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: Parasites & Vectors

Article Title: Development and evaluation of an assay for the detection of tick-borne encephalitis virus RNA via real-time PCR with reverse transcription

doi: 10.1186/s13071-026-07366-5

Figure Lengend Snippet: Partial nucleotide sequence alignment of the TBEV NS3 gene

Article Snippet: The Saint Petersburg Pasteur Institute is responsible for the TBEV surveillance program in this region.

Techniques: Sequencing

Journal: Parasites & Vectors

Article Title: Development and evaluation of an assay for the detection of tick-borne encephalitis virus RNA via real-time PCR with reverse transcription

doi: 10.1186/s13071-026-07366-5

Figure Lengend Snippet: Cycle threshold value ( C t ) of RNA from TBEV-positive ticks via different test systems. The following systems were used: commercial kit A (“TBEV, B. burgdorferi s.l., A. phagocytophillum , E. chaffeensis / E. muris -FL kit,” AmpliSens, Moscow, Russia) (blue); commercial kit B (“RealBest DNA Borrelia burgdorferi s.l./RNA TBEV kit,” AO Vector-Best, Russia) (red); and the developed TBEV AmpPS assay (green)

Article Snippet: The Saint Petersburg Pasteur Institute is responsible for the TBEV surveillance program in this region.

Techniques: Plasmid Preparation

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: bioRxiv

Article Title: Bemnifosbuvir and remdesivir inhibit tick-borne encephalitis virus infection in complementary in vitro and ex vivo disease models

doi: 10.1101/2025.11.13.688315

Figure Lengend Snippet: A549 cells were infected with TBEV, treated with selected NAs at 50 µM and incubated for 48 hours. A, Virus titers were determined using plaque assays. Data are shown as mean ± s.d. n = 5 independent experiments. Statistical analysis was performed using Welch’s t-test. *p ≤ 0.05, ****p ≤ 0.0001. B, A549 cells were fixed on slides, stained with a mouse orthoflavivirus E-protein–specific antibody (magenta), and counterstained with DAPI (blue). Scale bar, 500 μm.

Article Snippet: Treatment with 5 μM bemnifosbuvir was sufficient to eliminate detectable TBEV antigen , whereas for remdesivir, complete clearance required 100 μM; at 50 μM, viral antigen was reduced but still visible ( ).

Techniques: Infection, Incubation, Virus, Staining

Journal: bioRxiv

Article Title: Bemnifosbuvir and remdesivir inhibit tick-borne encephalitis virus infection in complementary in vitro and ex vivo disease models

doi: 10.1101/2025.11.13.688315

Figure Lengend Snippet: A-C, Cytotoxicity of the tested compounds measured in terms of cell viability using A549, Vero, and SK-N-SH cells. The cells were treated with increasing concentrations (0-50µM) of uprifosbuvir, remdesivir, sofosbuvir, and bemnifosbuvir and cultivated for 48 hours. D, F, H, L, Anti-TBEV activity of the tested compounds was tested by infecting the cells with TBEV. Cells were subsequently treated with increasing concentrations (0-50 µM) and cultivated for 48 hours. Viral titers were determined using plaque assays. Horizontal dotted line indicates the detection limit of the assays. E, G, I, M, Inhibition dose-response curves used to calculate the IC 50 using the nonlinear regression method. The dashed line in panels L and M indicates values measured in undifferentiated SH-SY5Y cells. Horizontal dotted line indicates 50% inhibition.

Article Snippet: Treatment with 5 μM bemnifosbuvir was sufficient to eliminate detectable TBEV antigen , whereas for remdesivir, complete clearance required 100 μM; at 50 μM, viral antigen was reduced but still visible ( ).

Techniques: Activity Assay, Inhibition

Journal: bioRxiv

Article Title: Bemnifosbuvir and remdesivir inhibit tick-borne encephalitis virus infection in complementary in vitro and ex vivo disease models

doi: 10.1101/2025.11.13.688315

Figure Lengend Snippet: A549 (A), Vero (B), and SK-N-SH cells (C) were infected with TBEV and treated with the indicated compounds. At 48 h post infection, the cells were fixed on slides, stained with a mouse orthoflavivirus E-protein–specific antibody (magenta), and counterstained with DAPI (blue). Scale bar, 500 μm.

Article Snippet: Treatment with 5 μM bemnifosbuvir was sufficient to eliminate detectable TBEV antigen , whereas for remdesivir, complete clearance required 100 μM; at 50 μM, viral antigen was reduced but still visible ( ).

