Journal: Viruses

Article Title: Genomic Organization of the Newly Discovered Cassava Congo Cheravirus Reveals a Unique Maf/HAM1 Motif in the C-Terminal Region of the RNA1 Polyprotein and Suggests the Presence of Two Protein Domains Upstream of the Putative Helicase Domain

doi: 10.3390/v18010084

Figure Lengend Snippet: ( A ). Electrophoretic separation (8–20% SDS-PAGE) of soluble leaf proteins from infected N. benthamiana (lanes 1–5, 7, and 8) and purified virus preparation (lane 6). ( B ), Nitrocellulose western immunoblot incubated with the antiserum DSMZ AS-0896, indicating virus coat protein units (lane 6).

Article Snippet: Following electrophoresis and transfer to nitrocellulose membranes, viral proteins were detected by incubating the membranes with the antiserum DSMZ AS-0896.

Techniques: SDS Page, Infection, Purification, Virus, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Serological cross-reactivity between Crimean-Congo haemorrhagic fever virus and Nairobi sheep disease virus glycoprotein C

doi: 10.3389/fimmu.2024.1423474

Figure Lengend Snippet: Orthonairovirus test antigens employed during experiments.

Article Snippet: CCHFV Gc , NP_950235 (IbAr10200, Nigeria) , HEK293 (Human) , The Native Antigen Company, UK #REC31696.

Techniques: Expressing, Plasmid Preparation

Journal: Cell Research

Article Title: LDLR is an entry receptor for Crimean-Congo hemorrhagic fever virus

