Journal: Frontiers in Immunology

Article Title: Induction of humoral and cell-mediated immunity to the NS1 protein of TBEV with recombinant Influenza virus and MVA affords partial protection against lethal TBEV infection in mice

doi: 10.3389/fimmu.2023.1177324

Figure Lengend Snippet: In vitro characterization of MVA-NS1. (A) Homologous and intragenomic homologous (marker gene deletion) recombinations lead to production of MVA-NS1 with TBEV NS1 expression under transcriptional control of VACV late promotor psynII. (B) PCR products specific for the six major deletion sites inside the MVA genome performed on MVA-NS1 (1% agarose TBE gel) (I: 291 bp, II: 354 bp, III: 447 bp, IV: 502 bp, V: 603 bp, VI: 702 bp). Integration of the NS1 gene in deletion site III was confirmed (III: 1596 bp). (C) Immunostaining of wtMVA or MVA-NS1 infected HeLa cells (MOI 1). 24 hpi, cells were fixed with 4% PFA, for intracellular staining permeabilized with TritonX®-100 and immunostained against VACV D8 and TBEV NS1 (20x magnification). (D) Western blot analysis of whole cell lysate from HeLa cells infected with wtMVA or MVA-NS1 for 24 h (MOI 5). Blots were stained against TBEV NS1. For control, antibodies against VACV D8 and GAPDH were included. (E) Growth curves of wtMVA (black) and MVA-NS1 (gray) on primary CEF cells (dotted line) and HeLa cells (solid line) (MOI 0.05). Mann-Whitney test was used for statistical comparison between CEF and HeLa cells (* p<0.05).

Article Snippet: Plasmid encoding for the Kozak sequence followed by the TBEV E gene signal peptide and entire TBEV NS1 were synthesized (based on TBEV Neudoerfl, UniProtKB: P14336; GenScript Biotech Corp) and cloned into MVA transfer plasmid pIIIsynIIred under transcriptional control of VACV late promotor psynII ( ). pIIIsynIIred contains mCherry as marker gene which is flanked by short repetitive regions.

Techniques: In Vitro, Marker, Expressing, Control, Immunostaining, Infection, Staining, Western Blot, MANN-WHITNEY, Comparison

Journal: Frontiers in Immunology

Article Title: Induction of humoral and cell-mediated immunity to the NS1 protein of TBEV with recombinant Influenza virus and MVA affords partial protection against lethal TBEV infection in mice

doi: 10.3389/fimmu.2023.1177324

Figure Lengend Snippet: In vitro characterization of IAV-NS1. (A) Schematic representation of the NA-TBEV NS1 gene segment, NCR = non-coding region, ATG = start codon, *Stop = stop codon. (B) PCR products specific for IAV NA performed on I = rPR8, II = IAV-RBD, III = IAV-NS1 and IV = negative control (1% agarose TBE gel, M = 1 kb ladder). (C) Immunostaining of rPR8, IAV-RBD or IAV-NS1 infected MDCK cells (MOI 0.01). 24 hpi, cells were fixed with 4% PFA, for intracellular staining permeabilized with TritonX®-100 and immunostained against IAV NP or TBEV NS1 (10x magnification). (D) Western blot analysis of whole cell lysate from MDCK cells infected with rPR8, IAV-RBD or IAV-NS1 for 24 h (MOI 0.01). Blots were stained against TBEV NS1. For control, antibodies against IAV nucleoprotein (NP) and GAPDH were included. (E) Growth curves for rPR8 (black), IAV-RBD (gray) and IAV-NS1 (red) in the presence (solid line) or absence (dotted line) of eNA on MDCK cells (MOI 0.001). Mann-Whitney test was used for statistical comparison between rPR8 –eNA and IAV-RBD –eNA or IAV-NS1 –eNA, respectively (* p<0.05).

Article Snippet: Plasmid encoding for the Kozak sequence followed by the TBEV E gene signal peptide and entire TBEV NS1 were synthesized (based on TBEV Neudoerfl, UniProtKB: P14336; GenScript Biotech Corp) and cloned into MVA transfer plasmid pIIIsynIIred under transcriptional control of VACV late promotor psynII ( ). pIIIsynIIred contains mCherry as marker gene which is flanked by short repetitive regions.

