C) was performed as described for panel A. The protein mobility estimation in panels A and D was done using Precision Plus protein standards (Bio-Rad). The band labeled (Gn)n may also contain Gc.

Article Snippet: The Puumala virus (PUUV) Sotkamo strain and Tula virus (TULV) Moravia strain 5302 were cultivated in Vero E6 green monkey kidney epithelial cells (ATCC 94 CRL-1586).

Techniques: Immunoprecipitation, Autoradiography, Labeling, Control, Binding Assay, SDS Page, Virus, Metabolic Labelling

Journal: Journal of Virology

Article Title: Interactions and Oligomerization of Hantavirus Glycoproteins

doi: 10.1128/jvi.00481-09

Figure Lengend Snippet: FIG. 7. 3D structure model of PUUV Gc with SFV E1 as a template. The Swiss-Model-generated PDB coordinate file of PUUV Gc and the SFV E1 used as a template (PBD code, 2ALA) (45) were visualized with YASARA View. The model and template were superimposed, and the images shown were captured in the same respective angles. DI, DII, and DIII are shown in red, yellow, and blue, respectively, as suggested by Rey and collaborators (41). The PDB coordinates of the created Gc model were analyzed using VADAR, version 1.5, and the Ramachandran plots of the PUUV Gc model and the SFV E1 template obtained from the VADAR server (http://redpoll.pharmacy.ualberta.ca/vadar) are presented below for the respective structures.

Article Snippet: The Puumala virus (PUUV) Sotkamo strain and Tula virus (TULV) Moravia strain 5302 were cultivated in Vero E6 green monkey kidney epithelial cells (ATCC 94 CRL-1586).

Techniques: Generated

Journal: International Journal of Molecular Sciences

Article Title: Differential Cytokine Responses and the Clinical Severity of Adult and Pediatric Nephropathia Epidemica

doi: 10.3390/ijms24087016

Figure Lengend Snippet: The sites of orthohantavirus exposure. The green circles and names (PUUV strains Hu466, Hu475, Hu493, Hu497, Hu500, Hu505, Hu518, Hu520, Hu523, Hu546, Hu563, Hu566, Hu587, Hu599, Hu608, HU_611 and Hu614 from NE patients). The brown circles (Hu526, Hu545, Hu574, Hu 600 and Hu604 strains were closely related to PUUV strains from habitats of bank voles in the east and southeast suburbs of Kazan). The blue circles and names (Hu464, Hu549, Hu598 and HU_639 strains were closely related to PUUV strains from Mamadysh in the east of the Pre-Kama area). The red circles and names for (Hu465). The purple circles (PUUV HU_585, HU_626 and HU_627, Hu461, Hu471, Hu487, Hu488, Hu542, Hu578, Hu602, Hu603, Hu638 and Hu 624 strains).

Article Snippet: Multiple nucleotide sequences of the PUUV strains were first aligned using the MegAlign program (Clustal W algorithm) from the DNASTAR software v. 7.1.0 package Lasergene (DNASTAR, Madison, WI, USA; accessed on 12 February 2022 [ ], and then used for phylogenetic analysis with MEGA v6.0 [ ].

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Differential Cytokine Responses and the Clinical Severity of Adult and Pediatric Nephropathia Epidemica

doi: 10.3390/ijms24087016

Figure Lengend Snippet: Phylogenetic tree. Sequencing and phylogenetic analysis of PUUV strains from humans and rodents.

Article Snippet: Multiple nucleotide sequences of the PUUV strains were first aligned using the MegAlign program (Clustal W algorithm) from the DNASTAR software v. 7.1.0 package Lasergene (DNASTAR, Madison, WI, USA; accessed on 12 February 2022 [ ], and then used for phylogenetic analysis with MEGA v6.0 [ ].

Techniques: Sequencing

Journal: Frontiers in Microbiology

Article Title: Gastrointestinal Tract As Entry Route for Hantavirus Infection

doi: 10.3389/fmicb.2017.01721

Figure Lengend Snippet: Hantavirus survival in gastric juice. Infectious dose of 10 6 Puumala virus particles was suspended to pure gastric juice set to pH 1–7 with NaOH for 1, 10, or 15 min. After given incubation intervals the gastric juice was neutralized with NaOH and the virus suspension was used to infect VERO-E6 cells for focus titration of infectious particles ( n = 4). As at low pH the inactivation activity was expected to be high pH 2 was omitted to save valuable sample volume.

Article Snippet: For in vitro studies: PUUV strain Sotkamo was grown on Vero-E6 cells (ATCC CRL-1586; American Type Culture Collection, Manassas, USA) under standard cell culture conditions.

