Journal: One Health

Article Title: Multiplex serological screening of wild boar as sentinels of emerging zoonoses: HEV, WNV, and TBEV distribution in Saxony, Germany

doi: 10.1016/j.onehlt.2025.101283

Figure Lengend Snippet: BMBA assay performance characteristics: Inter- and intra-assay variability, dynamic range and receiver operating characteristic (ROC) curve analysis. (A) Mean fluorescence intensity (MFI) of ≥7 independent measurement runs with domestic pig serum Pig 23 (HEV), monoclonal anti-Flavivirus NS1 antibody (WNV), or monoclonal anti-TBEV NS1 antibody (TBEV). CV: coefficient of variation between independent runs. (B) Samples described in A were tested in duplicate in ≥3 independent measurement runs. CVs for duplicates of each run are indicated. (C) Duplicate MFI data from 2-fold serial dilutions (1:100 to 1:12,800) of Pig 23 serum (HEV) or wild boar sera WS/20/43 (WNV) and WS/21/119 (TBEV) with results of a five-parameter logistic model (5-PL) non-linear regression curve and R 2 values as measure for goodness-of-fit. (D) Shown are ROC curves depicting the area under the curve (AUC; shown by the blue line) as well as the calculated cutoff values, sensitivities, specificities and AUC values. Statistical data on assay performance are summarized in Supplementary Table S3, Supplementary material 1. Analysis of monoplex reactions (one bead-antigen type per assay). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: In addition, mouse monoclonal anti-Flavivirus NS1 antibody [D/2/D6/B7] (1:100; Abcam, Cambridge, UK) and mouse monoclonal anti-tick-borne encephalitis virus NS1 (M838) antibody (1:500; The Native Antigen Company, Kidlington, UK) were used as WNV and TBEV positive control, respectively.

Techniques: Intra Assay, Fluorescence

Journal: One Health

Article Title: Multiplex serological screening of wild boar as sentinels of emerging zoonoses: HEV, WNV, and TBEV distribution in Saxony, Germany

doi: 10.1016/j.onehlt.2025.101283

Figure Lengend Snippet: BMBA assay performance characteristics: Inter- and intra-assay variability, dynamic range and receiver operating characteristic (ROC) curve analysis. (A) Mean fluorescence intensity (MFI) of ≥7 independent measurement runs with domestic pig serum Pig 23 (HEV), monoclonal anti-Flavivirus NS1 antibody (WNV), or monoclonal anti-TBEV NS1 antibody (TBEV). CV: coefficient of variation between independent runs. (B) Samples described in A were tested in duplicate in ≥3 independent measurement runs. CVs for duplicates of each run are indicated. (C) Duplicate MFI data from 2-fold serial dilutions (1:100 to 1:12,800) of Pig 23 serum (HEV) or wild boar sera WS/20/43 (WNV) and WS/21/119 (TBEV) with results of a five-parameter logistic model (5-PL) non-linear regression curve and R 2 values as measure for goodness-of-fit. (D) Shown are ROC curves depicting the area under the curve (AUC; shown by the blue line) as well as the calculated cutoff values, sensitivities, specificities and AUC values. Statistical data on assay performance are summarized in Supplementary Table S3, Supplementary material 1. Analysis of monoplex reactions (one bead-antigen type per assay). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: WNV non-structural protein 1 (NS1) (strain NY99, amino acids 768–1143) and TBEV NS1 (Ncbi NP_043135.1 , amino acids 773–1128) were expressed in mammalian HEK293 cells, incorporating a C-terminal 6× His-tag (The Native Antigen Company, Kidlington, UK).

Techniques: Intra Assay, Fluorescence

Journal: One Health

Article Title: Multiplex serological screening of wild boar as sentinels of emerging zoonoses: HEV, WNV, and TBEV distribution in Saxony, Germany

doi: 10.1016/j.onehlt.2025.101283

Figure Lengend Snippet: Distribution of seroprevalences of wild boar in different districts of Saxony. (A) Map of Germany with federal state borders (red lines) and district borders (black lines). Wild boar serum samples from eight districts (orange) in the German federal state Saxony were analyzed. HEV- (B), WNV- (C) or TBEV- (D) specific antibodies were detected by BMBA screening and results are depicted as % of positive sera per Saxonian district. Raw data are available in Table S4 and S5, Supplementary material 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: WNV non-structural protein 1 (NS1) (strain NY99, amino acids 768–1143) and TBEV NS1 (Ncbi NP_043135.1 , amino acids 773–1128) were expressed in mammalian HEK293 cells, incorporating a C-terminal 6× His-tag (The Native Antigen Company, Kidlington, UK).