Techniques: In Vitro, Negative Control, Immunostaining, Infection, Staining, Western Blot, Control, MANN-WHITNEY, Comparison

Journal: Frontiers in Immunology

Article Title: Induction of humoral and cell-mediated immunity to the NS1 protein of TBEV with recombinant Influenza virus and MVA affords partial protection against lethal TBEV infection in mice

doi: 10.3389/fimmu.2023.1177324

Figure Lengend Snippet: Antibody response against TBEV NS1. (A) Quantitative measurement of TBEV NS1-specific IgG by NS1 ELISA of sera samples collected 56 days post prime immunization. Results are reported as arbitrary units per ml (U/ml). Cut-off values were calculated according to the manufacturer’s instructions (1 U/ml). One-way ANOVA with Tukey’s multiple-comparison test was used for statistical analysis. Significances are shown for all NS1-specific vaccine groups (* p<0.05, **** p<0.0001). (B) Semi-quantitative measurement of TBEV NS1-specific antibodies by LIPS assay with mouse sera from day 0, 28 and 56 post prime immunization in relative light units (RLU). Mean values with SD are shown from 2-5 independent experiments (n=4 mice/group). Two-way ANOVA with Tukey’s multiple-comparison test was used for statistical analysis. Significances are shown for RLU values on d28 vs. d56 (solid line) and for all NS1-specific vaccine groups compared at d56 (dotted line) (** p<0.01, **** p<0.0001).

Article Snippet: Plasmid encoding for the Kozak sequence followed by the TBEV E gene signal peptide and entire TBEV NS1 were synthesized (based on TBEV Neudoerfl, UniProtKB: P14336; GenScript Biotech Corp) and cloned into MVA transfer plasmid pIIIsynIIred under transcriptional control of VACV late promotor psynII ( ). pIIIsynIIred contains mCherry as marker gene which is flanked by short repetitive regions.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Lips Assay

Journal: Frontiers in Immunology

Article Title: Induction of humoral and cell-mediated immunity to the NS1 protein of TBEV with recombinant Influenza virus and MVA affords partial protection against lethal TBEV infection in mice

doi: 10.3389/fimmu.2023.1177324

Figure Lengend Snippet: T cell response against TBEV NS1. (A) For IFN-γ ELISpot, mouse splenocytes were restimulated overnight with TBEV NS1 peptide pools NS1 1-183 and NS1 173-352 . Mean values with SD are shown as IFN-γ spot-forming cells (SFC) per one million splenocytes after background subtraction. For statistical analysis, unpaired t-test was used (**p<0.01). (B) Calculated IFN-γ SFC per million splenocytes for total NS1 (NS1 1-183 + NS1 173-352 ). One-way ANOVA with Tukey’s multiple-comparison test was used for statistical analysis. Significances are only shown for NS1-vaccine groups (ns = not significant, ** p<0.01, **** p<0.0001). (C–G) Flow cytometric analysis of mouse splenocytes. Frequency of CD3 + subpopulations gated on CD4 + (C, D) and CD8 + (E–G) T cells positive for CD69, IFN-γ and Granzyme B (GrzB) upon restimulation with NS1 1-183 (black circle) or NS1 173-352 (unfilled square) (n = 4). Bars represent mean with SD. Data is shown after background subtraction.

Article Snippet: Plasmid encoding for the Kozak sequence followed by the TBEV E gene signal peptide and entire TBEV NS1 were synthesized (based on TBEV Neudoerfl, UniProtKB: P14336; GenScript Biotech Corp) and cloned into MVA transfer plasmid pIIIsynIIred under transcriptional control of VACV late promotor psynII ( ). pIIIsynIIred contains mCherry as marker gene which is flanked by short repetitive regions.