doi: 10.1038/s41422-023-00917-w

Figure Lengend Snippet: a Screening of LDLR and LDLR-related proteins (LRPs) that are crucial for CCHFV infection. The HEK293T cells were edited with a control or sgRNAs targeting the genes encoding LDLR and LRPs (two sgRNAs for each gene). After puromycin selection, the cell pools were infected with CCHFV (MOI = 0.05) for 24 h before RT-qPCR was performed. Data were normalized to the relative mRNA level of CCHFV S in the control sgRNA-edited cells. b Surface expression of LDLR in different cell lines. The indicated cell lines were assessed by flow cytometry using the anti-LDLR mAb (R301). c CCHFV infectivity of different cell lines. The indicated cell lines were infected with CCHFV (MOI = 0.05) for 48 h. The CCHFV Gn-positive cells were examined by flow cytometry with a customized anti-Gn monoclonal antibody (7A11). d Overexpression of LDLR enhances CCHFV infection in DLD1 cells. Control and LDLR-overexpressing DLD1 cells were infected with CCHFV (MOI = 0.05) for 24 h (left panel) or 48 h (right panel). The levels of CCHFV S mRNA and NP protein were determined by RT-qPCR (left panel) and immunoblots (right panel), respectively. e Effects of LDLR-deficiency on CCHFV infection in SW13 cells. SW13 cells were edited with a control (g NC ) or three individual sgRNAs targeting different regions of LDLR coding sequence (g LDLR ). The control and LDLR sgRNA-edited SW13 cell pools were infected with CCHFV (MOI = 0.05). CCHFV NP expression (left panel, 48 hpi), mRNA level of CCHFV S segment (2 nd panel, 24 hpi), percentage of Gn-positive cells (3 rd panel, 48 hpi) and cell cytopathic effects (right panel, 72 hpi) was measured by immunoblots, RT-qPCR, flow cytometry and crystal violet staining, respectively. For bar graphs, data are normalized to that of the control gRNA-edited cells. f Effects of LDLR-deficiency on production of progeny viruses. SW13 cells were edited with a control (g NC ) or three individual sgRNAs targeting different regions of LDLR coding sequence (g LDLR ). The sgRNA-edited SW13 cell pools were then infected with CCHFV (MOI = 0.05) for 72 h. Titers of progeny viruses in the supernatants were measured by TCID 50 assay. Data are normalized to that of the control gRNA-edited cells. LOD, limit of detection. g Effects of LDLR-deficiency on CCHFV infection in various cells. Huh7, Vero E6 and Hepa1-6 cells were edited with a control gRNA or the indicated numbers of gRNAs targeting LDLR gene. Cells were infected with CCHFV (MOI = 0.05) for 24 h before RT-qPCR was performed. Data are normalized to the CCHFV S mRNA level in the control gRNA-edited cells. h CCHFV infectivity in LDLR-knockout SW13 and Huh7 cells. Single clones of LDLR-knockout SW13 and Huh7 were isolated and confirmed by immunoblots (left). The control (g NC ) or LDLR-deficient clone (g LDLR -C1) were infected with CCHFV (MOI = 0.05) for 24 h before RT-qPCR analysis. Data are normalized to that of each control gRNA-edited cells. i CCHFV infectivity in Ldlr −/− primary cells. Primary hepatocytes and lung fibroblasts (MLFs) prepared from WT and Ldlr −/− mice were incubated with CCHFV (MOI = 0.05). The mRNA level of CCHFV S segment (top, 48 hpi) and the viral genomic copies in the supernatant (bottom, 72 hpi) were measured by RT-qPCR. j Effects of LDLR-deficiency on CCHFV, RVFV, EBIV and VSV infection. The control (g NC ) or LDLR-deficient clone (g LDLR -C1) were inoculated with the indicated viruses for 24 h before RT-qPCR was performed. Data are represented as mean ± SD. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Mouse anti-Flag monoclonal antibody (#F3165-M2, Sigma-Aldrich), rabbit anti-β-actin monoclonal antibody (#AC026, ABclonal), rabbit anti-hLDLR monoclonal antibody (#10231-R301, SinoBiological), rabbit anti-hLDLR monoclonal antibody (#A14996, ABclonal), rabbit anti-mLDLR monoclonal antibody (#50305-R004, SinoBiological), mouse anti-IFNAR1 monoclonal antibody (MAR1-5A3, #BE0241, BioXCell), mouse anti-Gn monoclonal antibody (JE12, #MAB12317, The Native Antigen Company), phycoerythrin (PE)-conjugated anti-hLDLR monoclonal antibody (#10231-R301-P, SinoBiological), Alexa Fluor™ 488-conjugated anti-mouse IgG (#R37114, Thermo Fisher), human control IgG (#12000C, Invitrogen), mouse control IgG (#I5381, Sigma-Aldrich), rabbit control IgG (#CR1, SinoBiological) and goat control IgG (#CR2, SinoBiological) were purchased from the indicated companies.

Techniques: Infection, Control, Selection, Quantitative RT-PCR, Expressing, Flow Cytometry, Over Expression, Western Blot, Sequencing, Staining, Knock-Out, Clone Assay, Isolation, Incubation

Journal: Cell Research

Article Title: LDLR is an entry receptor for Crimean-Congo hemorrhagic fever virus