Techniques: Virus, Incubation, Suspension, Titration, Activity Assay

Journal: Frontiers in Microbiology

Article Title: Gastrointestinal Tract As Entry Route for Hantavirus Infection

doi: 10.3389/fmicb.2017.01721

Figure Lengend Snippet: (A) Growth kinetics of hantaviruses in Caco-2 cells. Caco-2 cells growing on cell culture slides were infected with Puumala virus at MOI of 0.1. Viral replication was monitored by qPCR in culture medium and cells ( n = 3). (B) Translocation of hantavirus through polarized Caco-2 monolayers. Caco-2 cells growing on filter inserts for at least 3 weeks were infected by Puumala virus at MOI 0.1. Apical and basal medium were collected at 24, 120, and 240 h p.i. and investigated for viral replication by qPCR ( n = 3).

Article Snippet: For in vitro studies: PUUV strain Sotkamo was grown on Vero-E6 cells (ATCC CRL-1586; American Type Culture Collection, Manassas, USA) under standard cell culture conditions.

Techniques: Cell Culture, Infection, Virus, Translocation Assay

Journal: Frontiers in Microbiology

Article Title: Gastrointestinal Tract As Entry Route for Hantavirus Infection

doi: 10.3389/fmicb.2017.01721

Figure Lengend Snippet: Intracellular localization of hantavirus in Caco-2 cells. Confocal laser-scanning microscopy of infected cell monolayers. Caco-2 cells growing on coverslips were infected with Puumala virus at MOI of 0.1. The cells were fixed with methanol-free formaldehyde and stained with antibodies against PUUV nucleocapsid protein (red) and early endosomal antigen 1 (EEA1, green). Cell nuclei were stained by DAPI (blue).

Article Snippet: For in vitro studies: PUUV strain Sotkamo was grown on Vero-E6 cells (ATCC CRL-1586; American Type Culture Collection, Manassas, USA) under standard cell culture conditions.

Techniques: Confocal Laser Scanning Microscopy, Infection, Virus, Staining

Journal: Frontiers in Microbiology

Article Title: Gastrointestinal Tract As Entry Route for Hantavirus Infection

doi: 10.3389/fmicb.2017.01721

Figure Lengend Snippet: Epithelial barrier dysfunction in infected Caco-2 monolayers. Caco-2 cells growing on filter inserts were infected by Puumala virus at MOI 0.1. Transepithelial electrical resistance (TER) was measured during infection with chopstick electrodes ( n = 4).

Article Snippet: For in vitro studies: PUUV strain Sotkamo was grown on Vero-E6 cells (ATCC CRL-1586; American Type Culture Collection, Manassas, USA) under standard cell culture conditions.

Techniques: Infection, Virus

Journal: Frontiers in Microbiology

Article Title: Gastrointestinal Tract As Entry Route for Hantavirus Infection

doi: 10.3389/fmicb.2017.01721

Figure Lengend Snippet: (A) Tight junction disturbance. Caco-2 cells grown on filter inserts were infected with Puumala virus at MOI of 0.1 and analyzed by confocal laser-scanning microscopy. At 48 h cells were fixed and stained for PUUV nucleocapsid protein (red) and zonula occludens protein 1 (green). Cell nuclei were stained by DAPI (blue). (B) Cytoskeleton rearrangements in infected Caco-2 monolayers. Caco-2 cells were infected with PUUV at MOI of 0.1. At different time points the cells were fixed with methanol-free formaldehyde and stained with antibodies against PUUV nucleocapsid protein (red) and actin (white). Cell nuclei were stained by DAPI (blue). (C) Leaks in Caco-2 cells monolayers at high MOI. Caco-2 cells grown on coverslips were infected with Puumala virus at MOI of 1.0. At different time points the cells were fixed and stained with antibodies against Puumala virus nucleocapsid protein (red) and zonula occludens protein 1 (green) and analyzed by confocal laser-scanning microscopy. Mock control is shown at 96 h.

Article Snippet: For in vitro studies: PUUV strain Sotkamo was grown on Vero-E6 cells (ATCC CRL-1586; American Type Culture Collection, Manassas, USA) under standard cell culture conditions.

Techniques: Infection, Virus, Confocal Laser Scanning Microscopy, Staining, Control

Journal: Frontiers in Microbiology

Article Title: Gastrointestinal Tract As Entry Route for Hantavirus Infection

doi: 10.3389/fmicb.2017.01721

Figure Lengend Snippet: (A) Intragastral infection of hamster by Puumala virus. Syrian hamsters (animal identification numbers given on the x-axis) were infected with either 1,000 plaque forming units (PFU), 10,000 PFU or 10,000 PFU γ-irradiated PUUV. Thirty five days post infection hamsters were bled to test for seroconversion by N-ELISA. Dots represent hamsters for which subsequent Andes virus challenge was lethal. Specific OD sum is the sum of the optical densities (ODs) greater than background, and represents the area under the titer curve. (B) Survival curves for hamsters “vaccinated” intragastrically with Puumala virus. Forty two days post intragastric Puumala virus challenge, the same hamsters were challenged with 200 PFU ANDV i.m.

Article Snippet: For in vitro studies: PUUV strain Sotkamo was grown on Vero-E6 cells (ATCC CRL-1586; American Type Culture Collection, Manassas, USA) under standard cell culture conditions.

Techniques: Infection, Virus, Irradiation, Enzyme-linked Immunosorbent Assay