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

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: Pathogens

Article Title: Enhanced Seroconversion to West Nile Virus Proteins in Mice by West Nile Kunjin Replicon Virus-like Particles Expressing Glycoproteins from Crimean–Congo Hemorrhagic Fever Virus

doi: 10.3390/pathogens11020233

Figure Lengend Snippet: Expression of Crimean–Congo hemorrhagic fever virus (CCHFV) glycoproteins Gn and GC proteins using the West Nile Kunjin (WNV KUN ) replicon. ( A ) The reporter luciferase (Luc) gene was substituted with genes encoding CCHFV Gn and Gc in the WNV KUN DNA replicon. In short, the replicon is driven by the cytomegalovirus (CMV) promoter expressing an open reading frame flanked by the 5′- untranslated region (UTR) and the 3′-UTR comprising: first, partial capsid (C) gene fused in frame with the Luc, the foot and mouth disease virus autoprotease 2a (FMDV2A) then partial envelop (E) gene, and all the nonstructural proteins. The hepatitis delta virus antigenomic ribozyme (HDVr) sequence was inserted immediately downstream of the WNV KUN 3′-UTR followed by the Simian virus 40 (SV40) polyadenylation signal (pA). ( B ) Immunoblotting of cell lysates 2 days after transfection of the WNV KUN CCHFV Gn–Gc replicons versus the WNV KUN Luc replicon into the BHK-21 cell line expressing the WNV KUN C–prM–E.

Article Snippet: The following proteins and antibodies were used in this study: the WNV NY99 E protein, the WNV NY99 NS1 protein, the CCHFV Gn protein, the CCHFV Gc protein human Fc tag, the mouse anti-CCHFV Gn (The Native Antigen Company, Oxford, UK), the mouse anti-flavivirus NS1 (Abcam, Cambridge, UK), the mouse J2 anti dsRNA (Scicons, Szirák, Hungary), the mouse anti-CCHFV Gn–Gc (Friedrich-Loeffler-Institute, Greifswald, Germany), the Alexa Fluor 594-conjugated anti-mouse goat antibody (Invitrogen, Vilnius, Lithuania), and the HPR-conjugated anti-mouse goat antibody (Invitrogen).

Techniques: Expressing, Virus, Luciferase, Sequencing, Western Blot, Transfection

Journal: Pathogens

Article Title: Enhanced Seroconversion to West Nile Virus Proteins in Mice by West Nile Kunjin Replicon Virus-like Particles Expressing Glycoproteins from Crimean–Congo Hemorrhagic Fever Virus

doi: 10.3390/pathogens11020233

Figure Lengend Snippet: Immunofluorescence labeling of BHK-21 C-prM-E cells after transfection with the Gn–Gc replicon, followed by two consecutive cycles of RVP infection into naïve BHK-21 cells. The cells were visualized with the antibodies anti-CCHFV Gn–Gc (red), anti-WNV KUN NS1 (red), and anti-dsRNA (red). The nucleus was counterstained with DAPI (blue). Bar scales represent 20 µm.

Article Snippet: The following proteins and antibodies were used in this study: the WNV NY99 E protein, the WNV NY99 NS1 protein, the CCHFV Gn protein, the CCHFV Gc protein human Fc tag, the mouse anti-CCHFV Gn (The Native Antigen Company, Oxford, UK), the mouse anti-flavivirus NS1 (Abcam, Cambridge, UK), the mouse J2 anti dsRNA (Scicons, Szirák, Hungary), the mouse anti-CCHFV Gn–Gc (Friedrich-Loeffler-Institute, Greifswald, Germany), the Alexa Fluor 594-conjugated anti-mouse goat antibody (Invitrogen, Vilnius, Lithuania), and the HPR-conjugated anti-mouse goat antibody (Invitrogen).

Techniques: Immunofluorescence, Labeling, Transfection, Infection

Journal: Pathogens

Article Title: Enhanced Seroconversion to West Nile Virus Proteins in Mice by West Nile Kunjin Replicon Virus-like Particles Expressing Glycoproteins from Crimean–Congo Hemorrhagic Fever Virus

doi: 10.3390/pathogens11020233

Figure Lengend Snippet: CCHFV Gn–Gc RVPs induced seroconversion to CCHFV Gn and Gc and enhanced seroconversion to WNV NY99 NS1, E. ( A ) Schematic illustration of the mice immunization schedule. Mice were subcutaneously immunized three times at weeks 0, 2, and 5 with RVPs expressing CCHFV Gn–Gc (6 mice), Luc (6 mice), or phosphate-buffered saline (PBS) (3 mice). ( B ) Mouse weight from one week before the experiment to the mouse-euthanized day. Mice sera from the study groups were diluted and assayed with enzyme-linked immunosorbent assays (ELISA) to measure antibody titers against CCHFV Gn ( C ), CCHFV Gc ( D ) WNV NY99 E ( E ), and WNV NY99 NS1 ( F ). The end-point titers were determined as there was no difference in the measured optical density values at 450 nm (OD450) between the vaccinated group and the control group. The experiments were conducted with two technical repeats. The p values are indicated using * p < 0.05 and ** p < 0.01. ( G ) Serum titers that elicited 50% reduction in the WNV KUN plaque number. Sera from experimented animal were combined before assaying.

