sars cov nucleoprotein np antibody rabbit pab  (Sino Biological)


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    Name:
    SARS CoV Nucleoprotein NP Antibody Rabbit PAb Antigen Affinity Purified
    Description:
    Produced in rabbits immunized with purified recombinant SARS CoV Nucleoprotein NP Catalog 40143 V08B NP 828858 1 Met1 Ala422 SARS CoV Nucleoprotein NP specific IgG was purified by SARS CoV Nucleoprotein NP affinity chromatography
    Catalog Number:
    40143-T62
    Price:
    None
    Category:
    Primary Antibody
    Reactivity:
    SARS
    Applications:
    WB,ELISA
    Immunogen:
    Recombinant SARS-CoV Nucleoprotein / NP Protein (Catalog#40143-V08B)
    Antibody Type:
    PAb
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    Sino Biological sars cov nucleoprotein np antibody rabbit pab
    In vivo <t>anti-SARS-CoV-2</t> efficacy of GS-441524 in mouse AAV-hACE2 model. AAV-hACE2 transduced mice were infected with SARA-CoV-2. The mice were administrated with either vehicle or GS-441524 25 mg/kg/day at −1 days pi (post innoculation) and the treatment was continued for a total of 8 days. Body weights were monitored every day ( A ). Lung tissues of 3 mice in each group were harvested and the viral titers were analyzed by qRT-PCR at 2 Dpi ( B ). ( C ) Representative Hematoxylin-eosin (HE) staining of lungs from hACE2 transduced mice Scale bars, 500mm (top) and 111 mm (bottom). *p values ≤ 0.05; **p values ≤ 0.005; ***p values ≤ 0.0005.
    Produced in rabbits immunized with purified recombinant SARS CoV Nucleoprotein NP Catalog 40143 V08B NP 828858 1 Met1 Ala422 SARS CoV Nucleoprotein NP specific IgG was purified by SARS CoV Nucleoprotein NP affinity chromatography
    https://www.bioz.com/result/sars cov nucleoprotein np antibody rabbit pab/product/Sino Biological
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    Price from $9.99 to $1999.99
    sars cov nucleoprotein np antibody rabbit pab - by Bioz Stars, 2021-09
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    Images

    1) Product Images from "Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mice Models"

    Article Title: Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mice Models

    Journal: bioRxiv

    doi: 10.1101/2020.10.26.353300

    In vivo anti-SARS-CoV-2 efficacy of GS-441524 in mouse AAV-hACE2 model. AAV-hACE2 transduced mice were infected with SARA-CoV-2. The mice were administrated with either vehicle or GS-441524 25 mg/kg/day at −1 days pi (post innoculation) and the treatment was continued for a total of 8 days. Body weights were monitored every day ( A ). Lung tissues of 3 mice in each group were harvested and the viral titers were analyzed by qRT-PCR at 2 Dpi ( B ). ( C ) Representative Hematoxylin-eosin (HE) staining of lungs from hACE2 transduced mice Scale bars, 500mm (top) and 111 mm (bottom). *p values ≤ 0.05; **p values ≤ 0.005; ***p values ≤ 0.0005.
    Figure Legend Snippet: In vivo anti-SARS-CoV-2 efficacy of GS-441524 in mouse AAV-hACE2 model. AAV-hACE2 transduced mice were infected with SARA-CoV-2. The mice were administrated with either vehicle or GS-441524 25 mg/kg/day at −1 days pi (post innoculation) and the treatment was continued for a total of 8 days. Body weights were monitored every day ( A ). Lung tissues of 3 mice in each group were harvested and the viral titers were analyzed by qRT-PCR at 2 Dpi ( B ). ( C ) Representative Hematoxylin-eosin (HE) staining of lungs from hACE2 transduced mice Scale bars, 500mm (top) and 111 mm (bottom). *p values ≤ 0.05; **p values ≤ 0.005; ***p values ≤ 0.0005.

    Techniques Used: In Vivo, Mouse Assay, Infection, Quantitative RT-PCR, Staining

    The prodrug remdesivir and parent nucleoside GS-441524 potently inhibit SARS-CoV-2 replication in vitro . Vero-E6 ( A ), calu-3 ( B ) and caco-2 ( C ) were infected with SARS-CoV-2 at an MOI of 0.05 and treated with different drugs (GS-441524 and Remdesivir) at different doses (0, 0.01, 0.1, 1, 5, 10, 50 μM) for 48 h. The viral yield in the cell supernatant was then quantified by qRT-PCR. Data represented are the mean value of % inhibition of SARS-CoV-2 on cells. At the same time, the cytotoxicity at different concentrations of drugs was tested. Vero-E6 cells were infected with SARS-CoV-2 at an MOI of 0.05 and treated with different doses (0, 5, 10 μM) of the indicated compounds for 48 h. The viral RNA in the cell supernatant ( D ) and intracellular ( E ) was then quantified by qRT-PCR.
    Figure Legend Snippet: The prodrug remdesivir and parent nucleoside GS-441524 potently inhibit SARS-CoV-2 replication in vitro . Vero-E6 ( A ), calu-3 ( B ) and caco-2 ( C ) were infected with SARS-CoV-2 at an MOI of 0.05 and treated with different drugs (GS-441524 and Remdesivir) at different doses (0, 0.01, 0.1, 1, 5, 10, 50 μM) for 48 h. The viral yield in the cell supernatant was then quantified by qRT-PCR. Data represented are the mean value of % inhibition of SARS-CoV-2 on cells. At the same time, the cytotoxicity at different concentrations of drugs was tested. Vero-E6 cells were infected with SARS-CoV-2 at an MOI of 0.05 and treated with different doses (0, 5, 10 μM) of the indicated compounds for 48 h. The viral RNA in the cell supernatant ( D ) and intracellular ( E ) was then quantified by qRT-PCR.

    Techniques Used: In Vitro, Infection, Quantitative RT-PCR, Inhibition

    2) Product Images from "Development of a Broadly Applicable Cas12a-Linked Beam Unlocking Reaction for Sensitive and Specific Detection of Respiratory Pathogens Including SARS-CoV-2"

    Article Title: Development of a Broadly Applicable Cas12a-Linked Beam Unlocking Reaction for Sensitive and Specific Detection of Respiratory Pathogens Including SARS-CoV-2

    Journal: ACS Chemical Biology

    doi: 10.1021/acschembio.0c00840

    Sensitivity and specificity of CALIBURN for SARS-CoV-2 detection. (A) Types of specimens. (B) Sensitivity of CALIBURN. Positive groups are defined as those with all three CALIBURN replicates reporting a positive signal. Presumptive positive groups are defined as those with one or two CALIBURN replicates reporting a positive signal. False negative groups are defined as those with no CALIBURN replicates reporting a positive signal. (C) Composition of specimen types in the positive, presumptive positive, and false negative groups. (D) Specificity of CALIBURN. The RT-RPA primers and crRNA probes for SARS-CoV-2 are identical to those in Figure 1 .
    Figure Legend Snippet: Sensitivity and specificity of CALIBURN for SARS-CoV-2 detection. (A) Types of specimens. (B) Sensitivity of CALIBURN. Positive groups are defined as those with all three CALIBURN replicates reporting a positive signal. Presumptive positive groups are defined as those with one or two CALIBURN replicates reporting a positive signal. False negative groups are defined as those with no CALIBURN replicates reporting a positive signal. (C) Composition of specimen types in the positive, presumptive positive, and false negative groups. (D) Specificity of CALIBURN. The RT-RPA primers and crRNA probes for SARS-CoV-2 are identical to those in Figure 1 .

    Techniques Used: Recombinase Polymerase Amplification

    CALIBURN detection of multiple pathogens in SARS-CoV-2 mouse model. (A) Flowchart showing experimental design. (B) Focus forming assay to determine SARS-CoV-2 titers at different time points. LOD, limit of detection. (C) RT-PCR quantification of SARS-CoV-2 viral loads. (D,E) CALIBURN detection of SARS-CoV-2 (D) and Ad5 transcript (E). For the data in C–E, the results from three biological replicates of infection are shown as mean ± SD ( n = 3). Each data point represents the mean value of three replicates of detection. The RT-RPA primers and crRNA probes for SARS-CoV-2 are identical to those in Figure 1 .
    Figure Legend Snippet: CALIBURN detection of multiple pathogens in SARS-CoV-2 mouse model. (A) Flowchart showing experimental design. (B) Focus forming assay to determine SARS-CoV-2 titers at different time points. LOD, limit of detection. (C) RT-PCR quantification of SARS-CoV-2 viral loads. (D,E) CALIBURN detection of SARS-CoV-2 (D) and Ad5 transcript (E). For the data in C–E, the results from three biological replicates of infection are shown as mean ± SD ( n = 3). Each data point represents the mean value of three replicates of detection. The RT-RPA primers and crRNA probes for SARS-CoV-2 are identical to those in Figure 1 .