Techniques: Enzyme-linked Immunospot, Comparison

Journal: Frontiers in Immunology

Article Title: Induction of humoral and cell-mediated immunity to the NS1 protein of TBEV with recombinant Influenza virus and MVA affords partial protection against lethal TBEV infection in mice

doi: 10.3389/fimmu.2023.1177324

Figure Lengend Snippet: Protective efficacy of TBEV NS1-based vector constructs. Body weights and survival of BL6 mice immunized with controls (A, B) , IAV-NS1 or MVA-NS1 (C, D) , MVA-NS1/IAV-NS1 (E, F) or IAV-NS1/MVA-NS1 (G, F) after challenge infection with TBEV Neudoerfl. In (A) and (C) mean body weights are shown (n = 6). For (E) and (G) body weights from individual mice are shown. Weights of PBS mice are shown as mean (n = 6). (B, D, F) Kaplan-Meier curves were analyzed by log-rank test (*p<0.05, **p<0.01). (H) Pearson correlation of TBEV NS1-specific antibodies on day 56 before challenge infection measured by LIPS with days of survival post TBEV infection for all groups (FSME-IMMUN® group was excluded). Linear regression is depicted by gray line (r = 0.7964, p<0.0001).

Article Snippet: Plasmid encoding for the Kozak sequence followed by the TBEV E gene signal peptide and entire TBEV NS1 were synthesized (based on TBEV Neudoerfl, UniProtKB: P14336; GenScript Biotech Corp) and cloned into MVA transfer plasmid pIIIsynIIred under transcriptional control of VACV late promotor psynII ( ). pIIIsynIIred contains mCherry as marker gene which is flanked by short repetitive regions.

Techniques: Plasmid Preparation, Construct, Infection

Journal: Frontiers in Immunology

Article Title: Induction of humoral and cell-mediated immunity to the NS1 protein of TBEV with recombinant Influenza virus and MVA affords partial protection against lethal TBEV infection in mice

doi: 10.3389/fimmu.2023.1177324

Figure Lengend Snippet: Viral loads in the periphery, CNS and GIT. Presence of TBEV RNA in spleen (A) , serum (B) , spinal cord (C) , brain (D) , ileum (E) and colon (F) was determined by performing real time RT-qPCR on cleared organ homogenates or serum from TBEV challenged mice sacrificed at 8 dpi (n = 6). Bars depict geometric means.

Article Snippet: Plasmid encoding for the Kozak sequence followed by the TBEV E gene signal peptide and entire TBEV NS1 were synthesized (based on TBEV Neudoerfl, UniProtKB: P14336; GenScript Biotech Corp) and cloned into MVA transfer plasmid pIIIsynIIred under transcriptional control of VACV late promotor psynII ( ). pIIIsynIIred contains mCherry as marker gene which is flanked by short repetitive regions.

Techniques: Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Induction of humoral and cell-mediated immunity to the NS1 protein of TBEV with recombinant Influenza virus and MVA affords partial protection against lethal TBEV infection in mice

doi: 10.3389/fimmu.2023.1177324

Figure Lengend Snippet: Histological and immunohistochemical changes in the olfactory bulb at 8 dpi. (A–H) Hematoxylin and eosin (H&E) stained sections of the olfactory bulb of a mouse treated with PBS (A) or vaccinated with FSME-IMMUN® (B) , wtMVA (C) , IAV-RBD (D) , IAV-NS1 (E) , MVA-NS1 (F) , MVA-NS1/IAV-NS1 (G) or IAV-NS1/MVA-NS1 (H) , respectively. (A) Olfactory bulb of the PBS-treated mouse displays marked cellular necrosis with karyorrhectic, karyolytic and pyknotic cells (insert, arrowhead) and shrunken, hypereosinophilic, triangular shaped necrotic neurons as well as inflammatory cell infiltrates. (B–H) In FSME-IMMUN®- (B) , wtMVA- (C) , IAV-NS1- (E) , MVA-NS1- (F) , MVA-NS1/IAV-NS1- (G) or IAV-NS1/MVA-NS1- (H) vaccinated mice, no significant microscopic lesions within the olfactory bulb are visible. In the IAV-RBD (D) vaccinated mouse, single necrotic cells are present. (I–P) Immunohistochemistry for TBEV E antigen of the olfactory bulb of a mouse treated with PBS (I) or vaccinated with FSME-IMMUN® (J) , wtMVA (K) , IAV-RBD (L) , IAV-NS1 (M) , MVA-NS1 (N) , MVA-NS1/IAV-NS1 (O) or IAV-NS1/MVA-NS1 (P) , respectively. (I) Immunohistochemically, a cytoplasmic TBEV immunoreactivity (arrowhead) is present in multiple cells of the olfactory bulb from the PBS-treated mouse. (J–P) Olfactory bulbs of FSME-IMMUN®- (J) , wtMVA- (K) , IAV-NS1- (M) , MVA-NS1- (N) , MVA-NS1/IAV-NS1- (O) or IAV-NS1/MVA-NS1- (P) vaccinated mice do not show immunoreactivity for TBEV, while the IAV-RBD- (N) vaccinated mouse shows single TBEV-positive cells (arrowhead). Scale bars: 20µm.