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: Bemnifosbuvir and remdesivir inhibit tick-borne encephalitis virus infection in complementary in vitro and ex vivo disease models

doi: 10.1101/2025.11.13.688315

Figure Lengend Snippet: A , Infectious released virus dynamics in TBEV challenged hNOs with or without the addition of NAs at 50 μM. Points indicate mean values ± s.d. n = 3 independent organoid batches. Horizontal dotted line indicates assay detection limit. B , Maximum reached TBEV titers across all sampling time points in TBEV-infected hNOs with or without NAs treatment at 50 μM. Bars indicate mean values ± s.d. n = 3 independent organoid batches. Horizontal dotted line indicates assay detection limit. Statistical analysis was performed using Welch’s t-test. **p≤0.01, ****p≤0.0001. C , Representative tile-scan confocal micrographs of mock-infected and TBEV-challenged hNOs with or without NA treatment after 8 days of infection. n = 3 independent organoid batches DAPI, blue; TBEV E-protein (4G2), green; TUJ1, orange; SOX2, magenta. Scale bars, 500 µm.

Article Snippet: Treatment with 5 μM bemnifosbuvir was sufficient to eliminate detectable TBEV antigen , whereas for remdesivir, complete clearance required 100 μM; at 50 μM, viral antigen was reduced but still visible ( ).

Techniques: Virus, Sampling, Infection

Journal: bioRxiv

Article Title: Bemnifosbuvir and remdesivir inhibit tick-borne encephalitis virus infection in complementary in vitro and ex vivo disease models

doi: 10.1101/2025.11.13.688315

Figure Lengend Snippet: Representative tile-scan confocal micrographs of mock- and TBEV-challenged organoids at 2 days p.i. ( A ) and 4 days p.i. ( B ) with or without treatment with 50 μM of selected NAs. n = 3 independent organoid batches. DAPI, blue; TBEV E-protein (4G2), green; TUJ1, orange; SOX2, magenta. Scale bars, 500 µm.

Article Snippet: Treatment with 5 μM bemnifosbuvir was sufficient to eliminate detectable TBEV antigen , whereas for remdesivir, complete clearance required 100 μM; at 50 μM, viral antigen was reduced but still visible ( ).

Techniques:

Journal: bioRxiv

Article Title: Bemnifosbuvir and remdesivir inhibit tick-borne encephalitis virus infection in complementary in vitro and ex vivo disease models

doi: 10.1101/2025.11.13.688315

Figure Lengend Snippet: A , Infectious released virus dynamics in TBEV challenged hNOs with and without addition of 100 μM-0.39 μM bemnifosbuvir. Points indicate mean values ± s.d. n = 3 independent organoid batches. Horizontal dotted line indicates assay detection limit. B , Mean Log 2 fold change of maximum released infectious virus titers following TBEV challenge and treatment with 100 μM-0.39 μM of bemnifosbuvir. Mean values displayed ± s.d. n = 3 independent organoid batches. C , Representative tile-scan confocal micrographs of mock-infected and TBEV-challenged hNOs with or without treatment 100 μM-0.39 μM bemnifosbuvir after 8 days of infection. n = 3 independent organoid batches. DAPI, blue; TBEV E-protein (4G2), green; TUJ1, orange; SOX2, magenta. Scale bars, 500 µm. D , Percentage of virus-harboring cells detected following 4G2-staining and flow cytometry analysis of TBEV-challenged and mock-infected organoids treated with 100 μM-0.39 μM of bemnifosbuvir after 8 days of infection. Points represent individual organoids (n = 1 independent organoid batch). E , Percentage of CC3-positive cells detected through flow cytometry analysis of TBEV-challenged and mock-infected organoids treated with 100 μM-0.39 μM of bemnifosbuvir after 8 days of infection. Points represent individual organoids (n = 1 independent organoid batch).

Article Snippet: Treatment with 5 μM bemnifosbuvir was sufficient to eliminate detectable TBEV antigen , whereas for remdesivir, complete clearance required 100 μM; at 50 μM, viral antigen was reduced but still visible ( ).

Techniques: Virus, Infection, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Bemnifosbuvir and remdesivir inhibit tick-borne encephalitis virus infection in complementary in vitro and ex vivo disease models

doi: 10.1101/2025.11.13.688315

Figure Lengend Snippet: Mean dose-dependent inhibition of infectious TBEV titers following treatment with remdesivir and bemnifosbuvir at concentrations between 100 μM and 0.39 μM. Dose response curves were obtained using a nonlinear regression method. Points indicate mean values ± s.d. n = 3 independent organoid batches. Horizontal dotted line indicates 50% inhibition.

Article Snippet: Treatment with 5 μM bemnifosbuvir was sufficient to eliminate detectable TBEV antigen , whereas for remdesivir, complete clearance required 100 μM; at 50 μM, viral antigen was reduced but still visible ( ).