doi: 10.1038/s41422-023-00917-w

Figure Lengend Snippet: a Effects of LDLR on CCHFV attachment and internalization. The control (g NC ), LDLR-deficient (g LDLR -C1) or LDLRAP1-deficient (g LDLRAP1 ) SW13 cells were incubated with CCHFV at 4 °C for 1 h (for binding assay), or followed with incubation at 37 °C for 1 h (for internalization assay). The cells were collected and CCHFV S mRNA level was measured by RT-qPCR. Data are normalized to the CCHFV S mRNA level in the control gRNA-edited cells. b Effects of LDLR blocking antibodies on CCHFV infection. SW13, Huh7, Vero E6, primary human PBMCs and HUVECs, and mouse Hepa1-6 cells were pre-incubated with a rabbit anti-hLDLR mAb (R301), a goat anti-hLDLR pAb (#AF2148), a rabbit anti-mLDLR mAb (R004), or their respective control IgGs as indicated for 1 h before CCHFV infection (MOI = 0.05). Twenty-four hours after infection, the cells were collected for RT-qPCR analysis for CCHFV S mRNA level. Data are normalized to that of cells treated with the respective control IgG at 0 μg/mL. c Effects of LDLR blocking antibodies on the entry of RVFV, EBIV and VSV. SW13 cells were pre-incubated with the indicated concentrations of a control rIgG or a rabbit anti-hLDLR mAb (R301) for 1 h before infection RVFV, EBIV or VSV. Twenty-four hours after infection, mRNA levels of RVFV M segment, EBIV S segment, or VSV L gene were measured by RT-qPCR analysis. Data are normalized to that of cells treated with the respective control IgG at 0 μg/mL. d LDL inhibits CCHFV infection. SW13 cells (left) and HUVEC cells (right) were pre-treated with the indicated concentrations of LDL for 1 h and then left uninfected or infected with CCHFV (MOI = 0.05). Twenty-four hours post infection, CCHFV S mRNA level was analyzed by RT-qPCR. Data are normalized to that of CCHFV infected cells without LDL treatment. e The soluble human LDLR-Fc fustion protein (sohLDLR-Fc) inhibits CCHFV infection. CCHFV (MOI = 0.05) was pre-incubated with the indicated concentrations of Fc or sohLDLR-Fc for 1 h before infection of SW13 and Huh7 cells. Twenty-four hours post infection, CCHFV S mRNA level was analyzed by RT-qPCR. Data are normalized to that of cells infected with un-pretreated viruses. f Effects of sohLDLR-Fc on RVFV, EBIV and VSV infection in SW13 cells. RVFV (MOI = 0.1), EBIV (MOI = 0.5) or VSV (MOI = 0.1) were pre-incubated with the indicated concentrations of sohLDLR-Fc or Fc for 1 h before infection of SW13 cells. Twenty-four hours post infection, mRNA levels of RVFV M segment, EBIV S segment, or VSV L gene were measured by RT-qPCR analysis. Data are normalized to that of cells infected with the respective un-pretreated viruses. Data are represented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Mouse anti-Flag monoclonal antibody (#F3165-M2, Sigma-Aldrich), rabbit anti-β-actin monoclonal antibody (#AC026, ABclonal), rabbit anti-hLDLR monoclonal antibody (#10231-R301, SinoBiological), rabbit anti-hLDLR monoclonal antibody (#A14996, ABclonal), rabbit anti-mLDLR monoclonal antibody (#50305-R004, SinoBiological), mouse anti-IFNAR1 monoclonal antibody (MAR1-5A3, #BE0241, BioXCell), mouse anti-Gn monoclonal antibody (JE12, #MAB12317, The Native Antigen Company), phycoerythrin (PE)-conjugated anti-hLDLR monoclonal antibody (#10231-R301-P, SinoBiological), Alexa Fluor™ 488-conjugated anti-mouse IgG (#R37114, Thermo Fisher), human control IgG (#12000C, Invitrogen), mouse control IgG (#I5381, Sigma-Aldrich), rabbit control IgG (#CR1, SinoBiological) and goat control IgG (#CR2, SinoBiological) were purchased from the indicated companies.

Techniques: Control, Incubation, Binding Assay, Quantitative RT-PCR, Blocking Assay, Infection

Journal: Cell Research

Article Title: LDLR is an entry receptor for Crimean-Congo hemorrhagic fever virus