Article Snippet: Plasmid encoding for the Kozak sequence followed by the TBEV E gene signal peptide and entire TBEV NS1 were synthesized (based on TBEV Neudoerfl, UniProtKB: P14336; GenScript Biotech Corp) and cloned into MVA transfer plasmid pIIIsynIIred under transcriptional control of VACV late promotor psynII ( ). pIIIsynIIred contains mCherry as marker gene which is flanked by short repetitive regions.

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Induction of humoral and cell-mediated immunity to the NS1 protein of TBEV with recombinant Influenza virus and MVA affords partial protection against lethal TBEV infection in mice

doi: 10.3389/fimmu.2023.1177324

Figure Lengend Snippet: Histological and immunohistochemical analysis of colon at 8 dpi. (A–H) Hematoxylin and eosin (H&E) stained sections of the colon of a mouse treated with PBS (A) or vaccinated with FSME-IMMUN®, (B) , wtMVA (C) , IAV-RBD (D) , IAV-NS1 (E) , MVA-NS1 (F) , MVA-NS1/IAV-NS1 (G) or IAV-NS1/MVA-NS1 (H) , respectively. (A) Colon of the PBS-treated mouse displays minor to mild hypercellularity and cellular necrosis in the myenteric (arrowhead) and submucosal plexus (asterisk). (B–F) Colon of the FSME-IMMUN®- (B) , wtMVA- (C) , IAV-RBD- (D) , and MVA-NS1- (F) vaccinated mice show minimal to moderate histopathological changes mainly characterized by hypercellularity/inflammatory cell infiltrates in the myenteric and submucosal plexus. (E–H) In the IAV-NS1- (E) , MVA-NS1/IAV-NS1- (G) and IAV-NS1/MVA-NS1- (H) vaccinated mice, no histopathological changes in the plexi of the colon are observed. (I–P) Immunohistochemistry for TBEV E antigen of the colon of a mouse treated with PBS (I) or vaccinated with FSME-IMMUN® (J) , wtMVA (K) , IAV-RBD (L) , IAV-NS1 (M) , MVA-NS1 (N) , MVA-NS1/IAV-NS1 (O) or IAV-NS1/MVA-NS1 (P) , respectively. (I, K, L) Immunohistochemically, a cytoplasmic TBEV immunoreactivity is present in cells of the myenteric plexus (arrowhead) of the colon from the PBS-treated mouse (I) , the wtMVA-vaccinated mouse (K) and the IAV-RBD-vaccinated mouse (L) . A single TBEV-antigen positive cell is visible in the submucosal plexus of the colon from the MVA-NS1/IAV-NS1-vaccinated mouse ( O , asterisk). No TBEV-antigen positive cells are detectable in the submucosal (asterisk) or myenteric plexus (arrowhead) of FSME-IMMUN®- (J) , IAV-NS1- (M) , MVA-NS1- (N) and IAV-NS1/MVA-NS1-vaccinated (P) mice. Scale bars (A, B, D–H) : 20µm; Scale bar: (C, I–P) : 50µm.