    Techniques Used: Focus Forming Assay, Reverse Transcription Polymerase Chain Reaction, Infection, Recombinase Polymerase Amplification

    Specific detection of SARS-CoV-2, IAV, and IBV. (A) Schematic illustration of CALIBURN and coupled RT-RPA reaction. F, fluorophore. Q, quencher. (B) Investigation of the specificity of CALIBURN on each pathogen. For each virus, 5 μL of the extracted nucleic acids is added to each reaction without dilution. The viral copies are in the range of 10 6 –10 7 per reaction for different viruses. The RT-RPA primers are SARSCoV2-S-FWD-1/SARSCoV2-S-REV-1 for SARS-CoV-2, IAV-M-FWD/IAV-M-REV for IAV, and IBV-HA-FWD/IBV-HA-REV for IBV, respectively ( Table S1 ). The crRNA probes are SARSCoV2-S-crRNA2 for SARS-CoV-2, IAV-M-crRNA3 for IAV, and IBV-HA-crRNA4 for IBV, respectively ( Table S2 ). The data from three biological replicates are shown as mean ± SD.
    Figure Legend Snippet: Specific detection of SARS-CoV-2, IAV, and IBV. (A) Schematic illustration of CALIBURN and coupled RT-RPA reaction. F, fluorophore. Q, quencher. (B) Investigation of the specificity of CALIBURN on each pathogen. For each virus, 5 μL of the extracted nucleic acids is added to each reaction without dilution. The viral copies are in the range of 10 6 –10 7 per reaction for different viruses. The RT-RPA primers are SARSCoV2-S-FWD-1/SARSCoV2-S-REV-1 for SARS-CoV-2, IAV-M-FWD/IAV-M-REV for IAV, and IBV-HA-FWD/IBV-HA-REV for IBV, respectively ( Table S1 ). The crRNA probes are SARSCoV2-S-crRNA2 for SARS-CoV-2, IAV-M-crRNA3 for IAV, and IBV-HA-crRNA4 for IBV, respectively ( Table S2 ). The data from three biological replicates are shown as mean ± SD.

    Techniques Used: Recombinase Polymerase Amplification

    LODs of different methods on detecting laboratory strains of SARS-CoV-2, IAV, and IBV. (A) RT-PCR. Positive signal is determined by valid C t values. (B) CALIBURN. Viral RNA of indicated copies is added to 5 μL RT-RPA reactions and the reaction product is directly transferred to Cas12a reaction with a final volume of 20 μL. The threshold of positive signal is set as the mean plus 3-fold standard deviation (mean+3σ) of the mock sample. (C) Fluorescent RT-RPA alone without CALIBURN reaction for detection of SARS-CoV-2. Arrows denote LOD, as defined by the lowest input viral copies resulting in positive signal readouts with more than 95% confidence. The data are shown as mean ± SD ( n = 3). The RT-RPA primers and crRNA probes are identical to those in Figure 1 .
    Figure Legend Snippet: LODs of different methods on detecting laboratory strains of SARS-CoV-2, IAV, and IBV. (A) RT-PCR. Positive signal is determined by valid C t values. (B) CALIBURN. Viral RNA of indicated copies is added to 5 μL RT-RPA reactions and the reaction product is directly transferred to Cas12a reaction with a final volume of 20 μL. The threshold of positive signal is set as the mean plus 3-fold standard deviation (mean+3σ) of the mock sample. (C) Fluorescent RT-RPA alone without CALIBURN reaction for detection of SARS-CoV-2. Arrows denote LOD, as defined by the lowest input viral copies resulting in positive signal readouts with more than 95% confidence. The data are shown as mean ± SD ( n = 3). The RT-RPA primers and crRNA probes are identical to those in Figure 1 .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Recombinase Polymerase Amplification, Standard Deviation

    3) Product Images from "Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mouse Models"

    Article Title: Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mouse Models

    Journal: Journal of Medicinal Chemistry

    doi: 10.1021/acs.jmedchem.0c01929

    Anti-SARS-CoV-2 efficacy of GS-441524 in an AAV-hACE2 mouse model. AAV-hACE2 transduced mice were infected with SARS-CoV-2. Mice were administrated either vehicle or GS-441524 (25 mg/kg/day) at −1 dpi and were treated for a total of 8 d. (A) Changes in body weight for either vehicle (black) or GS-441524-treated (red) mice. (B) Viral titers from lung tissue of three mice per group were harvested at 2 dpi and analyzed by FFA. *** p -value ≤ 0.0005. (C) Representative H E staining of lungs from hACE2 transduced mice. (D) Scale bars, 500 μm (top) and 100 μm (bottom).
    Figure Legend Snippet: Anti-SARS-CoV-2 efficacy of GS-441524 in an AAV-hACE2 mouse model. AAV-hACE2 transduced mice were infected with SARS-CoV-2. Mice were administrated either vehicle or GS-441524 (25 mg/kg/day) at −1 dpi and were treated for a total of 8 d. (A) Changes in body weight for either vehicle (black) or GS-441524-treated (red) mice. (B) Viral titers from lung tissue of three mice per group were harvested at 2 dpi and analyzed by FFA. *** p -value ≤ 0.0005. (C) Representative H E staining of lungs from hACE2 transduced mice. (D) Scale bars, 500 μm (top) and 100 μm (bottom).

    Techniques Used: Mouse Assay, Infection, Staining

    Remdesivir and GS-441524 potently inhibit SARS-CoV-2 replication in vitro. Vero E6 (A), Calu-3 (B), and Caco-2 (C) were infected with SARS-CoV-2 at an MOI of 0.05 and treated with dilutions of either GS-441524 or remdesivir (0, 0.01, 0.1, 1, 5, 10, 50 μM) for 48 h. Viral yield in the cell supernatant was then quantified by qRT-PCR. Data represented are the mean value of % inhibition of SARS-CoV-2 in cells. Cytotoxicity of GS-441524 (green dots) and remdesivir (orange dots) was determined using a CCK-8 test. Vero E6 cells were infected with SARS-CoV-2 at an MOI of 0.05 and treated with dilutions of the indicated compounds for 48 h. Viral RNA in the cell supernatant (D) and pellet (E) was then quantified by qRT-PCR.
    Figure Legend Snippet: Remdesivir and GS-441524 potently inhibit SARS-CoV-2 replication in vitro. Vero E6 (A), Calu-3 (B), and Caco-2 (C) were infected with SARS-CoV-2 at an MOI of 0.05 and treated with dilutions of either GS-441524 or remdesivir (0, 0.01, 0.1, 1, 5, 10, 50 μM) for 48 h. Viral yield in the cell supernatant was then quantified by qRT-PCR. Data represented are the mean value of % inhibition of SARS-CoV-2 in cells. Cytotoxicity of GS-441524 (green dots) and remdesivir (orange dots) was determined using a CCK-8 test. Vero E6 cells were infected with SARS-CoV-2 at an MOI of 0.05 and treated with dilutions of the indicated compounds for 48 h. Viral RNA in the cell supernatant (D) and pellet (E) was then quantified by qRT-PCR.

    Techniques Used: In Vitro, Infection, Quantitative RT-PCR, Inhibition, CCK-8 Assay

    4) Product Images from "A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection"

    Article Title: A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection

    Journal: Nature Communications

    doi: 10.1038/s41467-021-24905-z

    Divalent engineering increases affinity and neutralizing activity. a , b Identification of two non-competing pairs, LR1/MR3 ( a ) and LR5/MR3 ( b ), for biparatopic constructs. For BLI assays, sensors coated with RBD were soaked in 200 nM of LR1 or LR5 before further soaked in LR1- or LR5-containing buffer with (magenta) or without (black) 100 nM of MR3. The MR3–RBD interaction profile was obtained in the absence of LR1 or LR5 (blue). c Neutralization assay of the biparatopic sybody LR5-MR3 with a Gly-Ser (GS) linker of 13 (blue), 19 (red), or 34 (black) amino acids as indicated. Brackets indicate IC 50 values in μg mL −1 . Data are from a representative of two independent experiments. d Neutralization assays of divalent sybodies. The original SARS-CoV-2 was used for all assays except that the D614G mutant 42 was additionally tested for MR3-MR3 (red asterisk). Color-coding of the tested sybodies is as indicated. For MR3-MR3(34GS) against 614G, data show a representative of three independent experiments, which were performed using non-overlapping concentrations. For the rest of the samples, mean ± standard deviation are plotted ( n = 3 independent experiments). Error bars are omitted where, in rare cases, available data points are less than three due to experimental design on concentration replicates. e Summary of binding kinetics and neutralizing activities of the divalent sybodies. Source data for a – d are provided as a Source data file. BLI biolayer interferometry, N.D. not determined.
    Figure Legend Snippet: Divalent engineering increases affinity and neutralizing activity. a , b Identification of two non-competing pairs, LR1/MR3 ( a ) and LR5/MR3 ( b ), for biparatopic constructs. For BLI assays, sensors coated with RBD were soaked in 200 nM of LR1 or LR5 before further soaked in LR1- or LR5-containing buffer with (magenta) or without (black) 100 nM of MR3. The MR3–RBD interaction profile was obtained in the absence of LR1 or LR5 (blue). c Neutralization assay of the biparatopic sybody LR5-MR3 with a Gly-Ser (GS) linker of 13 (blue), 19 (red), or 34 (black) amino acids as indicated. Brackets indicate IC 50 values in μg mL −1 . Data are from a representative of two independent experiments. d Neutralization assays of divalent sybodies. The original SARS-CoV-2 was used for all assays except that the D614G mutant 42 was additionally tested for MR3-MR3 (red asterisk). Color-coding of the tested sybodies is as indicated. For MR3-MR3(34GS) against 614G, data show a representative of three independent experiments, which were performed using non-overlapping concentrations. For the rest of the samples, mean ± standard deviation are plotted ( n = 3 independent experiments). Error bars are omitted where, in rare cases, available data points are less than three due to experimental design on concentration replicates. e Summary of binding kinetics and neutralizing activities of the divalent sybodies. Source data for a – d are provided as a Source data file. BLI biolayer interferometry, N.D. not determined.