Techniques:

Journal: Virologica Sinica

Article Title: Synthetic genomics-based generation of the tick-borne encephalitis virus Siberian subtype prototype strain and E51K-attenuated variant for vaccine development and antiviral screening

doi: 10.1016/j.virs.2025.09.010

Figure Lengend Snippet: Generation of a molecularly cloned tick-borne encephalitis virus (TBEV) and a culture-adapted variant. A TBEV genome map shows the structural (C, prM, E) and non-structural (NS1–NS5) genes and 5′- and 3′-untranslated regions (UTRs). B Schematic of the assembly of the infectious molecular clone (MIC) p.mcVs. The top line represents the TBEV genome with shown genomic termini (5′A, 3′T) and restriction sites ( Bsr GI, etc.) which define borders of DNA fragments selected for de novo synthesis. The DNA fragments were sequentially assembled into intermediate plasmids via restriction-ligation cloning, using the indicated restriction enzymes. The complete genome was cloned under the control of the CMV promoter, and the hepatitis D virus ribozyme (HDV Rz) was appended immediately after the genomic 3′-terminus, followed by a human growth hormone polyadenylation signal (PolyA HGH). Junction sequences at the genomic termini are shown at the bottom of panel ( B ). TSS: CMV promoter transcription start site.

Article Snippet: For immunofluorescence, infected BHK-21 cells were fixed, permeabilized with 1% Triton X-100, and stained with mouse anti-TBEV NS1 antibody (1:500, The Native Antigen Company, Cat. M838) followed by FITC-conjugated anti-mouse IgG (1:1000, ThermoScientific, Cat. A16091).

Techniques: Clone Assay, Virus, Variant Assay, Ligation, Cloning, Control

Journal: Virologica Sinica

Article Title: Synthetic genomics-based generation of the tick-borne encephalitis virus Siberian subtype prototype strain and E51K-attenuated variant for vaccine development and antiviral screening

doi: 10.1016/j.virs.2025.09.010

Figure Lengend Snippet: Cytopathic effect (CPE) in transfected culture and immunostaining of the TBEV NS1 antigen in infected cells. A CPE is evident on day 5 in a culture of BHK-21 cells transfected with TBEV MIC (right photograph). A photograph of the mock-transfected culture is in the left photograph. Objective magnification 10× . B BHK-21 cells were infected with the virus present in supernatants of a transfected culture (top panel) or mock-infected (bottom panel). On day 3 after infection the cultures were processed for immune fluorescence detection of TBEV NS1 using FITC-labeled secondary antibodies. Bright FITC fluorescence is evident in the perinuclear region of infected cells indicative of viral NS1-containing replicative complexes (photographs with objective magnification 20× ). Mock-infected cells upon staining show no fluorescence (photographs with objective magnification 10× ).

Article Snippet: For immunofluorescence, infected BHK-21 cells were fixed, permeabilized with 1% Triton X-100, and stained with mouse anti-TBEV NS1 antibody (1:500, The Native Antigen Company, Cat. M838) followed by FITC-conjugated anti-mouse IgG (1:1000, ThermoScientific, Cat. A16091).

Techniques: Transfection, Immunostaining, Infection, Virus, Fluorescence, Labeling, Staining

Journal: Virologica Sinica

Article Title: Synthetic genomics-based generation of the tick-borne encephalitis virus Siberian subtype prototype strain and E51K-attenuated variant for vaccine development and antiviral screening

doi: 10.1016/j.virs.2025.09.010

Figure Lengend Snippet: Generation and growth kinetics of the E51K-mutant virus. A Experimental workflow: TBEV MIC was transfected into BHK-21 cells yielding the molecularly cloned Vasilchenko strain (mcVs). Serial passages (P1–P4) in BHK-21 cells produced a culture-adapted variant. B The culture-adapted virus P4 has a Glu-to-Lys substitution at position 51 in the envelope (E) protein. Comparisons of amino acid sequences including the mutation in mcVs and the passaged virus P4 are shown. C BHK-21 cells were infected (MOI = 1) with parental TBEV (P0) or adapted passage (P4). Titers of the accumulating virus were measured using plaque assay. P4 showed significantly higher titers than P0 (∗∗ P < 0.01, ∗ P < 0.05). D Transfection assay: BHK-21 cells were transfected with MICs and the E51K-mutant exhibited enhanced replication (∗∗ P < 0.01). Dotted line indicates assay detection limit. E Viruses obtained post-transfection were used to infect PK-15 cells grown under an agar overlay for 5 days. During this period, plaques formed by the parental virus reached 3–5 mm in diameter, whereas those formed by the E51K mutant reached 10–14 mm.