Article Snippet: Plasmid encoding for the Kozak sequence followed by the TBEV E gene signal peptide and entire TBEV NS1 were synthesized (based on TBEV Neudoerfl, UniProtKB: P14336; GenScript Biotech Corp) and cloned into MVA transfer plasmid pIIIsynIIred under transcriptional control of VACV late promotor psynII ( ). pIIIsynIIred contains mCherry as marker gene which is flanked by short repetitive regions.

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Journal of Virology

Article Title: ADAM15 Participates in Tick-Borne Encephalitis Virus Replication

doi: 10.1128/JVI.01926-20

Figure Lengend Snippet: Effects of ADAM protein knockdown on TBEV infection. (A, C, and D) T98G cells were infected with TBEV for 48 h. (A) ADAM expression profiles were evaluated using RNA sequencing. For genomes used, see Data Set S1. (B to D) T98G cells were transfected with siRNAs targeting the indicated mRNA transcripts or with a nontargeting siRNA (control). (B) ADAM8 to -12, -15, -17, -19, and -22 mRNA transcript levels were analyzed by qPCR/RT-PCR, and values were corrected using β-actin. The graph shows average change compared to control for 3 independent experiments. (C) Cells were lysed for TBEV RNA analysis. The graph shows average change relative to control for 3 independent experiments. (D) NS1 protein levels were evaluated by Western blotting. Data are representative of 3 independent experiments; data in the graphs are means and SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, no significant difference.

Article Snippet: The antibody against NS1 of TBEV was custom produced by Abmart (Shanghai, China).

Techniques: Knockdown, Infection, Expressing, RNA Sequencing, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Virology

Article Title: ADAM15 Participates in Tick-Borne Encephalitis Virus Replication

doi: 10.1128/JVI.01926-20

Figure Lengend Snippet: Deficiency of ADAM15 reduces TBEV infection. (A to F) T98G cells were transfected with siRNAs targeting ADAM15 mRNA transcripts or a nontargeting siRNA (control). (A) At 48 h posttransfection, ADAM15 mRNA levels were evaluated in T98G cells. (A) Average change compared to control cells. (B to D) T98G cells were transfected with siNC or siADAM15 for 48 h and then infected with TBEV WH2012 for 48 h. The intracellular viral RNA (B), virus titer (C), and ADAM15/NS1 protein levels (D) were quantified. (B) Mean change in viral RNA levels relative to control cells. (C) Mean change in 50% tissue culture infective doses (TCID50) ml−1 relative to control cells. (D) WB showed changes in ADAM15 and NS1 protein levels relative to control cells. (E) Cell viability was evaluated in T98G cells. (F) T98G cells were infected with TBEV Neudoerfl for 48 h, and then virus titer was quantified. Data are mean change in TCID50 ml−1 relative to control cells. (G to I) TBEV WH2012 was used to infect control or ADAM15−/− T98G cells for 48 h. The intracellular viral RNA (G), virus titer (H), and ADAM15/NS1 protein levels (I) were quantified. (G) Mean change in viral RNA levels relative to control cells. (H) Mean change in TCID50 ml−1 relative to control cells. (I) WB showing changes in ADAM15 and NS1 protein levels relative to control cells. (J) Cell viability was evaluated in T98G cells or ADAM15−/− T98G cells. (K) T98G cells were infected with TBEV Neudoerfl for 48 h, and then virus titer was quantified. Data are mean change in TCID50 ml−1 relative to control cells. All graphs are representative of 3 independent experiments. Quantitative data are means and SD. *, P < 0.05; ***, P < 0.001.

Article Snippet: The antibody against NS1 of TBEV was custom produced by Abmart (Shanghai, China).

