Journal: Journal of Virological Methods

Article Title: Detection of human rhinoviruses by reverse transcription strand invasion based amplification method (RT-SIBA)

doi: 10.1016/j.jviromet.2018.10.015

Figure Lengend Snippet: List of microbes used for cross-reactivity testing.

Article Snippet: Enterovirus 71 ATCC VR-1432 , – , –.

Techniques: Virus

Journal: Nature Communications

Article Title: RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

doi: 10.1038/s41467-022-33805-9

Figure Lengend Snippet: A HIV-1 firefly luciferase reporter viruses pseudotyped with EBOV-GP 1,2 were produced from HEK293T WT, PDIA3, CALR or CANX-overexpressing, and PDIA3 -, CALR - or CANX -KO cells. After infecting HEK293T cells with an equal number of these different viruses, viral entry was determined by measuring intracellular luciferase activities. Viral entry is shown as relative values, with the entry of viruses produced from HEK293T WT cells in the presence of a control vector set to 100%. B EBOV replication and transcription-competent virus-like particles (trVLPs) were produced (p0) and passaged two times (p1, p2) in HEK293T cells in the presence or absence of PDIA3, CALR, or CANX. EBOV replication was determined by measuring intracellular Renilla luciferase activity. Viral replication was also measured in the absence of EBOV-L [L(-)], which served as a negative control. Results from three independent experiments are presented. C Huh7 cells were transfected with increasing amounts of vectors expressing PDIA3, CALR, or CANX, and infected with EBOV Mayinga strain at 0.01 multiplicity of infection (MOI). Viral RNAs were extracted from the supernatants at indicated times and quantified by real-time PCR. D EBOV virus-like particles (VLPs) were produced from HEK293T WT, CALR or CANX-overexpressing, and CALR - or CANX -KO cells after expression of EBOV-GP 1,2 with EBOV-VP40. VLPs were purified by ultra-centrifugation and protein expression in cell lysate and virions was analyzed by WB. GP, VP40, and endogenous CALR and CANX were detected by their specific antibodies; ectopic CALR and CANX were detected by anti-Myc or anti-HA. E Viral fusion proteins from indicated viruses were expressed with CALR in HEK293T cells (lanes 1–14). Alternatively, they were also expressed in HEK293T WT or CALR -KO cells (lanes 15-28). Protein expression was detected by WB. EBOV-GP 1,2 , MERS-S, SRAS1-S, and SARS2-S were detected by anti-FLAG; IAV (H5N5) HA, VSV-G, HIV-1 Env, and CALR were detected by their specific antibodies. F Viral fusion proteins from indicated viruses were expressed with CANX in HEK293T cells (lanes 1-14). Alternatively, they were also expressed in HEK293T WT or CANX -KO cells (lanes 15-28). Protein expression was detected by WB as in D , except that CANX was detected by its specific antibody. Error bars in A , B , and C represent the standard error of measurements (SEMs) calculated from three independent experiments.

Article Snippet: Rabbit polyclonal anti-Zaire EBOV-GP (40442-T48, 1:5000), anti-EBOV-VP40 (40446-T48, 1:10,000), anti-IAV H5HA (11048-RP02, 1:5000), anti-HDAC6 (100768-T08, 1:1000), and anti-RNF26 (203155-T32, 1:500) were purchased from Sino Biological.

Techniques: Luciferase, Produced, Plasmid Preparation, Activity Assay, Negative Control, Transfection, Expressing, Infection, Real-time Polymerase Chain Reaction, Purification, Centrifugation