Article Snippet: The following proteins and antibodies were used in this study: the WNV NY99 E protein, the WNV NY99 NS1 protein, the CCHFV Gn protein, the CCHFV Gc protein human Fc tag, the mouse anti-CCHFV Gn (The Native Antigen Company, Oxford, UK), the mouse anti-flavivirus NS1 (Abcam, Cambridge, UK), the mouse J2 anti dsRNA (Scicons, Szirák, Hungary), the mouse anti-CCHFV Gn–Gc (Friedrich-Loeffler-Institute, Greifswald, Germany), the Alexa Fluor 594-conjugated anti-mouse goat antibody (Invitrogen, Vilnius, Lithuania), and the HPR-conjugated anti-mouse goat antibody (Invitrogen).

Techniques: Expressing, Saline, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Immunization with DNA Plasmids Coding for Crimean-Congo Hemorrhagic Fever Virus Capsid and Envelope Proteins and/or Virus-Like Particles Induces Protection and Survival in Challenged Mice

doi: 10.1128/JVI.02076-16

Figure Lengend Snippet: Serum IgG subclass reactivity for each pooled A129 IFNAR −/− serum sample to CCHFV antigen. In the top panel, the subclass pattern of the serum pools (indicated on the x axis) is shown prior to CCHFV challenge, and in the bottom panel the serum IgG subclass pattern is shown at 10 days post-CCHFV challenge (at day of sacrifice).

Article Snippet: An inactivated CCHFV antigen was used for coating at a 1:300 dilution in 0.05 M Na 2 CO 3 -NaHCO 3 buffer, pH 9.6, in flat-bottomed 96-well plates (PolySorp, Nunc, Thermo Scientific).

Techniques:

Journal: Frontiers in Veterinary Science

Article Title: Development of anti-Crimean-Congo hemorrhagic fever virus Gc and NP-specific ELISA for detection of antibodies in domestic animal sera

doi: 10.3389/fvets.2022.913046

Figure Lengend Snippet: CCHFV Gc-specific and CCHFV NP-specific IgG sero-reactivity in sheep sera from endemic CCHFV areas. Sheep sera were collected as part of a cross-sectional study in an endemic CCHFV area (Bulgaria, n = 1,200) and tested for anti-CCHFV Gc-specific IgG levels (A) and anti-CCHFV NP-specific IgG levels (B) by in-house ELISAs. Data are represented as a histogram of the distribution of the OD 450 values frequency (gray bars). Non-parametric estimation of the distribution (solid blue lines) and finite-mixture model (dashed blue lines) with estimated cut-off (red solid line). (C) Relationship between levels of anti-CCHFV Gc-specific and anti-CCHFV NP-specific IgG represented as correlation analysis (Spearman rank test) with red solid lines representing the estimated cut-off by finite mixed model. (D) Heatmap of data normalized across anti-CCHFV Gc and anti-CCHFV NP-specifc IgG using min-max normalization where minimum was defined as the cut-off specific for the assay as descriptive representation of the correlation at individual level. Each row represents data from one animal. Animals below the cut-off represented as gray and animals above the cut-off as different shades of red defined in the legend.

Article Snippet: CCHFV Gc protein and CCHFV NP protein (strain IbAr10200, Nigeria, 1996) were produced in HEK293 cells (The Native Antigen Company, REC31696 and REC31639, respectively).

Techniques:

Journal: Frontiers in Veterinary Science

Article Title: Development of anti-Crimean-Congo hemorrhagic fever virus Gc and NP-specific ELISA for detection of antibodies in domestic animal sera

doi: 10.3389/fvets.2022.913046

Figure Lengend Snippet: Correlation and receiver operating characteristic (ROC) analysis of anti-CCHFV Gc-specific and anti-CCHFV NP-specific IgG in-house ELISAs vs. VectoCrimean-CCHF IgG and ID Screen ® CCHF Double Antigen Multi-species commercial kits. A subset of sheep sera collected as part of a cross-sectional study in an endemic CCHFV area ( n =8 0) was tested for anti-CCHFV Gc-specific IgG and anti-CCHFV NP-specific IgG by in-house ELISAs and with VectoCrimean-CHF IgG and ID Screen ® CCHF Double Antigen Multi-species commercial kits. Left panel shows spearman correlations between responses evaluated by: (A) anti-CCHFV Gc-specific IgG in-house ELISA and VectoCrimean-CHF IgG, (B) anti-CCHFV NP-specific IgG in-house ELISA and VectoCrimean-CHF IgG, or (C) ID Screen ® CCHF Double Antigen Multi-species. Right panel shows ROC curves generated using: (A) anti-CCHFV Gc-specific in-house ELISA OD measured at 450 nm and positive ( n = 41) and negative ( n = 39) sera results obtained with VectoCrimean-CHF IgG; (B) anti-CCHFV NP-specific in-house ELISA OD measured at 450 nm and positive ( n = 41) and negative ( n = 39) sera results obtained with VectoCrimean-CHF IgG, or (C) positive ( n = 35) and negative ( n = 45) sera results obtained with ID Screen ® CCHF Double Antigen Multi-species.

Article Snippet: CCHFV Gc protein and CCHFV NP protein (strain IbAr10200, Nigeria, 1996) were produced in HEK293 cells (The Native Antigen Company, REC31696 and REC31639, respectively).

Techniques: Enzyme-linked Immunosorbent Assay, Generated