Article Snippet: For immunofluorescence, infected BHK-21 cells were fixed, permeabilized with 1% Triton X-100, and stained with mouse anti-TBEV NS1 antibody (1:500, The Native Antigen Company, Cat. M838) followed by FITC-conjugated anti-mouse IgG (1:1000, ThermoScientific, Cat. A16091).

Techniques: Mutagenesis, Virus, Transfection, Clone Assay, Produced, Variant Assay, Infection, Plaque Assay

Journal: Virologica Sinica

Article Title: Synthetic genomics-based generation of the tick-borne encephalitis virus Siberian subtype prototype strain and E51K-attenuated variant for vaccine development and antiviral screening

doi: 10.1016/j.virs.2025.09.010

Figure Lengend Snippet: Virulence of parental TBEV strain Vasilchenko and the E51K-mutant virus. A Survival of BALB/c mice (n = 10/group) following intramuscular infection with 10,000 PFU of wild-type TBEV (mcVs), E51K-mutant, or control (saline). All wild-type-infected mice succumbed before day 13, while all mutant-infected mice survived. B Body weight changes over 15 days. Wild-type infection caused progressive weight loss from day 5, whereas mutant-infected and control mice showed weight gain. Data represent means ± SD of surviving mice. C Viremia kinetics in BALB/c mice (n = 60/group). Serum viral titers were measured at different time points, with each data point averaging two mice. Error bars indicate SD.

Article Snippet: For immunofluorescence, infected BHK-21 cells were fixed, permeabilized with 1% Triton X-100, and stained with mouse anti-TBEV NS1 antibody (1:500, The Native Antigen Company, Cat. M838) followed by FITC-conjugated anti-mouse IgG (1:1000, ThermoScientific, Cat. A16091).

Techniques: Mutagenesis, Virus, Infection, Control, Saline

Journal: Virologica Sinica

Article Title: Synthetic genomics-based generation of the tick-borne encephalitis virus Siberian subtype prototype strain and E51K-attenuated variant for vaccine development and antiviral screening

doi: 10.1016/j.virs.2025.09.010

Figure Lengend Snippet: Molecular design and characterization of a GFP-expressing reporter virus. A Genome of the E51K TBEV mutant and cloning strategy for inserting a GFP gene into the TBEV genome. Designations as in . The star marks the E51K mutation in the envelope ( E ) gene. The coding sequence placed upstream of the main ORF consists of a 51-bp sequence of the capsid gene presumably containing replication signals, followed by the GFP gene and a self-cleaving FMDV 2A peptide. Sequence at GFP-2A/viral polyprotein is shown. B Photographs of BHK-21 cells transfected with TBEV/GFP MIC. Objective magnification 20 × . C Comparison of viral growth kinetics following infection of BHK-21 cells at an MOI of 1. The TBEV/GFP reporter virus shows similar titers as the parental E51K-mutant. Error bars indicate SD. D Percentage of GFP-positive foci among total foci formed under agar overlay following infection with TBEV/GFP passages P1, P2, and P3.

Article Snippet: For immunofluorescence, infected BHK-21 cells were fixed, permeabilized with 1% Triton X-100, and stained with mouse anti-TBEV NS1 antibody (1:500, The Native Antigen Company, Cat. M838) followed by FITC-conjugated anti-mouse IgG (1:1000, ThermoScientific, Cat. A16091).

Techniques: Expressing, Virus, Mutagenesis, Cloning, Sequencing, Transfection, Comparison, Infection

Journal: bioRxiv

Article Title: Interactions between tick-borne encephalitis virus non-structural protein 1 and blood-brain barrier tight junction proteins: potential clues to strain-specific neuropathogenicity

doi: 10.1101/2025.08.12.669603

Figure Lengend Snippet: (A) The NS1 dimer is shown in cartoon representation. The ribbon diagram highlights the β-roll, wing, β-ladder, and connector domains (1 and 2), which are coloured blue, yellow, pink, green, and purple, respectively, in one monomer. The dengue virus (DENV) NS1 dimer (PDB ID: 4OIG) is shown interacting with a phospholipid membrane. (B) Percent identity matrix for the NS1 (B) or (C) (envelop) E proteins among the indicated 14 tick-borne Orthoflaviviruses (TBFV), generated by ClustalW. The each in the matrix represents the percentage of identical residues between each pair of sequences in the alignment, calculated by the aligned positions. The identity values are shown as an inferno heatmap with the gradient from highest (light yellow; 100%) to lowest (dark purple; <74%).

Article Snippet: After washing (3× with PBS-T), bound NS1 was detected using a mouse anti-TBEV NS1 monoclonal antibody (R&D Systems, MAB10106; 0.05[μg/mL), followed by incubation with HRP-conjugated anti-mouse IgG (Sigma, A9044; 1:40,000) for 1[h at RT.