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Mutations on the spike glycoprotein of SARS-CoV-2 Omicron subvariants. Residues that are not identical among Omicron subvariants are colored red. b , Workflow to generate cryo-EM structure of BA.2, BA.3, BA.2.13, BA.2.12.1, BA.4/5 spike glycoprotein trimer with S6P and R683A, R685A substitutions. c , Binding affinities of Omicron variants spike trimers to hACE2 measured by SPR. SPR analyses were conducted in biological duplicates. d , MD simulated interactions between hACE2 and RBD of Omicron variants. Structures of the RBD from Omicron variants and hACE2 are shown as ribbons.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , FACS analysis of pooled memory B cells (IgM − CD27 + ) from plasma of individuals who have recovered from BA.1 breakthrough infection after vaccination, vaccinated individuals and unvaccinated individuals who have recovered from BA.1 breakthrough infection. The percentage of cells recognizing WT or BA.1 RBD are shown. b , The heavy chain V domain somatic hypermutation (SHM) rate of BA.1-specific ( n = 968) and BA.1–WT cross-reactive ( n = 4,782) BCRs obtained from 10X scVDJ-seq from individuals who have recovered from BA.1 breakthrough infection after vaccination. Two-tailed Wilcoxon rank-sum test. Boxes show 25th percentile, median and 75th percentile, and violin plots show kernel density estimation curves of the distribution. c , t -SNE and unsupervised clustering of antibodies that bind WT SARS-CoV-2 RBD. Twelve epitope groups were identified on the basis of DMS of 1,538 antibodies. d , e , Epitope distribution and projection of antibodies from plasma of individuals who had recovered from infection with the WT virus, individuals who have recovered from BA.1 breakthrough infection after vaccination, and vaccinated individuals who had recovered from SARS. f , ACE2 competition level determined by competition ELISA ( n = 1,286) were projected onto the t -SNE. g , Neutralizing activity against SARS-CoV-2 D614G ( n = 1,509) and SARS-CoV-1 (HKU-39849; n = 1,457). h , Average mutational escape score projection of each epitope group on SARS-CoV-2 RBD (Protein Data Bank (PDB): 6M0J). All neutralization assays were performed as biological duplicates.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Infection, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Activity Assay, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a – c , Neutralizing activity against SARS-CoV-1 and SARS-CoV-2 subvariants by NAbs in group E1 ( a ; n = 70), F2 ( b ; n = 171) and F3 ( c ; n = 69). The geometric mean of the fold change in IC 50 relative to BA.2 is shown above each plot. P -values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples, compared with the IC 50 for BA.2. d , The epitope of Group E1 antibody BD55-3152 on the BA.1 RBD. e , Overlay of BD55-5840 in the complex with BA.1 or BA.2 RBD. f , g , The epitope and interactions on the binding interface of BD55-1239 (group F2) ( f ) and BD55-3372 (group F3) ( g ). Antibody residues are shown in blue, and RBD residues are in black or red. Residues highlighted in red indicate sites that are mutated in Omicron variants. h , Average escape maps of antibodies in epitope groups E1, F2 and F3, and the corresponding multiple sequence alignment of various sarbecovirus RBDs. The height of each amino acid in the escape map represents its mutation escape score. Sites that are mutated in Omicron subvariants are marked in bold. All neutralization assays were performed as biological duplicates.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Activity Assay, Two Tailed Test, Binding Assay, Sequencing, Mutagenesis, Neutralization

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Cartoon models of Cryo-EM structures of BD55-3152 in complex of BA.1 RBD, BD55-1239 in complex of BA.1 RBD, and BD55-3372 in complex of Delta RBD. b , Workflow to generate refined structural model of BD55-3152 and BD55-1239 in complex of BA.1 RBD, BD55-3372 in complex of Delta RBD, and BD55-5840 in complex of BA.2 RBD. c , Neutralizing activity of representative NAbs in group E1 (n = 68), F2 (n = 139) and F3 (n = 61) against SARS-CoV-2 D614G, in addition to D614G+D405N and D614G+R408S. Geometric mean of IC50 fold changes compared to IC50 against D614G are annotated above the bars. P-values were calculated using a two-tailed Wilcoxon signed-rank test of paired samples. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant, p > 0.05. All neutralization assays were conducted in biological duplicates. d , Conformational comparison between BA.1 and BA.2 RBD regarding the 366-377 hairpin. e , Biolayer interferometry analysis of Group E1 antibodies S309 and BD55-5840 binding to Omicron BA.1 and BA.2 Spike trimer. Biolayer interferometry analyses were conducted in biological duplicates.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Cryo-EM Sample Prep, Activity Assay, Two Tailed Test, Neutralization, Binding Assay