    Techniques Used: Activity Assay, Construct, Neutralization, Mutagenesis, Standard Deviation, Concentration Assay, Binding Assay

    Biochemical and structural characterization of neutralizing synthetic nanobodies (sybodies). a Summary of the characterization. Yield refers to purification from 1 L of culture. b Neutralization assay. SARS-CoV-2 pseudoviruses were preincubated with different concentrations of sybodies before infection of VeroE6-hACE2 cells. The rate of infection was measured by FACS. IC 50 was obtained by Sigmoidal fitting of the percentage of neutralization. Mean ± standard deviation are plotted ( n = 3 or 4 independent experiments). Error bars are omitted where, in rare cases, available data points are less than three due to experimental design on concentration replicates. Color coding of the sybodies is as indicated. c The overall structure of SR4 (pink cartoon) in complex with RBD (green surface), which resembles a short backrest high chair. The binding surface is highlighted red. d SR4 CDR1 (yellow), CDR2 (magenta), and CDR3 (cyan) all contribute to the binding. Note that Tyr37 is a framework residue. e The overall structure of the MR17 (pink cartoon) in complex with RBD (green surface). The binding surface is highlighted red. f The overlap (magenta) between the SR4 (blue) and MR17 (red) interacting surfaces on RBD. g All three CDRs contribute to the binding with the receptor-binding domain (RBD) (green). Lys65 and Tyr60 are from the framework region. CDRs are color-coded as indicated in d . Dashed lines indicate H-bonding or salt bridges between atoms that are
    Figure Legend Snippet: Biochemical and structural characterization of neutralizing synthetic nanobodies (sybodies). a Summary of the characterization. Yield refers to purification from 1 L of culture. b Neutralization assay. SARS-CoV-2 pseudoviruses were preincubated with different concentrations of sybodies before infection of VeroE6-hACE2 cells. The rate of infection was measured by FACS. IC 50 was obtained by Sigmoidal fitting of the percentage of neutralization. Mean ± standard deviation are plotted ( n = 3 or 4 independent experiments). Error bars are omitted where, in rare cases, available data points are less than three due to experimental design on concentration replicates. Color coding of the sybodies is as indicated. c The overall structure of SR4 (pink cartoon) in complex with RBD (green surface), which resembles a short backrest high chair. The binding surface is highlighted red. d SR4 CDR1 (yellow), CDR2 (magenta), and CDR3 (cyan) all contribute to the binding. Note that Tyr37 is a framework residue. e The overall structure of the MR17 (pink cartoon) in complex with RBD (green surface). The binding surface is highlighted red. f The overlap (magenta) between the SR4 (blue) and MR17 (red) interacting surfaces on RBD. g All three CDRs contribute to the binding with the receptor-binding domain (RBD) (green). Lys65 and Tyr60 are from the framework region. CDRs are color-coded as indicated in d . Dashed lines indicate H-bonding or salt bridges between atoms that are

    Techniques Used: Purification, Neutralization, Infection, FACS, Standard Deviation, Concentration Assay, Binding Assay

    Potent divalent MR3 protects hamsters against weight loss and viral replication. a Neutralization assay of MR3-MR3-ABD using pseudovirus bearing the wild-type Spike (614D, black square) or the D614G mutant Spike (614G, red circle). Data are from one representative experiment of three (614 G) or two (614D) independent experiments. Statistics were not performed for 614G because the three experiments were performed with different sybody concentrations. b Neutralization of authentic SARS-CoV-2 by Fc-MR3 (red square) and MR3-MR3-ABD (blue circle) measured using a plaque-reduction assay. Mean ± standard deviation are plotted ( n = 3 independent experiments). c Body weights of hamsters treated with antibodies (color-coded as indicated) were measured at the indicated days after inoculation with SARS-CoV-2. Statistics were performed using two-way ANOVA followed by Sidak’s multiple comparisons test. ** p
    Figure Legend Snippet: Potent divalent MR3 protects hamsters against weight loss and viral replication. a Neutralization assay of MR3-MR3-ABD using pseudovirus bearing the wild-type Spike (614D, black square) or the D614G mutant Spike (614G, red circle). Data are from one representative experiment of three (614 G) or two (614D) independent experiments. Statistics were not performed for 614G because the three experiments were performed with different sybody concentrations. b Neutralization of authentic SARS-CoV-2 by Fc-MR3 (red square) and MR3-MR3-ABD (blue circle) measured using a plaque-reduction assay. Mean ± standard deviation are plotted ( n = 3 independent experiments). c Body weights of hamsters treated with antibodies (color-coded as indicated) were measured at the indicated days after inoculation with SARS-CoV-2. Statistics were performed using two-way ANOVA followed by Sidak’s multiple comparisons test. ** p

    Techniques Used: Neutralization, Mutagenesis, Standard Deviation

    5) Product Images from "Neuropilin-1 Mediates SARS-CoV-2 Infection in Bone Marrow-derived Macrophages"

    Article Title: Neuropilin-1 Mediates SARS-CoV-2 Infection in Bone Marrow-derived Macrophages

    Journal: bioRxiv

    doi: 10.1101/2021.04.14.439793

    Decreased SARS-CoV-2 infection in mBMM-derived osteoclasts is associated with the loss of NRP1 expression. a , Flowchart showing the procedure of BMM-osteoclast differentiation, pseudovirus infection and RT-qPCR quantification. b-c , RT-qPCR quantification of ACE2 and NRP1 expression in BMMs (Day 0), deafferenting osteoclasts (Day 3) and mature osteoclasts (Day 8). d , Immunoblotting of NRP1 expression in BMMs (Day 0), deafferenting osteoclasts (Day 3) and mature osteoclasts (Day 8). e , RT-qPCR quantification of SARS-CoV-2 pseudovirus infection in BMMs (Day 0), differentiating osteoclasts (Day 3) and mature osteoclasts (Day 8), determined by tdTomato transgene expression. f , Confocal imaging showing SARS-CoV-2 pseudovirus infection in BMMs (Day 0), deafferenting osteoclasts (Day 3) and mature osteoclasts (Day 8). b-c and e , The data are shown as mean ± s.d. Statistical difference is determined using two-tailed Student’s t test.
    Figure Legend Snippet: Decreased SARS-CoV-2 infection in mBMM-derived osteoclasts is associated with the loss of NRP1 expression. a , Flowchart showing the procedure of BMM-osteoclast differentiation, pseudovirus infection and RT-qPCR quantification. b-c , RT-qPCR quantification of ACE2 and NRP1 expression in BMMs (Day 0), deafferenting osteoclasts (Day 3) and mature osteoclasts (Day 8). d , Immunoblotting of NRP1 expression in BMMs (Day 0), deafferenting osteoclasts (Day 3) and mature osteoclasts (Day 8). e , RT-qPCR quantification of SARS-CoV-2 pseudovirus infection in BMMs (Day 0), differentiating osteoclasts (Day 3) and mature osteoclasts (Day 8), determined by tdTomato transgene expression. f , Confocal imaging showing SARS-CoV-2 pseudovirus infection in BMMs (Day 0), deafferenting osteoclasts (Day 3) and mature osteoclasts (Day 8). b-c and e , The data are shown as mean ± s.d. Statistical difference is determined using two-tailed Student’s t test.

    Techniques Used: Infection, Derivative Assay, Expressing, Quantitative RT-PCR, Imaging, Two Tailed Test

    Authentic SARS-CoV-2 infects bone marrow-derived macrophages (BMMs). a-b , Confocal images showing the infection of SARS-CoV-2 in human ( a ) and mouse ( b ) BMMs. c-i , RNA-Seq analysis of SARS-CoV-2 infection in mouse BMMs. c , Heat map showing the correlation analysis of the SMART RNA-Seq results. d , Analysis of the expression of viral genes. e , Volcano plot showing the profile of altered gene expression. f , Top 20 enriched gene ontology (GO) terms. g , Significantly enriched GO terms on macrophage and osteoclast-related genes. h , Altered macrophage-related genes. i , Altered osteoclast-related genes.
    Figure Legend Snippet: Authentic SARS-CoV-2 infects bone marrow-derived macrophages (BMMs). a-b , Confocal images showing the infection of SARS-CoV-2 in human ( a ) and mouse ( b ) BMMs. c-i , RNA-Seq analysis of SARS-CoV-2 infection in mouse BMMs. c , Heat map showing the correlation analysis of the SMART RNA-Seq results. d , Analysis of the expression of viral genes. e , Volcano plot showing the profile of altered gene expression. f , Top 20 enriched gene ontology (GO) terms. g , Significantly enriched GO terms on macrophage and osteoclast-related genes. h , Altered macrophage-related genes. i , Altered osteoclast-related genes.