Techniques: Infection, Transfection, Control, Virus

Journal: Journal of Virology

Article Title: ADAM15 Participates in Tick-Borne Encephalitis Virus Replication

doi: 10.1128/JVI.01926-20

Figure Lengend Snippet: ADAM15 modulates TBEV replication and assembly. (A to C) Cells were collected and RNA was measured by qPCR/RT-PCR. Mean change in viral RNA levels relative to control cells is shown. (A and B) TBEV was incubated with control and ADAM15−/− cells at 4°C (A) or 37°C (B) as described in Materials and Methods. (C) Control and ADAM15−/− cells were incubated with anti-ADAM15 or isotype control MAbs before infection. (D) Transfection of TBEV subgenomic RNA into control or ADAM15−/− cells. Cells were analyzed for Renilla luciferase and NS1 protein expression levels. Mean change relative to control cells is shown. (E) Control and ADAM15−/− cells were infected with TBEV for 48 h, and then viral RNA levels in cells and culture supernatants were quantified. Mean change in assembly efficiency (RNA copies in supernatant/RNA copies in cells) relative to the control cells is shown. (F) After 48 h infection, cells were fixed with glutaraldehyde and processed for viewing by TEM. Mature particles (black arrowheads) are indicated. Bars, 1 μm (left), 500 nm (middle), and 200 nm (right). (G) Viral particles or vesicle packets were determined by counting using ImageJ software, and mean values are presented (error bars represent SD). Data are representative of 3 independent experiments. Quantitative data are means and SD. **, P < 0.01; ***, P < 0.001; NS, no significant difference.

Article Snippet: The antibody against NS1 of TBEV was custom produced by Abmart (Shanghai, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Incubation, Infection, Transfection, Luciferase, Expressing, Software

Journal: Journal of Virology

Article Title: ADAM15 Participates in Tick-Borne Encephalitis Virus Replication

doi: 10.1128/JVI.01926-20

Figure Lengend Snippet: TBEV infection does not affect ADAM15 expression but changes the localization and distribution of ADAM15. Cells were infected with TBEV for the indicated times. (A) Mean change in ADAM15 mRNA and protein levels compared to uninfected cells. (B) After 48 h infection, lipid raft flotation analysis of ADAM15 localization in the RIPA-soluble fraction or DRM was carried out. Data are changes compared to uninfected cells. (C) After 48 h infection, cells were fixed with paraformaldehyde and stained for ADAM15, NS1, ER, or trans-Golgi. Cells were analyzed by confocal microscopy. Bar, 10 μm. (C and D) Control and ADAM15−/− cells were grown on glass dishes. Cells were fixed followed by staining for visualization and imaging by confocal microscopy. After 48 h infection, cells were fixed with paraformaldehyde and stained with ER tracker, endogenous ADAM15, or the Golgi marker syntaxin 6. Cells were then analyzed by confocal microscopy. Images were representative of 3 independent samples. Results are representative of 3 independent experiments. Quantitative data are means and SD.

Article Snippet: The antibody against NS1 of TBEV was custom produced by Abmart (Shanghai, China).

Techniques: Infection, Expressing, Staining, Confocal Microscopy, Control, Imaging, Marker

Journal: Journal of Virology

Article Title: Model System for the Formation of Tick-Borne Encephalitis Virus Replication Compartments without Viral RNA Replication