Journal: Nature

Article Title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

doi: 10.1038/s41586-022-04980-y

Figure Lengend Snippet: a , Four epitope groups were identified among 102 BA.1-specific NAbs via k -means clustering and t -SNE of BA.1 RBD-based DMS profiles. b , c , Distribution of ACE2 competition level ( b ) and neutralizing activities ( c ) against BA.1. d , Neutralizing activities of BA.1-specific antibodies against pseudovirus with SARS-CoV-1 and SARS-CoV-2 spike variants (A Omi , n = 18; B Omi , n = 30; D Omi , n = 22; F3 Omi , n = 32). The geometric mean of the fold change in IC 50 relative to BA.1 is shown above each plot. e , Average mutational escape score projection of each BA.1-specific epitope group on SARS-CoV-2 RBD (PDB: 7WPB). f , Averaged escape maps at escape hotspots of the 102 NAbs in the four epitope groups, and corresponding multiple sequence alignment of various sarbecovirus RBDs. The height of each amino acid in the escape map represents its mutation escape score. Sites that are mutated in Omicron variants are marked in bold. WT-related escaping mutations are highlighted. g , Neutralizing activities of BA.1-specific NAbs against BA.1- or BA.2-based pseudoviruses carrying single substitutions (A Omi , n = 18; B Omi , n = 30; D Omi , n = 22; F3 Omi , n = 32). The geometric mean of the fold change in IC 50 relative to BA.1 is shown above each plot. Wilcoxon signed-rank test of paired samples, compared with IC 50 for BA.1. All neutralization assays were performed as biological duplicates.

Article Snippet: WT RBD used here was SARS-CoV-2 (2019-nCoV) Spike RBD-His Recombinant Protein (Sino Biological, 40592-V08H).

Techniques: Sequencing, Mutagenesis, Neutralization

Journal: bioRxiv

Article Title: Jamaican fruit bats’ ( Artibeus jamaicensis ) competence for Ebola virus but not Marburg virus is driven by intrinsic differences in viral entry and IFN-I signaling antagonism

doi: 10.1101/2024.10.17.618736

Figure Lengend Snippet: (A) Schematic mechanisms of EBOV VP24 and MARV VP40 antagonism of the type-I interferon signaling pathway. Image created using Biorender. (B) Immunoblot of cytoplasmic and nuclear fractions of Jamaican fruit bat kidney cells, AjKi_RML2, infected with MOI 1.5 of EBOV-Mayinga or MARV-Ozolin for 24 hours prior to the addition of exogenous recombinant Chiroptera IFN-β (100ng/mL) for 30 minutes. Cells were treated with type I interferon induction inhibitor BAY-985 (25 nM) at the time of infection. The presented panel is representative of three independent immunoblots. (C) Quantification of phosphorylated STAT1 in the cytoplasmic and nuclear fractions. Normalized expression was determined using the following formula: (AUC pSTAT1)/(AUC β-tubulin (cytoplasmic) or Lamin A/C (nuclear)). To calculate relative expression, the normalized sample value is divided by the normalized expression in uninfected cells treated with IFNβ. Three independent experiments were performed. Percentage of pSTAT1 in each of the fractions for EBOV and MARV-infected cells was compared mock using a two-way ANOVA with Dunnett’s multiple test comparison. (D) AjKi_RML2 cells were infected with MOI 0.1 EBOV-Mayinga or MARV-Ozolin for 1 hour and treated with vehicle control DMSO or itacitinib (0.25-25 nM) immediately after infection. Fresh media with inhibitor was replaced at 24- and 48-hours post infection. Supernatants collected at 72 hours post-infection were titrated. To calculate the relative viral load, the viral titers were normalized to the average titer of EBOV-infected cells treated with DMSO. The experiment was performed in triplicate. The effect of itacitinb on EBOV and MARV replication was determined using a one-way ANOVA with Dunnett’s multiple test comparison. (E) Immunoblot showing the effect of itacitinib treatment on the activation of the immune response and VP40 expression. The panel is representative of two independent immunoblots. (F) Quantification of VP40 for the immunoblot presented in panel D. Normalized expression was determined using the following formula: (AUC VP40)/(AUC β-tubulin). To calculate relative expression, the normalized sample value is divided by the normalized expression in DMSO-treated cells infected with either EBOV or MARV. (G) Infectious titers of EBOV-Mayinga or MARV-Ozolin at 48 hours post-infection. AjKi_RML2 cells were treated with recombinant Chiroptera IFN-β in 10-fold serial dilution (0-100 ng/mL) for 24 hours prior to infection with MOI 0.1. Data presented as log 10 transformed values. The experiment was performed in triplicate. To test a change in viral titer at each dose compared to mock treated cells, we performed a two-way ANOVA with Dunnett’s multiple test comparison.

Article Snippet: Primary antibodies used: pSTAT1 – Y701 (Cell Signaling Technology, 9167S), pSTAT2 – Y690 (Cell Signaling Technology, 88410S), total STAT1 (Cell Signaling Technology, 14994S), total STAT2 (Cell Signaling Technology, 72604S), RIG-I (Kerafast, 1C3), Lamin A/C (Cell Signaling Technology, 4777S), β-tubulin (Sigma Aldrich, T8328), EBOV VP24 (Sino Biological 40454-T46), EBOV VP40 (GeneTex, GTX134034), MARV VP40 (The Native Antigen Company, MAV12450-100).