    Techniques Used: Derivative Assay, Infection, RNA Sequencing Assay, Expressing

    In situ immunofluorescence analysis of the in vivo infection of authentic SARS-CoV-2 in BMMs of Ad5-hACE2 transduced BALB/c mice. a , Schematic illustration of experimental procedures. b-d , Confocal images showing the infection of SARS-CoV-2 in BMMs from different femoral segments. SARS-CoV-2 nucleocapsid protein (N protein) and macrophage major marker F4/80 are immunostained.
    Figure Legend Snippet: In situ immunofluorescence analysis of the in vivo infection of authentic SARS-CoV-2 in BMMs of Ad5-hACE2 transduced BALB/c mice. a , Schematic illustration of experimental procedures. b-d , Confocal images showing the infection of SARS-CoV-2 in BMMs from different femoral segments. SARS-CoV-2 nucleocapsid protein (N protein) and macrophage major marker F4/80 are immunostained.

    Techniques Used: In Situ, Immunofluorescence, In Vivo, Infection, Mouse Assay, Marker

    NRP1 facilitates SARS-CoV-2 pseudovirus infection in mBMMs. a-c , Single-cell transcriptome analysis of BMMs directly isolated from 1−, 6− or 20-month mice. a , Overview of cell clusters in the integrated cell population. Violin plot showing the expression of ACE2 ( b ) and NRP1 ( c ) in each cell type. d-e , RT-qPCR quantification of ACE2 and NRP1 expression in cultured hBMMs ( d ) and mBMMs ( e ). f , Human NRP1b1-S1 CendR peptide complex superposed with mouse NRP1b1-S1 CendR peptide complex (PDB ID:7JJC and 4GZ9). Binding peptide is shown in stick representation. RMSD, root mean square deviation. Enlarged view highlights the binding of S1 CendR peptide. g , Amino acid sequence alignment of human and mouse NRP1b1. h , UMAP of tdTomato positive and negative mBMMs. i , NRP1 expression in tdTomato positive and negative mBMMs. j , Western blot confirming NRP1 knockout in cultured mouse BMMs. Mock, non-targeting sgRNA. k , SARS-CoV-2 pseudovirus infection in cultured WT and NRP1 KO mBMMs. l-m , Confocal images showing SARS-CoV-2 pseudovirus infection in mock and NRP1 KO mBMMs. l , Representative images. m , Quantification of the infection efficiency. k and m , The data are represented as mean ± standard deviation. Statistical difference between mock and NRP1 KO cells is determined using two-tailed Student’s t test.
    Figure Legend Snippet: NRP1 facilitates SARS-CoV-2 pseudovirus infection in mBMMs. a-c , Single-cell transcriptome analysis of BMMs directly isolated from 1−, 6− or 20-month mice. a , Overview of cell clusters in the integrated cell population. Violin plot showing the expression of ACE2 ( b ) and NRP1 ( c ) in each cell type. d-e , RT-qPCR quantification of ACE2 and NRP1 expression in cultured hBMMs ( d ) and mBMMs ( e ). f , Human NRP1b1-S1 CendR peptide complex superposed with mouse NRP1b1-S1 CendR peptide complex (PDB ID:7JJC and 4GZ9). Binding peptide is shown in stick representation. RMSD, root mean square deviation. Enlarged view highlights the binding of S1 CendR peptide. g , Amino acid sequence alignment of human and mouse NRP1b1. h , UMAP of tdTomato positive and negative mBMMs. i , NRP1 expression in tdTomato positive and negative mBMMs. j , Western blot confirming NRP1 knockout in cultured mouse BMMs. Mock, non-targeting sgRNA. k , SARS-CoV-2 pseudovirus infection in cultured WT and NRP1 KO mBMMs. l-m , Confocal images showing SARS-CoV-2 pseudovirus infection in mock and NRP1 KO mBMMs. l , Representative images. m , Quantification of the infection efficiency. k and m , The data are represented as mean ± standard deviation. Statistical difference between mock and NRP1 KO cells is determined using two-tailed Student’s t test.

    Techniques Used: Infection, Isolation, Mouse Assay, Expressing, Quantitative RT-PCR, Cell Culture, Binding Assay, Sequencing, Western Blot, Knock-Out, Standard Deviation, Two Tailed Test

    SARS-CoV-2 pseudovirus infects human and mouse BMMs. a , Schematic illustration of the design of SARS-CoV-2 pseudovirus. NTD, N terminal domain; RBD, receptor binding domain; PA, poly (A); LTR, long terminal repeats; CMV, cytomegalovirus promoter; EF-1a, elongation factor-1a promoter. b , Flowchart showing the procedures of SARS-CoV-2 pseudovirus infection in BMMs. c-d , RT-qPCR quantification of SARS-CoV-2 pseudovirus infection in cultured human ( c ) and mouse ( d ) BMMs. e-l , Single-cell transcriptome analysis of SARS-CoV-2 pseudovirus infection in cultured BMMs that are derived from 1-month and 18-month mice. e , Overview of cell clusters in integrated cell population. f , Analysis of cell type ratio, showing that macrophages are the predominant population. g , Analysis of SARS-CoV-2 pseudovirus-infected and -uninfected cells, determined by tdTomato transgene expression. h-i , Re-cluster of SARS-CoV-2 pseudovirus-infected cells ( h ) and cell number quantitation ( i ), showing that macrophages are the predominant population in SARS-CoV-2 pseudovirus-infected cells. j-l , Re-cluster of macrophages. j , Cell clusters and analysis of SARS-CoV-2 pseudovirus-infected and -uninfected cells, determined by tdTomato transgene expression. k , Cluster 7 shows the highest efficiency of SARS-CoV-2 pseudovirus infection. l , Cluster 7 exhibits highest correlation with COVID-19 and osteoclast differentiation.
    Figure Legend Snippet: SARS-CoV-2 pseudovirus infects human and mouse BMMs. a , Schematic illustration of the design of SARS-CoV-2 pseudovirus. NTD, N terminal domain; RBD, receptor binding domain; PA, poly (A); LTR, long terminal repeats; CMV, cytomegalovirus promoter; EF-1a, elongation factor-1a promoter. b , Flowchart showing the procedures of SARS-CoV-2 pseudovirus infection in BMMs. c-d , RT-qPCR quantification of SARS-CoV-2 pseudovirus infection in cultured human ( c ) and mouse ( d ) BMMs. e-l , Single-cell transcriptome analysis of SARS-CoV-2 pseudovirus infection in cultured BMMs that are derived from 1-month and 18-month mice. e , Overview of cell clusters in integrated cell population. f , Analysis of cell type ratio, showing that macrophages are the predominant population. g , Analysis of SARS-CoV-2 pseudovirus-infected and -uninfected cells, determined by tdTomato transgene expression. h-i , Re-cluster of SARS-CoV-2 pseudovirus-infected cells ( h ) and cell number quantitation ( i ), showing that macrophages are the predominant population in SARS-CoV-2 pseudovirus-infected cells. j-l , Re-cluster of macrophages. j , Cell clusters and analysis of SARS-CoV-2 pseudovirus-infected and -uninfected cells, determined by tdTomato transgene expression. k , Cluster 7 shows the highest efficiency of SARS-CoV-2 pseudovirus infection. l , Cluster 7 exhibits highest correlation with COVID-19 and osteoclast differentiation.

    Techniques Used: Binding Assay, Infection, Quantitative RT-PCR, Cell Culture, Derivative Assay, Mouse Assay, Expressing, Quantitation Assay

    6) Product Images from "Characterization of Virus Replication, Pathogenesis, and Cytokine Responses in Syrian Hamsters Inoculated with SARS-CoV-2"

    Article Title: Characterization of Virus Replication, Pathogenesis, and Cytokine Responses in Syrian Hamsters Inoculated with SARS-CoV-2

    Journal: Journal of Inflammation Research

    doi: 10.2147/JIR.S323026

    SARS-CoV nucleoprotein expression in SARS-CoV-2-infected hamsters post-infection day 3 and 6. ( A ) Syrian hamsters aged 6 to 8 weeks (n=5 for SARS-CoV-2, and n=3 for non-infected control) were infected with 5×10 5 TCID 50 of SARS-CoV-2. Liver, pancreas, heart and kidney sections were stained by H E on day 3 and 6. ( B ) IHC staining for SARS-CoV nucleoprotein expression in the tissue sections. Scale bar for 400× panels = 20 µm.
    Figure Legend Snippet: SARS-CoV nucleoprotein expression in SARS-CoV-2-infected hamsters post-infection day 3 and 6. ( A ) Syrian hamsters aged 6 to 8 weeks (n=5 for SARS-CoV-2, and n=3 for non-infected control) were infected with 5×10 5 TCID 50 of SARS-CoV-2. Liver, pancreas, heart and kidney sections were stained by H E on day 3 and 6. ( B ) IHC staining for SARS-CoV nucleoprotein expression in the tissue sections. Scale bar for 400× panels = 20 µm.

    Techniques Used: Expressing, Infection, Staining, Immunohistochemistry

    Body weight change, viral load, and pathological changes of SARS-CoV-2-infected Syrian hamsters following different challenge doses. ( A ) Body weights of hamsters infected with 10 5 , 5 × 10 5 , and 10 6 TCID 50 of SARS-CoV-2 (n=5 per group, and non-infected control, n=5) over 14 days (compared with the weight on day 0). Differences among groups were determined using two-way ANOVA with a Bonferroni post hoc test (* P
    Figure Legend Snippet: Body weight change, viral load, and pathological changes of SARS-CoV-2-infected Syrian hamsters following different challenge doses. ( A ) Body weights of hamsters infected with 10 5 , 5 × 10 5 , and 10 6 TCID 50 of SARS-CoV-2 (n=5 per group, and non-infected control, n=5) over 14 days (compared with the weight on day 0). Differences among groups were determined using two-way ANOVA with a Bonferroni post hoc test (* P

    Techniques Used: Infection

    Pathological changes in SARS-CoV-2-infected hamster lung on days 3, 6, and 9 post-infection. Syrian hamsters aged 6 to 8 weeks (n=5 for SARS-CoV-2, and n=3 for non-infected control) were infected with 5×10 5 TCID 50 of SARS-CoV-2 on day 0. On days 3, 6, and 9, samples of lung and trachea tissue were removed, fixed in formalin and embedded in paraffin using routine methods, and then processed for staining. ( A ) Samples of lung sections stained with H E. The blue dotted circle outlines hyaline membrane formation. The yellow arrows present macrophage aggregates in the airway and alveolar spaces. ( B ) Lung sections immunostained with anti-SARS-CoV nucleoprotein antibody. ( C ) PAS staining showing mucus expression in hamster lung. Small box frames show macrophages that scavenged the extra mucus near the trachea. Large box frames show enlargements of macrophage phagocytosis. Scale bar for the 400× panels = 20 µm.
    Figure Legend Snippet: Pathological changes in SARS-CoV-2-infected hamster lung on days 3, 6, and 9 post-infection. Syrian hamsters aged 6 to 8 weeks (n=5 for SARS-CoV-2, and n=3 for non-infected control) were infected with 5×10 5 TCID 50 of SARS-CoV-2 on day 0. On days 3, 6, and 9, samples of lung and trachea tissue were removed, fixed in formalin and embedded in paraffin using routine methods, and then processed for staining. ( A ) Samples of lung sections stained with H E. The blue dotted circle outlines hyaline membrane formation. The yellow arrows present macrophage aggregates in the airway and alveolar spaces. ( B ) Lung sections immunostained with anti-SARS-CoV nucleoprotein antibody. ( C ) PAS staining showing mucus expression in hamster lung. Small box frames show macrophages that scavenged the extra mucus near the trachea. Large box frames show enlargements of macrophage phagocytosis. Scale bar for the 400× panels = 20 µm.

    Techniques Used: Infection, Staining, Expressing

    Viral load changes and tissue tropism in SARS-CoV-2-infected Syrian hamsters on days 3, 6, and 9 post-infection. ( A ) Viral loads (TCID 50 /lung) of hamsters infected with 5×10 5 TCID 50 of SARS-CoV-2 (n=5 for SARS-CoV-2, and n=3 for non-infected controls) on days 3, 6, and 9 ( B, E – H ). N=4 for panel ( C and D ). Quantitative PCR of hamster lung ( B ), liver ( C ), pancreas ( D ), heart ( E ), kidney ( F ), PBMC ( G ), and bone marrow (BM) ( H ) of the SARS-CoV-2 N gene (from CCDC) on days 3, 6, and 9 post-infection. Differences among groups were determined using two-way ANOVA with a Bonferroni post hoc test (* P
    Figure Legend Snippet: Viral load changes and tissue tropism in SARS-CoV-2-infected Syrian hamsters on days 3, 6, and 9 post-infection. ( A ) Viral loads (TCID 50 /lung) of hamsters infected with 5×10 5 TCID 50 of SARS-CoV-2 (n=5 for SARS-CoV-2, and n=3 for non-infected controls) on days 3, 6, and 9 ( B, E – H ). N=4 for panel ( C and D ). Quantitative PCR of hamster lung ( B ), liver ( C ), pancreas ( D ), heart ( E ), kidney ( F ), PBMC ( G ), and bone marrow (BM) ( H ) of the SARS-CoV-2 N gene (from CCDC) on days 3, 6, and 9 post-infection. Differences among groups were determined using two-way ANOVA with a Bonferroni post hoc test (* P

    Techniques Used: Infection, Real-time Polymerase Chain Reaction

    Cytokine expression in lungs of SARS-CoV-2-infected hamsters on days 3, 6, and 9 post-infection. Hamsters were infected with 5×10 5 TCID 50 of SARS-CoV-2 (n=5 for SARS-CoV-2, and n=3 for non-infected control). On days 3, 6, and 9 post-infection, the hamsters were sacrificed, lung RNA was extracted, and cytokine gene expression profiles were determined by using qPCR. Relative index is presented as 2 −ΔΔCT and the mean ± SEM of the numeric values in each SARS-CoV-2 group is also presented below at 3, 6, and 9 d.p.i., respectively. ( A ) IL-1β (4.787±0.592, 0.892±0.159, 1.054±0.270), ( B ) IL-4 (0.665±0.157, 0.190±0.043, 0.238±0.082), ( C ) IL-6 (5.682±1.304, 2.038±0.368, 0.666±0.198), ( D ) IL-10 (2.701±0.466, 1.004±0.143, 1.112±0.251), ( E ) IL-12 (3.610±0.653, 1.684±0.499, 0.198±0.024), ( F ) IL-17 (0.804±0.087, 0.493±0.0455, 0.456±0.0792), ( G ) IFN-γ (2.479±0.209, 2.106±0.328, 1.094±0.242), ( H ) iNOS (0.883±0.128, 0.513±0.024, 0.560±0.054), ( I ) MCP-1 (2.097±0.543, 1.696±0.171, 0.362±0.049), ( J ) MIP-1α (37.216±6.960, 4.745±0.496, 2.589±0.409), ( K ) PD-L1 (4.754±0.775, 1.221±0.168, 1.025±0.201), ( L ) RANTES (45.247±16.554, 25.458±2.261, 5.574±0.834, ( M ) TGF-β (0.672±0.073, 0.507±0.0231, 0.716±0.1228), ( N ) TNF-α (1.288±0.1220, 0.68±0.0517, 0.968±0.1251). Differences among groups were determined using two-way ANOVA with a Bonferroni post hoc test in panels ( A–N ) (* P
    Figure Legend Snippet: Cytokine expression in lungs of SARS-CoV-2-infected hamsters on days 3, 6, and 9 post-infection. Hamsters were infected with 5×10 5 TCID 50 of SARS-CoV-2 (n=5 for SARS-CoV-2, and n=3 for non-infected control). On days 3, 6, and 9 post-infection, the hamsters were sacrificed, lung RNA was extracted, and cytokine gene expression profiles were determined by using qPCR. Relative index is presented as 2 −ΔΔCT and the mean ± SEM of the numeric values in each SARS-CoV-2 group is also presented below at 3, 6, and 9 d.p.i., respectively. ( A ) IL-1β (4.787±0.592, 0.892±0.159, 1.054±0.270), ( B ) IL-4 (0.665±0.157, 0.190±0.043, 0.238±0.082), ( C ) IL-6 (5.682±1.304, 2.038±0.368, 0.666±0.198), ( D ) IL-10 (2.701±0.466, 1.004±0.143, 1.112±0.251), ( E ) IL-12 (3.610±0.653, 1.684±0.499, 0.198±0.024), ( F ) IL-17 (0.804±0.087, 0.493±0.0455, 0.456±0.0792), ( G ) IFN-γ (2.479±0.209, 2.106±0.328, 1.094±0.242), ( H ) iNOS (0.883±0.128, 0.513±0.024, 0.560±0.054), ( I ) MCP-1 (2.097±0.543, 1.696±0.171, 0.362±0.049), ( J ) MIP-1α (37.216±6.960, 4.745±0.496, 2.589±0.409), ( K ) PD-L1 (4.754±0.775, 1.221±0.168, 1.025±0.201), ( L ) RANTES (45.247±16.554, 25.458±2.261, 5.574±0.834, ( M ) TGF-β (0.672±0.073, 0.507±0.0231, 0.716±0.1228), ( N ) TNF-α (1.288±0.1220, 0.68±0.0517, 0.968±0.1251). Differences among groups were determined using two-way ANOVA with a Bonferroni post hoc test in panels ( A–N ) (* P

    Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction

    SARS-CoV-2 nucleoprotein expression and mucosal mucus levels in SARS-CoV-2-infected Syrian hamsters following different challenge doses. Hamsters aged 6 to 8 weeks (n=5) were infected with 10 5 , 5×10 5 , and 10 6 TCID 50 of SARS-CoV-2 on day 0. ( A ) On days 3 and 14, samples of lung tissue were dissected, fixed in formalin and embedded in paraffin using routine methods, and the sections were then stained with anti-SARS-CoV nucleoprotein antibody. ( B ) PAS staining was performed on the same paraffin-embedded sections. The small box frame in each panel shows the macrophages that scavenged the extra mucus near the trachea. The large box frame shows an enlargement of macrophage phagocytosis. Scale bar for the 100× panels = 200 µm, for the 400× panels = 20 µm.
    Figure Legend Snippet: SARS-CoV-2 nucleoprotein expression and mucosal mucus levels in SARS-CoV-2-infected Syrian hamsters following different challenge doses. Hamsters aged 6 to 8 weeks (n=5) were infected with 10 5 , 5×10 5 , and 10 6 TCID 50 of SARS-CoV-2 on day 0. ( A ) On days 3 and 14, samples of lung tissue were dissected, fixed in formalin and embedded in paraffin using routine methods, and the sections were then stained with anti-SARS-CoV nucleoprotein antibody. ( B ) PAS staining was performed on the same paraffin-embedded sections. The small box frame in each panel shows the macrophages that scavenged the extra mucus near the trachea. The large box frame shows an enlargement of macrophage phagocytosis. Scale bar for the 100× panels = 200 µm, for the 400× panels = 20 µm.

    Techniques Used: Expressing, Infection, Staining

    Anti-spike neutralizing antibody in SARS-CoV-2-infected hamsters. ( A ) Syrian hamsters aged 6 to 8 weeks (n=5 for SARS-CoV-2, and n=3 for non-infected control) were infected with 10 5 , 5×10 5 , and 10 6 TCID 50 of SARS-CoV-2 on day 0. Anti-spike neutralizing antibody of plasma was detected by ELISA following the manufacturer’s protocol. Differences among groups were determined using one-way ANOVA with a Tukey post hoc test, P > 0.05, no significant among all groups. ( B ) Syrian hamsters aged 6 to 8 weeks (n=5 for SARS-CoV-2, and n=3 for non-infected control) were infected with 5×10 5 TCID 50 of SARS-CoV-2 on days 3, 6, and 9. Anti-spike neutralizing antibody of plasma was detected by ELISA following the manufacturer’s protocol. Differences among groups were determined using two-way ANOVA with a Bonferroni post hoc test (*** P
    Figure Legend Snippet: Anti-spike neutralizing antibody in SARS-CoV-2-infected hamsters. ( A ) Syrian hamsters aged 6 to 8 weeks (n=5 for SARS-CoV-2, and n=3 for non-infected control) were infected with 10 5 , 5×10 5 , and 10 6 TCID 50 of SARS-CoV-2 on day 0. Anti-spike neutralizing antibody of plasma was detected by ELISA following the manufacturer’s protocol. Differences among groups were determined using one-way ANOVA with a Tukey post hoc test, P > 0.05, no significant among all groups. ( B ) Syrian hamsters aged 6 to 8 weeks (n=5 for SARS-CoV-2, and n=3 for non-infected control) were infected with 5×10 5 TCID 50 of SARS-CoV-2 on days 3, 6, and 9. Anti-spike neutralizing antibody of plasma was detected by ELISA following the manufacturer’s protocol. Differences among groups were determined using two-way ANOVA with a Bonferroni post hoc test (*** P

    Techniques Used: Infection, Enzyme-linked Immunosorbent Assay

    Blood biological and chemistry parameters in SARS-CoV-2-infected hamsters on days 3, 6, and 9 post-infection. Hamsters were infected with 5×10 5 TCID 50 of SARS-CoV-2 (n=5 for SARS-CoV-2, and n=3 for non-infected control). On days 3, 6, and 9 post-infection, plasma was collected and stored at −80°C until use. Multiple biological and chemistry parameters were analyzed with Fuji Dri-Chem slides on a Fujifilm Dri-Chem 4000 analyzer. ( A ) Amylase (Amyl). ( B ) Lipase (LIP). ( C ) Creatine phosphokinase isozyme KB (CKMB). ( D ) Creatine phosphokinase (CPK). ( E ) Ratio of glutamic oxalacetic transaminase/glutamic pyruvic transaminase (GOT/GPT). ( F ) Alkaline phosphatase (ALP). ( G ) Direct Bilirubin (D-BIL). ( H ) γ-Glutamyltransferase (GGT). ( I ) Urea nitrogen (BUN). ( J ) Creatinine (CRE). Differences among groups were determined using two-way ANOVA with a Bonferroni post hoc test in panels ( A–J ) (** P
    Figure Legend Snippet: Blood biological and chemistry parameters in SARS-CoV-2-infected hamsters on days 3, 6, and 9 post-infection. Hamsters were infected with 5×10 5 TCID 50 of SARS-CoV-2 (n=5 for SARS-CoV-2, and n=3 for non-infected control). On days 3, 6, and 9 post-infection, plasma was collected and stored at −80°C until use. Multiple biological and chemistry parameters were analyzed with Fuji Dri-Chem slides on a Fujifilm Dri-Chem 4000 analyzer. ( A ) Amylase (Amyl). ( B ) Lipase (LIP). ( C ) Creatine phosphokinase isozyme KB (CKMB). ( D ) Creatine phosphokinase (CPK). ( E ) Ratio of glutamic oxalacetic transaminase/glutamic pyruvic transaminase (GOT/GPT). ( F ) Alkaline phosphatase (ALP). ( G ) Direct Bilirubin (D-BIL). ( H ) γ-Glutamyltransferase (GGT). ( I ) Urea nitrogen (BUN). ( J ) Creatinine (CRE). Differences among groups were determined using two-way ANOVA with a Bonferroni post hoc test in panels ( A–J ) (** P

    Techniques Used: Infection

    7) Product Images from "SARS-CoV-2 Causes Acute Kidney Injury by Directly Infecting Renal Tubules"

    Article Title: SARS-CoV-2 Causes Acute Kidney Injury by Directly Infecting Renal Tubules

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.664868

    Histological location of ACE2 and SARS-CoV-2 in the human kidney. (A) The public single-cell RNA sequencing datasets based on two different platforms (10X Genomics and Microwell-seq) were analyzed. After quality control, dimension descending, and cell type identification, major cell types in the kidney were shown in the t-SNE plot. ACE2 was highly expressed in the proximal tubular cells, whereas TMPRSS2 was highly expressed in the intercalated cells. (B) ACE2 and TMPRSS2 protein levels were analyzed in normal kidney tissues (n = 3). ACE2 signature was detected mainly on the apical side of the proximal tubular cells. In contrast, TMPRSS2 was mainly localized at the distal tubular cells. (C) SARS-CoV-2 nucleoprotein was co-stained with ACE2 or TMPRSS2 by Immunofluorescence (IFC) staining in 10 COVID-19 patients’ kidney samples. SARS-CoV-2 nucleoprotein was detected in ACE2 + or TMPRSS2 + renal tubular cells. (D) A novel in situ hybridization assay (RNAscope ® Assay) targeting the SARS-CoV-2 Spike gene was positive in the kidney distal tubular cells of COVID-19 patients.
    Figure Legend Snippet: Histological location of ACE2 and SARS-CoV-2 in the human kidney. (A) The public single-cell RNA sequencing datasets based on two different platforms (10X Genomics and Microwell-seq) were analyzed. After quality control, dimension descending, and cell type identification, major cell types in the kidney were shown in the t-SNE plot. ACE2 was highly expressed in the proximal tubular cells, whereas TMPRSS2 was highly expressed in the intercalated cells. (B) ACE2 and TMPRSS2 protein levels were analyzed in normal kidney tissues (n = 3). ACE2 signature was detected mainly on the apical side of the proximal tubular cells. In contrast, TMPRSS2 was mainly localized at the distal tubular cells. (C) SARS-CoV-2 nucleoprotein was co-stained with ACE2 or TMPRSS2 by Immunofluorescence (IFC) staining in 10 COVID-19 patients’ kidney samples. SARS-CoV-2 nucleoprotein was detected in ACE2 + or TMPRSS2 + renal tubular cells. (D) A novel in situ hybridization assay (RNAscope ® Assay) targeting the SARS-CoV-2 Spike gene was positive in the kidney distal tubular cells of COVID-19 patients.

    Techniques Used: RNA Sequencing Assay, Staining, Immunofluorescence, In Situ Hybridization

    Nucleic acid of SARS-CoV-2 was detected in urine of severe patients. (A) Color Doppler imaging showed reduced perfusion in severe COVID-19 patients. (B) SARS-CoV-2 RNA Real-Time PCR was performed on urine samples. SARS-CoV-2 nucleic acid was detected in 2 out of 15 (13.3%) severe samples, whereas none of the moderate samples ( n = 60) was positive.
    Figure Legend Snippet: Nucleic acid of SARS-CoV-2 was detected in urine of severe patients. (A) Color Doppler imaging showed reduced perfusion in severe COVID-19 patients. (B) SARS-CoV-2 RNA Real-Time PCR was performed on urine samples. SARS-CoV-2 nucleic acid was detected in 2 out of 15 (13.3%) severe samples, whereas none of the moderate samples ( n = 60) was positive.

