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