doi: 10.1038/s41422-023-00917-w

Figure Lengend Snippet: a Pull-down of CCHFV virions by sohLDLR. CCHFV, biotinylated sohLDLR and magnetic streptavidin beads were co-incubated as indicated. Pull-down was performed with a magnet and the pellet was subjected to RT-qPCR analysis. Data are represented as mean ± SD. b Monoclonal antibodies against CCHFV Gc (ADI 36121), Gn (JE12) or their respective control IgG was immobilized on plates. ELISA-based binding assays were performed with CCHFV, biotinylated sohLDLR and Avidin-HRP. Data are represented as mean ± SD. c Recombinant Gc of CCHFV YL16070 and IbAr 10200 strains and biotinylated sohLDLR were co-incubated as indicated. Pull-down assay was performed with magnetic streptavidin beads and the pellets were subjected to immunoblots with the indicated antibodies. d Biotinylated sohLDLR was immobilized onto the streptavidin biosensors. Binding parameters of recombinant Gc or Gn of the YL16070 and IbAr 10200 strain and VSV-G to LDLR were measured by Bio-Layer Interferometry (BLI) in the indicated PBS buffers. Fitted curves are shown with dotted lines. A 1:1 binding model was used to calculate the K D .

Article Snippet: Mouse anti-Flag monoclonal antibody (#F3165-M2, Sigma-Aldrich), rabbit anti-β-actin monoclonal antibody (#AC026, ABclonal), rabbit anti-hLDLR monoclonal antibody (#10231-R301, SinoBiological), rabbit anti-hLDLR monoclonal antibody (#A14996, ABclonal), rabbit anti-mLDLR monoclonal antibody (#50305-R004, SinoBiological), mouse anti-IFNAR1 monoclonal antibody (MAR1-5A3, #BE0241, BioXCell), mouse anti-Gn monoclonal antibody (JE12, #MAB12317, The Native Antigen Company), phycoerythrin (PE)-conjugated anti-hLDLR monoclonal antibody (#10231-R301-P, SinoBiological), Alexa Fluor™ 488-conjugated anti-mouse IgG (#R37114, Thermo Fisher), human control IgG (#12000C, Invitrogen), mouse control IgG (#I5381, Sigma-Aldrich), rabbit control IgG (#CR1, SinoBiological) and goat control IgG (#CR2, SinoBiological) were purchased from the indicated companies.

Techniques: Incubation, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Avidin-Biotin Assay, Recombinant, Pull Down Assay, Western Blot

Journal: Cell Research

Article Title: LDLR is an entry receptor for Crimean-Congo hemorrhagic fever virus

doi: 10.1038/s41422-023-00917-w

Figure Lengend Snippet: a , b WT ( n = 11) and LDLR-knockout ( n = 11) mice were pretreated with anti-IFNAR1 monoclonal antibody MAR1-5A3 (200 µg/mouse) 24 h before infection and then infected with CCHFV (10 TCID 50 ) via the intraperitoneal route. Forty-eight hours post infection, 200 µg of MAR1-5A3 was administrated. The body weight ( a ) and survival ( b ) of the mice were monitored daily. c , d WT ( n = 7) and LDLR-knockout ( n = 7) mice were pretreated with anti-IFNAR1 monoclonal antibody MAR1-5A3 (200 µg/mouse) 24 h before infection and then infected with CCHFV (10 TCID 50 ) via the intraperitoneal route. Forty-eight hours post infection, 200 µg of MAR1-5A3 was administrated. Mice were necropsied and the livers and spleens were collected at day 3 and 5 post infection. Viral loads were quantified by RT-qPCR and shown as the number of viral RNA copies per microgram of organs or per mL of sera ( c ). H&E staining and immunostaining with anti-Gn mAb (7A11) were performed and the pathological changes were indicated ( d ). The extensive necrosis (white arrowheads) and necrotic cellular debris (white arrows) in the liver were indicated; white pulps in the spleen (white asterisks) were marked. The bars represent 100 µm. Data are represented as mean ± SD. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Mouse anti-Flag monoclonal antibody (#F3165-M2, Sigma-Aldrich), rabbit anti-β-actin monoclonal antibody (#AC026, ABclonal), rabbit anti-hLDLR monoclonal antibody (#10231-R301, SinoBiological), rabbit anti-hLDLR monoclonal antibody (#A14996, ABclonal), rabbit anti-mLDLR monoclonal antibody (#50305-R004, SinoBiological), mouse anti-IFNAR1 monoclonal antibody (MAR1-5A3, #BE0241, BioXCell), mouse anti-Gn monoclonal antibody (JE12, #MAB12317, The Native Antigen Company), phycoerythrin (PE)-conjugated anti-hLDLR monoclonal antibody (#10231-R301-P, SinoBiological), Alexa Fluor™ 488-conjugated anti-mouse IgG (#R37114, Thermo Fisher), human control IgG (#12000C, Invitrogen), mouse control IgG (#I5381, Sigma-Aldrich), rabbit control IgG (#CR1, SinoBiological) and goat control IgG (#CR2, SinoBiological) were purchased from the indicated companies.