Techniques: Virus, Membrane, Generated

Journal: bioRxiv

Article Title: Interactions between tick-borne encephalitis virus non-structural protein 1 and blood-brain barrier tight junction proteins: potential clues to strain-specific neuropathogenicity

doi: 10.1101/2025.08.12.669603

Figure Lengend Snippet: Localisation of amino acid (AA) changes in representative low- and high-pathogenic strains of TBEV (A), ZIKA (B), JEV (C) and WNV (D) NS1. The domains are colour corded as in . Initial atomic positions were based on (PDB ID: 4OIG) for all. (A) Position of 22-AA changes in the three-dimensional structure of the Vs (low-path)- and Hypr (high-path)-NS1 monomer prediction by AlphaFold3, visualised in PyMol. Focused view of the mutations in the Wing (yellow) or β-ladder (Pink) domain are indicated. The mutated amino acid is shown in atomic stick representation. (B-D) NS1 of high- and low-pathogenic strains are indicated in left and right, respectively. (B) Dimer of Asian (PDB: 5GS6) (left) and African (PDB:5K6K)-ZIKA (right) virus NS1, showing all 9 AA changes (orange). The domains are colour corded as in . (C) Dimer of JEV genotype III-and genotype I-NS1 with 13 site-specific amino acid changes highlight in orange colour. (D) NS1 dimer of lineage 1a- and lineage 2-WNV NS1, indicating 30 site-specific amino acid mutations.

Article Snippet: After washing (3× with PBS-T), bound NS1 was detected using a mouse anti-TBEV NS1 monoclonal antibody (R&D Systems, MAB10106; 0.05[μg/mL), followed by incubation with HRP-conjugated anti-mouse IgG (Sigma, A9044; 1:40,000) for 1[h at RT.

Techniques: Virus

Journal: bioRxiv

Article Title: Interactions between tick-borne encephalitis virus non-structural protein 1 and blood-brain barrier tight junction proteins: potential clues to strain-specific neuropathogenicity

doi: 10.1101/2025.08.12.669603

Figure Lengend Snippet: Predicted NS1–TJP interaction models generated by AlphaFold3 (AF3). Interface interacting residues are shown for TBEV Hypr-NS1 (A) or Vs-NS1 (B) with JAM-A. Hypr-NS1 interactions with JAM-B (C) and Vs-NS1 with Claudin 5 (D) are indicated. The predicted selected distances (range: ≤2.0–3.3 Å) between amino acid interface residues of NS1 and human TJPs are shown. A heatmap indicates interaction distances, with dark orange representing shorter distances and light orange representing longer ones. NS1 domains are colour coded as in .

Article Snippet: After washing (3× with PBS-T), bound NS1 was detected using a mouse anti-TBEV NS1 monoclonal antibody (R&D Systems, MAB10106; 0.05[μg/mL), followed by incubation with HRP-conjugated anti-mouse IgG (Sigma, A9044; 1:40,000) for 1[h at RT.

Techniques: Generated

Journal: bioRxiv

Article Title: Interactions between tick-borne encephalitis virus non-structural protein 1 and blood-brain barrier tight junction proteins: potential clues to strain-specific neuropathogenicity

doi: 10.1101/2025.08.12.669603

Figure Lengend Snippet: (A–C) Binding curves of NS1 proteins from TBEV-Hypr (green) and TBEV-Vs (gray) to immobilized human JAM-A, measured by ELISA across three independent experiments. NS1 at various starting concentrations ( [A] = 1 µg/mL, [B] = 1 µg/mL or [C] = 1µg/mL) was titrated in 2-fold serial dilutions. KD values are indicated. Data points represent mean OD450 ± SD from duplicate wells. Curves were fitted using a one-site binding model. (D) Quantification of NS1 binding at 0.01 µg/mL and 0.25 µg/mL to JAM-A. Bars represent mean OD450 ± SD. (E–G) Binding curves of Hypr and Vs NS1 to immobilized human ZO-1 across three independent ELISA experiments with variable NS1 starting concentrations ( [E] = 0.5 µg/mL, [F] = 1 µg/mL or [G] = 1 µg/mL). NS1 was titrated in 2-fold serial dilutions. Data represent mean OD450 ± SD from duplicate wells. Fitted curves and KD values are shown. (H) OD450 values at 0.01 µg/mL and 0.25 µg/mL NS1 concentrations. Bars represent mean ± SD.

Article Snippet: After washing (3× with PBS-T), bound NS1 was detected using a mouse anti-TBEV NS1 monoclonal antibody (R&D Systems, MAB10106; 0.05[μg/mL), followed by incubation with HRP-conjugated anti-mouse IgG (Sigma, A9044; 1:40,000) for 1[h at RT.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay