Journal: bioRxiv

Article Title: Side-by-side evaluation of two mouse models for Crimean-Congo Hemorrhagic Fever Virus infection

doi: 10.1101/2025.08.20.671421

Figure Lengend Snippet: IFNAR -/- mice were infected with either 100 TCID 50 CCHFV Afg09 (purple, n = 8) or Hoti (green, n = 6). The clinical score (A) , summarizing appearance, behaviour, body weight (B) , and body temperature (C) , was monitored daily. After reaching the clinical endpoint (score of ≥10 or ≥6 on two consecutive days), the mice were sacrificed or found dead (grey). Dotted lines mark the clinical endpoints.

Article Snippet: Microtiter plates were coated with CCHFV glycoprotein (Gc) protein (The Native Antigen Company, REC31696) diluted to 0.5 μg/ml in phosphate buffered saline (PBS) and incubated for 20 hours at 4°C.

Techniques: Infection

Journal: bioRxiv

Article Title: Side-by-side evaluation of two mouse models for Crimean-Congo Hemorrhagic Fever Virus infection

doi: 10.1101/2025.08.20.671421

Figure Lengend Snippet: IFNAR -/- mice were infected with either 100 TCID 50 CCHFV Afg09 (purple, n = 8) or Hoti (green, n = 6). A: CCHFV genome copies were measured by RT-qPCR in organs and serum samples (day 3 and final) of individual mice. B: Infectious CCHFV were determined by TCID 50 assay in organs and final serum samples of individual mice. Each data point represents a sample from an individual animal, data are shown as the means ± SD. Datasets were analyzed using the Šídák’s multiple comparisons test (A) or Tukey’s multiple comparisons test (B). Asterisks indicate statistical significance as detailed between CCHFV Afg09 and Kosovo Hoti group: ∗p ≤ 0.05; ∗∗∗∗p < 0.0001.

Article Snippet: Microtiter plates were coated with CCHFV glycoprotein (Gc) protein (The Native Antigen Company, REC31696) diluted to 0.5 μg/ml in phosphate buffered saline (PBS) and incubated for 20 hours at 4°C.

Techniques: Infection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Side-by-side evaluation of two mouse models for Crimean-Congo Hemorrhagic Fever Virus infection

doi: 10.1101/2025.08.20.671421

Figure Lengend Snippet: IFNAR -/- mice were infected with either 100 TCID 50 CCHFV Afg09 (purple) or Hoti (green). A: Final sera (Afg09 n = 6, Hoti n = 6) were analyzed with two virus-specific ELISAs using either inactivated CCHFV, measuring NP-specific antibodies or the major glycoprotein Gc for coating. Monoclonal antibodies detecting Gc or N protein were used as controls (not shown). Dotted lines indicate the respective lower limit of detection. B: Liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed in final serum samples (Afg09 n = 5, Hoti n = 3). Dotted lines indicate the physiological range of healthy C57BL/6 mice.

Article Snippet: Microtiter plates were coated with CCHFV glycoprotein (Gc) protein (The Native Antigen Company, REC31696) diluted to 0.5 μg/ml in phosphate buffered saline (PBS) and incubated for 20 hours at 4°C.

Techniques: Infection, Virus, Bioprocessing

Journal: bioRxiv

Article Title: Side-by-side evaluation of two mouse models for Crimean-Congo Hemorrhagic Fever Virus infection

doi: 10.1101/2025.08.20.671421

Figure Lengend Snippet: Liver, spleen and CNS of CCHFV Afg09 (purple) or Hoti-infected (green) IFNAR -/- mice were pathologically examined post-mortem. A: Shown are exemplary images of organ histopathology, H&E: hematoxylin and eosin staining. ISH: In situ Hybridization. Arrows indicate multifocal liver necrosis with loss of tissue architecture and lymphohistiocytic infiltrates. Arrowheads indicate apoptosis in germinal follicle centers. Scale bars: 100 µm. B: Quantification of ISH indicating percentage area of CCHFV-positive organ sections. Each data point represents a sample from an individual animal, horizonal lines represent the means ± SD. Datasets were analyzed using the Šídák’s multiple comparisons test. Asterisks indicate statistical significance as detailed between CCHFV Afg09 and Kosovo Hoti group: ∗∗∗∗p < 0.0001; ns = not significant.

Article Snippet: Microtiter plates were coated with CCHFV glycoprotein (Gc) protein (The Native Antigen Company, REC31696) diluted to 0.5 μg/ml in phosphate buffered saline (PBS) and incubated for 20 hours at 4°C.

Techniques: Infection, Histopathology, Staining, In Situ Hybridization

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Establishment of two serological methods for detecting IgG and neutralizing antibodies against Crimean-Congo hemorrhagic fever virus glycoprotein

doi: 10.3389/fcimb.2024.1341332

Figure Lengend Snippet: Expression of the truncated recombinant glycoprotein (rGP) and establishment of an IgG ELISA based on rGP. (A) A schematic representation of the glycoprotein open reading frame (ORF) of Crimean-Congo hemorrhagic fever virus (CCHFV) Ibar10200 strain. (B) Schematic diagram of expression, purification, and identification of recombinant protein in the prokaryotic expression system. (C) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) identification of the rGP. The rGP was detected at 23 kDa boxed out in red. (D) Western blotting analysis of the rGP protein expression in Escherichia coli . Expression was detected with a mouse anti-CCHFV pre-GC monoclonal antibody (mAb) clone 11E7. (D-1) Uninduced IPTG-pET30a(+) empty carrier control. (D-2) Induced IPTG-pET30a(+) empty carrier control. (D-3) Target protein expressed in induced IPTG-pET30a(+) boxed out in red. (E) ELISA analysis of the binding ability of truncated GP (purified rGP, 2-4-8-10 μg/mL) to mAb clone 11E7.

Article Snippet: The mouse monoclonal antibody clone 11E7 anti-CCHFV pre-Gc was bought from BEI Resources (Manassas, VA, USA), and mouse serum anti-CCHFV-eGN was prepared and stored in our laboratory.

Techniques: Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Virus, Purification, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Control, Binding Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Establishment of two serological methods for detecting IgG and neutralizing antibodies against Crimean-Congo hemorrhagic fever virus glycoprotein

doi: 10.3389/fcimb.2024.1341332

Figure Lengend Snippet: Identification analysis of recombinant vesicular stomatitis virus/Crimean-Congo hemorrhagic fever virus (rVSV/CCHFV). (A) The fluorescence and cytopathic effect (CPE) of rVSV-enhanced green fluorescence protein (eGFP) on Vero E6 cells. (B) The fluorescence and CPE of rVSV/CCHFV-P1 on BSR cells. (C) rVSV/CCHFV-P6 on Vero E6 cells at 48 h post-infection visualized under microscope. The fluorescence and CPE in panels (A–C) are indicated by a yellow arrowhead. (D, E) Electron microscopy (EM) detection of rVSVs. Transmission electron micrographs of rVSV-GFP (D) and replication-competent rVSV/CCHFV (E) . Supernatants were negatively stained with 2% aqueous uranyl acetate, and scale bars represent 200 nm. (F) Titer comparison of Vero E6 cells infected with rVSV-eGFP or rVSV/CCHFV at a multiplicity of infection (MOI) of 0.1 at hours 12, 24, 36, 48, 60, 72, 84, 96, 108, and 120. (G) Identification of CCHFV GPCΔ53aa gene in the rVSV/CCHFV (5th–10th generation) by RT-PCR. (H, I) Western blotting of approximately 30 μL rVSVs (10 6 TCID 50 /mL) mixed with 5× loading buffer on 10% gradient TGX gels. 1, rVSV-eGFP; 2, rVSV/CCHFV. Particle preparations were incubated with a mouse serum anti-CCHFV-eG N (H) , anti-CCHFV pre-G r monoclonal antibody (mAb) clone 11E7 (I) , and a horseradish peroxidase (HRP)-conjugated secondary.

Article Snippet: The mouse monoclonal antibody clone 11E7 anti-CCHFV pre-Gc was bought from BEI Resources (Manassas, VA, USA), and mouse serum anti-CCHFV-eGN was prepared and stored in our laboratory.

Techniques: Recombinant, Virus, Fluorescence, Infection, Microscopy, Electron Microscopy, Transmission Assay, Staining, Comparison, Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Establishment of two serological methods for detecting IgG and neutralizing antibodies against Crimean-Congo hemorrhagic fever virus glycoprotein

doi: 10.3389/fcimb.2024.1341332

Figure Lengend Snippet: Indirect immunofluorescence staining to detect the expression of Crimean-Congo hemorrhagic fever virus (CCHFV) GP (100×). After being infected with recombinant vesicular stomatitis virus (rVSV) for 48 h, Vero E6 cells were permeabilized and immunostained with specific antibodies (anti-CCHFV-eG N and anti-CCHFV pre-G C monoclonal antibody (mAb) clone 11E7) and the control antibody [serum in phosphate-buffered saline (PBS) group]. Then, Vero E6 cells were stained with the anti-mouse Cy3-conjugated secondary antibody and 4′,6-diamidino-2-phenylindole (DAPI). Merge: DAPI + Cy3. DAPI, 4′,6-diamidino-2-phenylindole; Cy3, cyanine. Cytomembranes were stained with Cy3 (red for cytomembrane localization). Nuclei were stained with DAPI (blue for nuclear localization).

Article Snippet: The mouse monoclonal antibody clone 11E7 anti-CCHFV pre-Gc was bought from BEI Resources (Manassas, VA, USA), and mouse serum anti-CCHFV-eGN was prepared and stored in our laboratory.

Techniques: Immunofluorescence, Staining, Expressing, Virus, Infection, Recombinant, Control, Saline

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Establishment of two serological methods for detecting IgG and neutralizing antibodies against Crimean-Congo hemorrhagic fever virus glycoprotein

doi: 10.3389/fcimb.2024.1341332

Figure Lengend Snippet: Establishment of recombinant vesicular stomatitis virus/Crimean-Congo hemorrhagic fever virus (rVSV/CCHFV)-based neutralization assay by evaluating the neutralization infectivity of monoclonal antibody (mAb) 11E7. (A) MAb 11E7 was unable to block rVSV-enhanced green fluorescence protein (eGFP) infection in Vero E6 cells [fluorescence, bottom; cytopathic effect (CPE), top]. (B) Sera in control group [serum in phosphate-buffered saline (PBS) group] were not able to inhibit the infection of rVSV/CCHFV particles in Vero E6 cells (fluorescence, bottom; CPE, top). (C) MAb 11E7 (at a dilution of 1:512) was able to completely inhibit the infectivity of rVSV/CCHFV particles (fluorescence, bottom; CPE, top). (D) MAb 11E7 (at a dilution of 1:1,024) cannot completely inhibit the infectivity of rVSV/CCHFV particles in Vero E6 cells (fluorescence, bottom; CPE, top); magnification of microscopy images, 100×.

Article Snippet: The mouse monoclonal antibody clone 11E7 anti-CCHFV pre-Gc was bought from BEI Resources (Manassas, VA, USA), and mouse serum anti-CCHFV-eGN was prepared and stored in our laboratory.

Techniques: Recombinant, Virus, Neutralization, Infection, Blocking Assay, Fluorescence, Control, Saline, Microscopy

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Establishment of two serological methods for detecting IgG and neutralizing antibodies against Crimean-Congo hemorrhagic fever virus glycoprotein

doi: 10.3389/fcimb.2024.1341332

Figure Lengend Snippet: In vivo experiment humoral immune responses and correlation between surrogate virus neutralization test (sVNT) and indirect IgG ELISA. Sera from mice or Syrian hamsters vaccinated with G C subunit candidate vaccine and Crimean-Congo hemorrhagic fever virus (CCHFV) candidate vaccine based on vesicular stomatitis virus (VSV). (A) Enzyme-linked immunosorbent assay (ELISA) using plates coated with 400 ng/well of purified recombinant glycoprotein (rGP) and diluted sera from mice or Syrian hamsters vaccinated with G C subunit candidate vaccine and CCHFV candidate vaccine based on VSV. (B) Surrogate virus neutralization test (sVNT) from diluted endpoint mouse or Syrian hamster sera with G C subunit candidate vaccine and CCHFV candidate vaccine based on VSV incubated on Vero E6 cells. Anti-CCHFV pre-GC monoclonal antibody (mAb) clone 11E7 (11E7) was used as a positive control and diluted in duplicate, together with mouse and Syrian hamster sera serially diluted twofold with DMEM. Data are shown as the mean ± SD and were analyzed using an unpaired t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001). G Csubunit i.s.-2/i.s.-3 : mice were vaccinated with subunit vaccine by subcutaneous injection after the second and third vaccinations. M-V-G i.s.-1 and M-V-G i.s.-2 , M-V-G i.P.-1 and M-V-G i.P.-2 : Mice were vaccinated with CCHFV candidate vaccine based on VSV by subcutaneous or intraperitoneal injection after the first and second vaccinations. S-V-G i.p.-1 and S-V-G i.p.-2 : Syrian hamsters were vaccinated with CCHFV candidate vaccine based on VSV by intraperitoneal injection after the first and second vaccinations. S-PBS/M-PBS: mice or Syrian hamsters were vaccinated with phosphate-buffered saline (PBS). (C) ELISA results with serum dilutions (x-axis) and the sVNT result/serum dilution (y-axis). There is an imperfect correlation (R 2 = 0.5058) between the titer of indirect IgG ELISA and neutralizing antibodies.

Article Snippet: The mouse monoclonal antibody clone 11E7 anti-CCHFV pre-Gc was bought from BEI Resources (Manassas, VA, USA), and mouse serum anti-CCHFV-eGN was prepared and stored in our laboratory.

Techniques: In Vivo, Virus, Neutralization, Enzyme-linked Immunosorbent Assay, Purification, Recombinant, Incubation, Positive Control, Injection, Saline

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Construction and evaluation of DNA vaccine encoding Crimean Congo hemorrhagic fever virus nucleocapsid protein, glycoprotein N-terminal and C-terminal fused with LAMP1

doi: 10.3389/fcimb.2023.1121163

Figure Lengend Snippet: Detection of specific antibodies and neutralizing antibodies in the sera of immunized mice. Mouse sera were collected as described in the Materials and Methods section. The horizontal lines denote the GMT values. (A) NP-specific antibody titers were detected using purified NP. (B) Gc-specific antibody titers were detected using purified Gc. (C) Gn-specific antibody titers were detected using purified Gn. (D) Neutralizing antibody titers were detected by determining the ability of the sera to neutralize the CCHFV tecVLPs. (* P < 0.05).

Article Snippet: Purified CCHFV Gn and CCHFV NP were purchased from Abcam (Boston, MA, USA), and purified CCHFV Gc was purchased from The Native Antigen Company (Kidlington, Oxford, UK).

Techniques: Purification