doi: 10.1128/JVI.00292-19

Figure Lengend Snippet: Dose- and time-dependent expression of NS1-NS4B-GFP in Flp-In T-REx HeLa cells. (A) Schematic illustration of the proposed membrane topology of the TBEV polyprotein, with color-coded representation of the individual structural (C, prM, and E) and nonstructural (NS1 to NS5) proteins. The GFP-tagged polyprotein (NS1 to NS4B) expressed following dox addition in the constructed NSP-GFP-Flp-In cells is shown below. (B) Immunoblotting expression analysis of the individual NS proteins, as indicated, following induction of NSP-GFP cells with different concentrations (0 to 10 ng/ml) of dox. Clathrin served as a loading control. (C) Immunoblotting analysis of the time-dependent expression of NS proteins, as indicated, following induction with 2 ng/ml dox in NSP-GFP cells (left), transfection with the TBEV DNA replicon (1 μg DNA per 5 × 10 6 cells) (middle), or infection with LGTV (MOI of 1) or TBEV Torö strain (MOI of 1) (right). Actin and clathrin served as loading controls. (D) Representative fluorescence micrographs of the time-dependent expression of NS4B-GFP in NSP-GFP cells induced by 2 ng/ml dox, as recorded by live cell imaging. Scale bars = 10 μm. (E) Schematic illustration of the organization of the TBEV DNA replicon. (F) Representative images from immunofluorescence analysis of Flp-In HeLa cells transfected with the TBEV DNA replicon. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and the ER marker calnexin was labeled with specific antibody, as indicated. dsRNA was labeled with the mouse anti-dsRNA monoclonal antibody J2. The GFP signal served as the reporter for replicon transfection. Scale bar = 10 μm. (G) Quantification of dsRNA in TBEV-replicon-transfected cells ( n = 25) and untransfected cells ( n = 25) from the immunofluorescence analysis in panel F. The dsRNA quantification was performed with Imaris v7.5 software (Bitplane). The statistical analysis was performed with GraphPad Prism software (GraphPad Software) ( n = 25). ***, P ≤ 0.01 ( t test).

Article Snippet: Rabbit anti-TBEV NS2B polyclonal antiserum, chicken anti-TBEV NS3 polyclonal antibody , and chicken anti-TBEV NS1 polyclonal antibody ( ) were produced by Agrisera (Vännäs, Sweden), using synthetic peptides (NS2B, GCVEWHPELVNEGGEVSLRVRQ; NS3, EGRDIKEFVAYASGRR) or purified NS1 protein (a kind gift from Alessandro Marcello, The International Center for Genetic Engineering and Biotechnology, Trieste, Italy) as immunogens.

Techniques: Expressing, Membrane, Construct, Western Blot, Control, Transfection, Infection, Fluorescence, Live Cell Imaging, Immunofluorescence, Staining, Marker, Labeling, Software

Journal: Journal of Virology

Article Title: Model System for the Formation of Tick-Borne Encephalitis Virus Replication Compartments without Viral RNA Replication

doi: 10.1128/JVI.00292-19

Figure Lengend Snippet: NS proteins form a protein complex in the ER of NSP-GFP cells. (A) Representative fluorescence micrographs of dox-induced NSP-GFP cells expressing NS4B-GFP and immunostained against calreticulin or Sec61A, as indicated. Scale bars = 10 μm. (B) FRAP analysis of the dynamics of NS4B-GFP in dox-induced NSP-GFP cells. ER networks were stained with ER-tracker. Regions enriched in both NS4B-GFP and ER-tracker were photobleached, and then the fluorescence signal of the regions was traced for 300 s. Means ± standard errors of the means (SEMs) are shown. (C) Immunoblot analysis of the separation of NS proteins in the supernatant (S) and pellet (P), as indicated, following lysis and differential centrifugation of induced NS1-NS4B cells at the indicated speed (1,000 to 100,000 × g ). Antibodies to Sec61A, calnexin, and calreticulin were used as markers of the ER. (D) OptiPrep density flotation analysis of the 20,000 × g pellet fraction in panel C, with or without pretreatment with1% Triton-X to dissolve membranes. Flotation of calnexin and individual NS proteins, as indicated, was analyzed by immunoblotting. (E) Immunoprecipitation of GFP and NS4B-GFP from dox-induced GFP (Ctl) or NS4B-GFP (4B) Flp-In cells coexpressing FLAG-tagged NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5, as indicated. Input and immunoprecipitated (IP) material was analyzed by immunoblotting as indicated.

Article Snippet: Rabbit anti-TBEV NS2B polyclonal antiserum, chicken anti-TBEV NS3 polyclonal antibody , and chicken anti-TBEV NS1 polyclonal antibody ( ) were produced by Agrisera (Vännäs, Sweden), using synthetic peptides (NS2B, GCVEWHPELVNEGGEVSLRVRQ; NS3, EGRDIKEFVAYASGRR) or purified NS1 protein (a kind gift from Alessandro Marcello, The International Center for Genetic Engineering and Biotechnology, Trieste, Italy) as immunogens.