    Techniques Used: Imaging, Real-time Polymerase Chain Reaction

    8) Product Images from "The SARS-CoV-2 multibasic cleavage site facilitates early serine protease-mediated entry into organoid-derived human airway cells"

    Article Title: The SARS-CoV-2 multibasic cleavage site facilitates early serine protease-mediated entry into organoid-derived human airway cells

    Journal: bioRxiv

    doi: 10.1101/2020.09.07.286120

    SARS-CoV-2 entry and replication is dependent on serine proteases in differentiated organoid-derived human airway cells. ( A and B ) Differentiated bronchiolar (A) or bronchial (B) airway spheroid cultures were infected at a MOI of 2. 16 hours ( A ) or 24 hours ( B ) post infection they were fixed and stained for viral nucleoprotein (red). Nuclei were stained with hoechst (blue) and actin was stained using phalloidin (white). AcTub stains ciliated cells (green). Scale bars indicate 200 μm in A and 50 μm in B . Representative images are shown from two independent experiments. ( C to E ) Replication kinetics of SARS-CoV-2 in bronchiolar airway spheroid cultures pretreated with camostat or carrier (DMSO). ( C and D ) TCID50 equivalents (eq.) per ml are shown in culture medium ( C ) and lysed organoids ( D ). ( E ) Live virus titers (TCID50/ml) in lysed organoids. Dotted line indicates limit of detection. ( F ) Replication kinetics of SARS-CoV-2 in 2D tracheal air-liquid interface airway cultures pretreated with camostat or carrier (DMSO). TCID50 eq. per ml in apical washes are shown. Error bars indicate SEM. H p.i. = hours post infection. Two-way ANOVA was performed for statistical analysis. * P
    Figure Legend Snippet: SARS-CoV-2 entry and replication is dependent on serine proteases in differentiated organoid-derived human airway cells. ( A and B ) Differentiated bronchiolar (A) or bronchial (B) airway spheroid cultures were infected at a MOI of 2. 16 hours ( A ) or 24 hours ( B ) post infection they were fixed and stained for viral nucleoprotein (red). Nuclei were stained with hoechst (blue) and actin was stained using phalloidin (white). AcTub stains ciliated cells (green). Scale bars indicate 200 μm in A and 50 μm in B . Representative images are shown from two independent experiments. ( C to E ) Replication kinetics of SARS-CoV-2 in bronchiolar airway spheroid cultures pretreated with camostat or carrier (DMSO). ( C and D ) TCID50 equivalents (eq.) per ml are shown in culture medium ( C ) and lysed organoids ( D ). ( E ) Live virus titers (TCID50/ml) in lysed organoids. Dotted line indicates limit of detection. ( F ) Replication kinetics of SARS-CoV-2 in 2D tracheal air-liquid interface airway cultures pretreated with camostat or carrier (DMSO). TCID50 eq. per ml in apical washes are shown. Error bars indicate SEM. H p.i. = hours post infection. Two-way ANOVA was performed for statistical analysis. * P

    Techniques Used: Derivative Assay, Infection, Staining

    The SARS-CoV-2 multibasic cleavage site increases serine protease usage. ( A and B ) SARS-CoV PP and SARS-CoV-2 PP entry route on VeroE6 cells pretreated with a concentration range of camostat ( A ) or E64D ( B ) to inhibit serine proteases and cathepsins, respectively. ( C and D ) SARS-CoV PP and SARS-CoV-2 PP entry route on VeroE6-TMPRSS2 cells pretreated with a concentration range of camostat ( C ) or E64D ( D ) to inhibit serine proteases and cathepsins, respectively. T-test was performed for statistical analysis at the highest concentration. * P
    Figure Legend Snippet: The SARS-CoV-2 multibasic cleavage site increases serine protease usage. ( A and B ) SARS-CoV PP and SARS-CoV-2 PP entry route on VeroE6 cells pretreated with a concentration range of camostat ( A ) or E64D ( B ) to inhibit serine proteases and cathepsins, respectively. ( C and D ) SARS-CoV PP and SARS-CoV-2 PP entry route on VeroE6-TMPRSS2 cells pretreated with a concentration range of camostat ( C ) or E64D ( D ) to inhibit serine proteases and cathepsins, respectively. T-test was performed for statistical analysis at the highest concentration. * P

    Techniques Used: Concentration Assay

    The SARS-CoV-2 multibasic cleavage site facilitates cell-cell fusion and SARS-CoV-2 is more fusogenic than SARS-CoV on differentiated organoid-derived human airway cells. ( A ) Proteolytic cleavage of SARS-CoV-2 S, SARS-CoV S, and S mutants was assessed by overexpression in HEK-293T cells and subsequent western blots for S1. GAPDH was used as a loading control. ( B and C ) Fusogenicity of SARS-CoV-2 S, SARS-CoV S, and S mutants was assessed after 18 hours by counting the number of nuclei per syncytium ( B ) and by measuring the sum of all GFP+ pixels per well ( C ). Statistical analysis was performed by one-way ANOVA on SARS-CoV or SARS-CoV-2 S-mediated fusion compared with its respective mutants. * P
    Figure Legend Snippet: The SARS-CoV-2 multibasic cleavage site facilitates cell-cell fusion and SARS-CoV-2 is more fusogenic than SARS-CoV on differentiated organoid-derived human airway cells. ( A ) Proteolytic cleavage of SARS-CoV-2 S, SARS-CoV S, and S mutants was assessed by overexpression in HEK-293T cells and subsequent western blots for S1. GAPDH was used as a loading control. ( B and C ) Fusogenicity of SARS-CoV-2 S, SARS-CoV S, and S mutants was assessed after 18 hours by counting the number of nuclei per syncytium ( B ) and by measuring the sum of all GFP+ pixels per well ( C ). Statistical analysis was performed by one-way ANOVA on SARS-CoV or SARS-CoV-2 S-mediated fusion compared with its respective mutants. * P

    Techniques Used: Derivative Assay, Over Expression, Western Blot

    The SARS-CoV-2 S multibasic cleavage site mediates entry into organoid-derived human airway cells. ( A ) Schematic overview of SARS-CoV-2 S protein mutants. Multibasic cleavage site residues are indicated in red; amino acid substitutions are indicated in green. Red arrows indicate cleavage sites. RBD = receptor binding domain, RBM = receptor binding motif. The SARS-CoV-2 S multibasic cleavage site was mutated to either remove the PRRA motif (SARS-2-Del-PRRA) or to substitute the R685 site (SARS-2-R685A and R685H). ( B ) Comparison of S cleavage of SARS-CoV-2 PPs and the multibasic cleavage site mutants. Western blots were performed against S1 with VSV-M silver stains as a production control. ( C and D ) PP infectivity of SARS-CoV-2 S and multibasic cleavage site mutants on VeroE6 ( C ) and Calu-3 ( D ) cells. ( E ) Differentiated airway spheroid cultures were infected with concentrated SARS-CoV-2 PPs containing a GFP reporter, indicated in green. Scale bar indicates 20 μm. ( F ) SARS-CoV-2 PP and multibasic cleavage site mutant infectivity on differentiated bronchiolar airway spheroid cultures. One-way ANOVA was performed for statistical analysis comparing all groups with SARS-CoV-2 PPs. * P
    Figure Legend Snippet: The SARS-CoV-2 S multibasic cleavage site mediates entry into organoid-derived human airway cells. ( A ) Schematic overview of SARS-CoV-2 S protein mutants. Multibasic cleavage site residues are indicated in red; amino acid substitutions are indicated in green. Red arrows indicate cleavage sites. RBD = receptor binding domain, RBM = receptor binding motif. The SARS-CoV-2 S multibasic cleavage site was mutated to either remove the PRRA motif (SARS-2-Del-PRRA) or to substitute the R685 site (SARS-2-R685A and R685H). ( B ) Comparison of S cleavage of SARS-CoV-2 PPs and the multibasic cleavage site mutants. Western blots were performed against S1 with VSV-M silver stains as a production control. ( C and D ) PP infectivity of SARS-CoV-2 S and multibasic cleavage site mutants on VeroE6 ( C ) and Calu-3 ( D ) cells. ( E ) Differentiated airway spheroid cultures were infected with concentrated SARS-CoV-2 PPs containing a GFP reporter, indicated in green. Scale bar indicates 20 μm. ( F ) SARS-CoV-2 PP and multibasic cleavage site mutant infectivity on differentiated bronchiolar airway spheroid cultures. One-way ANOVA was performed for statistical analysis comparing all groups with SARS-CoV-2 PPs. * P

    Techniques Used: Derivative Assay, Binding Assay, Western Blot, Infection, Mutagenesis