Techniques: Western Blot, Infection, Recombinant, Expressing, Comparison, Control, Activation Assay, Serial Dilution, Transformation Assay

Journal: Cell host & microbe

Article Title: Longitudinal human antibody repertoire against complete viral proteome from Ebola virus survivor reveals protective sites for vaccine design

doi: 10.1016/j.chom.2020.01.001

Figure Lengend Snippet: Serial dilutions of serum samples collected at different time points from the EBOV survivor were analyzed for antibody binding to purified proteins from EBOV/Makona strain by SPR. (A-H) Total antibody binding is represented in SPR resonance units (RU) in black for binding to NP (A), VP35 (B). VP40 (B), GP (B). sGP (E), VP30 (F), VP24 (G) and L polymerase (H). Total antibody binding show is calculated RU for an undiluted serum sample. (A-H) Polyclonal antibody affinity maturation to EBOV proteins following EBOV infection in survivor was determined by SPR. Binding affinity of serially diluted post-infection serum to EBOV proteins was measured and is plotted in blue for each of the proteins as mentioned above for total antibody binding. Antibody off-rate constants that describe the fraction of antibody-antigen complexes decaying per second were determined directly from the serum sample interaction with EBOV proteins using SPR in the dissociation phase as described in Materials and Methods. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variation for each sample in duplicate SPR runs was <5%. The data shown is average value of two experimental runs. The maximum resonance units (Max RU) data shown was the calculated RU signal for the undiluted serum sample. (I - P) Antibody isotype of EBOV/Makona protein binding antibodies following EBOV infection. The isotype composition of serum antibodies bound to different proteins of EBOV/Makona isolate as measured in SPR. The resonance units for each anti-Makona protein antibody isotype (IgM in black, IgG in green, and IgA in red) was divided by the total resonance units for all antibody isotypes combined to calculate the percentage of each antibody isotype. for individual serum sample.

Article Snippet: Zaire EBOV VP24 Protein , Sino Biologicals , 40454-V07E.

Techniques: Binding Assay, Purification, Infection, Protein Binding

Journal: Cell host & microbe

Article Title: Longitudinal human antibody repertoire against complete viral proteome from Ebola virus survivor reveals protective sites for vaccine design

doi: 10.1016/j.chom.2020.01.001

Figure Lengend Snippet: (A) IgM, IgG and IgA antibody epitope repertoire recognized in the EBOV infected sera at different days post-onset of symptoms ((D7, D13, D19, D31, D110 and D361) and their alignment to the whole proteome of EBOV showing different proteins (NP, VP35, VP40, GP, VP30, VP24 and L). Graphical distribution of representative clones with a frequency of ≥2, obtained after affinity selection, are shown. The horizontal position and the length of the bars indicate the peptide sequence displayed on the selected phage clone to its homologous sequence in the EBOV proteome on alignment. The thickness of each bar represents the frequency of repetitively isolated phage, with the scale shown below the alignment. Scale value for IgM, IgG and IgA is shown enclosed in a red box beneath the respective alignments. The GFPDL affinity selection data was performed in duplicate (two independent experiments by researcher in the lab, who was blinded to sample identity), and similar number of phage clones and epitope repertoire was observed in both phage display analysis. (B) Elucidation of antibody epitope profile against the EBOV proteome following EBOV infection. Antigenic sites within the EBOV proteins recognized by serum antibodies following EBOV infection (based on data presented in Fig. 1A). The amino acid designation is based on the EBOV protein sequence encoded by the complete EBOV/Makona genome. The antigenic regions/sites discovered in this study using the post-infection antibodies are depicted below the EBOV proteome schematic and are color coded. Epitopes of each protein are numbered in a sequential fashion indicated in black and the epitopes are color coded according to the protein color code in the proteome map.

Article Snippet: Zaire EBOV VP24 Protein , Sino Biologicals , 40454-V07E.

Techniques: Infection, Clone Assay, Selection, Sequencing, Isolation

Journal: Cell host & microbe

Article Title: Longitudinal human antibody repertoire against complete viral proteome from Ebola virus survivor reveals protective sites for vaccine design

doi: 10.1016/j.chom.2020.01.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Zaire EBOV VP24 Protein , Sino Biologicals , 40454-V07E.

Techniques: Recombinant