Techniques: Fluorescence, Expressing, Staining, Western Blot, Lysis, Centrifugation, Immunoprecipitation

Journal: Journal of Virology

Article Title: Model System for the Formation of Tick-Borne Encephalitis Virus Replication Compartments without Viral RNA Replication

doi: 10.1128/JVI.00292-19

Figure Lengend Snippet: Replication-independent expression of NS1 to NS4B in HeLa cells results in dilation of the ER membrane and generation of RC-like structures. (A) Representative TEM images of control HeLa cells, cells infected with LGTV (MOI of 1), cells transfected with the TBEV DNA replicon, and uninduced and induced NSP-GFP cells. White arrowheads show dilated ER areas; black arrowheads denote replication-vesicle-like structures inside the dilated ER areas. Insets show magnifications of the indicated areas. Scale bars = 1 μm (except in insets [scale bars = 0.1 μm]). (B) Quantification of the number of dilated ER areas in replicon-transfected cells, LGTV-infected cells, and dox-induced NSP-GFP cells, compared with control HeLa cells ( n = 5). Quantification was performed using ImageJ. **, P ≤ 0.05 ( t test). (C) Membrane profiles and sizes of replication-vesicle-like structures in replicon-transfected cells, LGTV-infected cells, and dox-induced NSP-GFP cells. The membrane profiles were outlined using Adobe Photoshop. The diameters of at least 30 randomly chosen vesicles from each group were measured using ImageJ.

Article Snippet: Rabbit anti-TBEV NS2B polyclonal antiserum, chicken anti-TBEV NS3 polyclonal antibody , and chicken anti-TBEV NS1 polyclonal antibody ( ) were produced by Agrisera (Vännäs, Sweden), using synthetic peptides (NS2B, GCVEWHPELVNEGGEVSLRVRQ; NS3, EGRDIKEFVAYASGRR) or purified NS1 protein (a kind gift from Alessandro Marcello, The International Center for Genetic Engineering and Biotechnology, Trieste, Italy) as immunogens.

Techniques: Expressing, Membrane, Control, Infection, Transfection

Journal: Journal of Virology

Article Title: Model System for the Formation of Tick-Borne Encephalitis Virus Replication Compartments without Viral RNA Replication

doi: 10.1128/JVI.00292-19

Figure Lengend Snippet: Localization of NS proteins in RC-like structures in NSP-GFP cells. (A) CLEM images of a NSP-GFP cell induced with dox. (Top) The left fluorescence micrograph shows NS4B-GFP (green) and ER-tracker (red), and the right panel shows the correlated electron micrograph, with arrows highlighting dilated ER membranes. (Bottom) The left panel shows an electron micrograph of the area indicated by the white square in higher magnification, and the right panel shows further magnification of the indicated area; the ER and dilated ER (dER) are indicated, and black arrowheads denote vesicle-like structures. Scale bars = 10 μm (top) or as indicated below the bars (bottom). (B) Immunofluorescence micrographs of the single and merged channels of a dox-induced NSP-GFP cell expressing NS4B-GFP and stained against NS1. Yellow and white arrowheads exemplify the punctuate NS1 localization on NS4B-GFP-positive ER tubules. Scale bar = 10 μm. (C) Immuno-EM images of NSP-GFP cells induced with dox. The cells were stained with anti-NS1 antibodies conjugated with gold particles. Scale bars = 200 nm.

Article Snippet: Rabbit anti-TBEV NS2B polyclonal antiserum, chicken anti-TBEV NS3 polyclonal antibody , and chicken anti-TBEV NS1 polyclonal antibody ( ) were produced by Agrisera (Vännäs, Sweden), using synthetic peptides (NS2B, GCVEWHPELVNEGGEVSLRVRQ; NS3, EGRDIKEFVAYASGRR) or purified NS1 protein (a kind gift from Alessandro Marcello, The International Center for Genetic Engineering and Biotechnology, Trieste, Italy) as immunogens.

Techniques: Fluorescence, Immunofluorescence, Expressing, Staining