Techniques: Knock-Out, Infection, Quantitative RT-PCR, Staining, Immunostaining

Journal: Cell Research

Article Title: LDLR is an entry receptor for Crimean-Congo hemorrhagic fever virus

doi: 10.1038/s41422-023-00917-w

Figure Lengend Snippet: a A flowchart of the experiment. C57BL/6 mice were pretreated with anti-IFNAR1 monoclonal antibody MAR1-5A3 (300 µg/mouse) plus control rIgG ( n = 10, 100 µg/mouse) or an LDLR blocking antibody (R004) ( n = 7, 100 µg/mouse) intraperitoneally 24 h before infection. Another dose of the rIgG or R004 mAb (100 µg) was administrated 1 h prior to infection. Mice were challenged with CCHFV (100 TCID 50 per mouse, subcutaneously). Twenty-four hours post infection, MAR1-5A3 (200 µg/mouse) was administrated. The rIgG or R004 mAb was administrated at 100 µg quaque die for 5 days post infection. b , c , Protective effects of LDLR blocking antibody on fatality caused by CCHFV infection. As described in ( a ), C57BL/6 mice were treated with rIgG ( n = 10) or the LDLR blocking antibody (R004) ( n = 7) and subjected to CCHFV challenge. Body weight ( b ) and survival ( c ) of the mice were monitored daily. d , e Protective effects of LDLR blocking antibody on CCHFV caused pathogenesis. As described in ( a ), C57BL/6 mice were treated with rIgG ( n = 6) or the LDLR blocking antibody (R004) ( n = 6) and subjected to CCHFV challenge. Mice were necropsied and the livers and spleens were collected at day 5 post infection. Viral loads were quantified by qRT-PCR and shown as the number of viral RNA copies per microgram of organs or per mL of sera ( d ). H&E staining and immunostaining with anti-Gn mAb (7A11) were performed and the pathological changes were indicated ( e ). The extensive necrosis (white arrowheads) and necrotic cellular debris (white arrows) in liver were indicated; white pulps in the spleen (white asterisks) were marked. The bars represent 100 µm. Data are represented as mean ± SD. * P < 0.05; ** P < 0.01.

Article Snippet: Mouse anti-Flag monoclonal antibody (#F3165-M2, Sigma-Aldrich), rabbit anti-β-actin monoclonal antibody (#AC026, ABclonal), rabbit anti-hLDLR monoclonal antibody (#10231-R301, SinoBiological), rabbit anti-hLDLR monoclonal antibody (#A14996, ABclonal), rabbit anti-mLDLR monoclonal antibody (#50305-R004, SinoBiological), mouse anti-IFNAR1 monoclonal antibody (MAR1-5A3, #BE0241, BioXCell), mouse anti-Gn monoclonal antibody (JE12, #MAB12317, The Native Antigen Company), phycoerythrin (PE)-conjugated anti-hLDLR monoclonal antibody (#10231-R301-P, SinoBiological), Alexa Fluor™ 488-conjugated anti-mouse IgG (#R37114, Thermo Fisher), human control IgG (#12000C, Invitrogen), mouse control IgG (#I5381, Sigma-Aldrich), rabbit control IgG (#CR1, SinoBiological) and goat control IgG (#CR2, SinoBiological) were purchased from the indicated companies.

Techniques: Control, Blocking Assay, Infection, Quantitative RT-PCR, Staining, Immunostaining