Techniques: Inhibition

Journal: bioRxiv

Article Title: Bemnifosbuvir and remdesivir inhibit tick-borne encephalitis virus infection in complementary in vitro and ex vivo disease models

doi: 10.1101/2025.11.13.688315

Figure Lengend Snippet: A , Exemplary gating strategy applied to flow cytometry data. The top graphs display a mock-infected and untreated, bottom gates a TBEV-infected and untreated organoid. B-C , Representative tile-scan confocal micrographs of mock- and TBEV-challenged organoids with or without treatment with 100 μM-0.39 μM of bemnifosbuvir ( B ) or remdesivir ( C ). n = 3 independent organoid batches. DAPI, blue; TBEV E-protein (4G2), green; TUJ1, orange; CC3, grey. Scale bars, 500 µm.

Article Snippet: Treatment with 5 μM bemnifosbuvir was sufficient to eliminate detectable TBEV antigen , whereas for remdesivir, complete clearance required 100 μM; at 50 μM, viral antigen was reduced but still visible ( ).

Techniques: Flow Cytometry, Infection

Journal: bioRxiv

Article Title: Bemnifosbuvir and remdesivir inhibit tick-borne encephalitis virus infection in complementary in vitro and ex vivo disease models

doi: 10.1101/2025.11.13.688315

Figure Lengend Snippet: A , Infectious released virus dynamics in TBEV-infected hNOs with and without the addition of 100 μM-0.39 μM remdesivir. Points indicate mean values ± s.d. n = 3 independent organoid batches. Horizontal dotted line indicates assay detection limit. B , Mean Log 2 fold change of maximum released infectious TBEV titers following hNO infection and treatment with 100 μM-0.39 μM of remdesivir. Mean values displayed ± s.d. n = 3 independent organoid batches. C , Representative tile-scan confocal micrographs of mock-infected and TBEV-challenged organoids with or without treatment 100 μM-0.39 μM remdesivir at 8 days p.i. n = 3 independent organoid batches. DAPI, blue; TBEV E-protein (4G2), green; TUJ1, orange; SOX2, magenta. Scale bars, 500 µm. D , Percentage of virus-harboring cells detected by 4G2-staining and flow cytometry analysis of TBEV- and mock-infected hNOs treated with 100 μM-0.39 μM of remdesivir at 8 days p.i. Points represent individual organoids (n = 1 independent organoid batch). E , Percentage of CC3-positive cells detected through flow cytometry analysis of TBEV- and mock-infected hNOs treated with 100 μM-0.39 μM of remdesivir at 8 days p.i. Points represent individual organoids (n = 1 independent organoid batch).

Article Snippet: Treatment with 5 μM bemnifosbuvir was sufficient to eliminate detectable TBEV antigen , whereas for remdesivir, complete clearance required 100 μM; at 50 μM, viral antigen was reduced but still visible ( ).

Techniques: Virus, Infection, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Bemnifosbuvir and remdesivir inhibit tick-borne encephalitis virus infection in complementary in vitro and ex vivo disease models

doi: 10.1101/2025.11.13.688315

Figure Lengend Snippet: A , Cerebellar brain slice cultures were infected with TBEV and simultaneously treated with selected NAs at 50 µM. Data are shown as mean ± s.d. n = 3 independent experiments. Statistical analysis was performed using Welch’s t-test. *p<0.05, ***p<0.001. B , Immunofluorescence staining of cerebellar brain slice cultures at 3 days p.i. TBEV E-protein (T036), green; Purkinje cells/Calbindin, purple. Scale bars, 1 mm.

Article Snippet: Treatment with 5 μM bemnifosbuvir was sufficient to eliminate detectable TBEV antigen , whereas for remdesivir, complete clearance required 100 μM; at 50 μM, viral antigen was reduced but still visible ( ).

Techniques: Slice Preparation, Infection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Bemnifosbuvir and remdesivir inhibit tick-borne encephalitis virus infection in complementary in vitro and ex vivo disease models

doi: 10.1101/2025.11.13.688315

Figure Lengend Snippet: A , Viral titers determined 3 days p.i. showing the inhibition of TBEV replication in OTCs treated with remdesivir and bemnifosbuvir (0–50 µM). Horizontal dotted line indicates the detection limit of the assays. B , Dose-response curves used to calculate the IC 50 using nonlinear regression method. Horizontal dotted line indicates 50% inhibition. C-D , bemnifosbuvir treatment in C and remdesivir treatment in D. Immunofluorescence staining of cerebellar brain slice cultures at 3 days p.i. TBEV E-protein (T036), green; Purkinje cells/Calbindin, purple. Scale bars, 1 mm.

Article Snippet: Treatment with 5 μM bemnifosbuvir was sufficient to eliminate detectable TBEV antigen , whereas for remdesivir, complete clearance required 100 μM; at 50 μM, viral antigen was reduced but still visible ( ).

Techniques: Inhibition, Immunofluorescence, Staining, Slice Preparation