    SARS-CoV-2 enters faster on Calu-3 cells than SARS-CoV and entry speed is increased by the multibasic cleavage site. ( A ) SARS-CoV PP and SARS-CoV-2 PP infectivity on VeroE6 and Calu-3 cells. ( B and C ) SARS-CoV PP and SARS-CoV-2 PP entry route on Calu-3 cells. Cells were pretreated with a concentration range of camostat ( B ) or E64D ( C ) to inhibit serine proteases and cathepsins, respectively. T-test was performed for statistical analysis at the highest concentration. * P
    Figure Legend Snippet: SARS-CoV-2 enters faster on Calu-3 cells than SARS-CoV and entry speed is increased by the multibasic cleavage site. ( A ) SARS-CoV PP and SARS-CoV-2 PP infectivity on VeroE6 and Calu-3 cells. ( B and C ) SARS-CoV PP and SARS-CoV-2 PP entry route on Calu-3 cells. Cells were pretreated with a concentration range of camostat ( B ) or E64D ( C ) to inhibit serine proteases and cathepsins, respectively. T-test was performed for statistical analysis at the highest concentration. * P

    Techniques Used: Infection, Concentration Assay

    9) Product Images from "SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model"

    Article Title: SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

    Journal: bioRxiv

    doi: 10.1101/2020.08.24.264630

    Gross pathological examination of the lungs of SARS-CoV-2 infected hamsters. Foci (arrowheads) of pulmonary consolidation in untreated SARS-CoV-2 infected animals (A) and animals treated with control MAb (D) or low dose plasma (F). Protection against pulmonary lesions in hamsters treated with MAb 47D11 (C) and high dose plasma (E), similar to mock infected animals (B). Images are from representative animals of each treatment group.
    Figure Legend Snippet: Gross pathological examination of the lungs of SARS-CoV-2 infected hamsters. Foci (arrowheads) of pulmonary consolidation in untreated SARS-CoV-2 infected animals (A) and animals treated with control MAb (D) or low dose plasma (F). Protection against pulmonary lesions in hamsters treated with MAb 47D11 (C) and high dose plasma (E), similar to mock infected animals (B). Images are from representative animals of each treatment group.

    Techniques Used: Infection

    Effect of preventive treatment with MAb or high dose convalescent plasma on severity of pneumonia and level of virus antigen expression in lung parenchyma of hamsters after challenge with SARS-CoV-2. Comparison of extent of histopathological changes (HE) and virus antigen expression (IHC) at four days after SARS-CoV-2 inoculation at low magnification (two left columns) and high magnification (two right columns) in hamsters treated 24 hours before virus inoculation with neutralizing antibodies (second, third and fourth rows) compared to no treatment before SARS-CoV-2 inoculation (first row) and sham inoculation (fifth row).
    Figure Legend Snippet: Effect of preventive treatment with MAb or high dose convalescent plasma on severity of pneumonia and level of virus antigen expression in lung parenchyma of hamsters after challenge with SARS-CoV-2. Comparison of extent of histopathological changes (HE) and virus antigen expression (IHC) at four days after SARS-CoV-2 inoculation at low magnification (two left columns) and high magnification (two right columns) in hamsters treated 24 hours before virus inoculation with neutralizing antibodies (second, third and fourth rows) compared to no treatment before SARS-CoV-2 inoculation (first row) and sham inoculation (fifth row).

    Techniques Used: Expressing, Immunohistochemistry

    Effect of prophylactic neutralizing antibody treatment on weight loss and virus replication following SARS-CoV-2 infection in hamsters. A. Body weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS-CoV-2. SARS-CoV-2 viral RNA (B, C, E and G) or infectious virus (D, F and H) was detected in throat (B), nasal washes (C and D), lung (E and F) and nasal turbinates (G and H). The mean % of starting weight, the mean copy number or the mean infectious titer is shown, error bars represent the standard error of mean. n = 4. * = P
    Figure Legend Snippet: Effect of prophylactic neutralizing antibody treatment on weight loss and virus replication following SARS-CoV-2 infection in hamsters. A. Body weights of hamsters treated with antibodies were measured at indicated days after inoculation with SARS-CoV-2. SARS-CoV-2 viral RNA (B, C, E and G) or infectious virus (D, F and H) was detected in throat (B), nasal washes (C and D), lung (E and F) and nasal turbinates (G and H). The mean % of starting weight, the mean copy number or the mean infectious titer is shown, error bars represent the standard error of mean. n = 4. * = P

    Techniques Used: Infection

    Quantitative assessment of histopathological changes and virus antigen expression. Percentage of inflamed lung tissue (A) and percentage of lung tissue expressing SARS-CoV-2 antigen (B) estimated by microscopic examination in different groups of hamsters at four days after SARS-CoV-2 inoculation. Individual (symbols) and mean (horizontal lines) percentages are shown. Error bars represent the standard error of mean. n = 4. * = P
    Figure Legend Snippet: Quantitative assessment of histopathological changes and virus antigen expression. Percentage of inflamed lung tissue (A) and percentage of lung tissue expressing SARS-CoV-2 antigen (B) estimated by microscopic examination in different groups of hamsters at four days after SARS-CoV-2 inoculation. Individual (symbols) and mean (horizontal lines) percentages are shown. Error bars represent the standard error of mean. n = 4. * = P

    Techniques Used: Expressing

    Histopathological changes and virus antigen expression in nasal turbinates of hamsters after challenge with SARS-CoV-2. In the nasal turbinate of a sham-inoculated hamster (left column), the nasal cavity is empty and the histology of the olfactory mucosa is normal (A). In a serial section, there is no SARS-CoV-2 antigen expression (C). In the nasal turbinate of a non-treated SARS-CoV-2-inoculated hamster (B and D), the nasal cavity is filled with edema fluid mixed with inflammatory cells and debris and the olfactory mucosa is infiltrated by neutrophils (B). A serial section of this tissue shows SARS-CoV-2 antigen expression in many olfactory mucosal cells, as well as in cells in the lumen (C).
    Figure Legend Snippet: Histopathological changes and virus antigen expression in nasal turbinates of hamsters after challenge with SARS-CoV-2. In the nasal turbinate of a sham-inoculated hamster (left column), the nasal cavity is empty and the histology of the olfactory mucosa is normal (A). In a serial section, there is no SARS-CoV-2 antigen expression (C). In the nasal turbinate of a non-treated SARS-CoV-2-inoculated hamster (B and D), the nasal cavity is filled with edema fluid mixed with inflammatory cells and debris and the olfactory mucosa is infiltrated by neutrophils (B). A serial section of this tissue shows SARS-CoV-2 antigen expression in many olfactory mucosal cells, as well as in cells in the lumen (C).

    Techniques Used: Expressing

    10) Product Images from "Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment"

    Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

    Journal: Cell

    doi: 10.1016/j.cell.2020.06.010

    Differentially Expressed Genes in the Lungs of SARS-CoV-2-Infected Mice (A) SARS-CoV-2 viral RNA detected by RNA-seq in Ad5-Empty- and Ad5-ACE2-transduced mouse lungs. Data are expressed as normalized read counts. (B) Volcano plot showing differentially expressed genes in the lungs of Ad5-ACE2-transduced mice compared with Ad5-Empty-transduced mice. A total of 3,056 transcripts were differentially regulated. (C) Gene ontology (GO) analysis showing the differentially expressed genes from (B). (D) CD4 + (Cd4), CD8 + (Cd8a) T cell and B cell (Cd79b), macrophage (Cd68), and monocyte (Cd14) lineage marker expression. The red lines are the means of the three biological replicates, and the error bars are the standard error of the mean. Data are expressed as normalized read counts. p values are from a one-tailed Student’s t test. (E) Selected cytokines and chemokines differentially regulated in the lungs of Ad5-Empty- and Ad5-ACE2-transduced BALB/c mice at 2 d.p.i., obtained from the RNA-seq data. The red lines are the means of the three biological replicates, and the error bars are the standard error of the mean. Data are expressed as normalized read counts. p values are from a one-tailed Student’s t test.
    Figure Legend Snippet: Differentially Expressed Genes in the Lungs of SARS-CoV-2-Infected Mice (A) SARS-CoV-2 viral RNA detected by RNA-seq in Ad5-Empty- and Ad5-ACE2-transduced mouse lungs. Data are expressed as normalized read counts. (B) Volcano plot showing differentially expressed genes in the lungs of Ad5-ACE2-transduced mice compared with Ad5-Empty-transduced mice. A total of 3,056 transcripts were differentially regulated. (C) Gene ontology (GO) analysis showing the differentially expressed genes from (B). (D) CD4 + (Cd4), CD8 + (Cd8a) T cell and B cell (Cd79b), macrophage (Cd68), and monocyte (Cd14) lineage marker expression. The red lines are the means of the three biological replicates, and the error bars are the standard error of the mean. Data are expressed as normalized read counts. p values are from a one-tailed Student’s t test. (E) Selected cytokines and chemokines differentially regulated in the lungs of Ad5-Empty- and Ad5-ACE2-transduced BALB/c mice at 2 d.p.i., obtained from the RNA-seq data. The red lines are the means of the three biological replicates, and the error bars are the standard error of the mean. Data are expressed as normalized read counts. p values are from a one-tailed Student’s t test.

    Techniques Used: Infection, Mouse Assay, RNA Sequencing Assay, Marker, Expressing, One-tailed Test

    Requirements for T Cells and Antibodies for SARS-CoV-2 Clearance and Protection from Subsequent Challenge Ad5-hACE2-transduced mice were infected with 1 × 10 5 PFU of SARS-CoV-2. (A) For systemic depletion of CD4 + or CD8 + T cells, mice were injected intraperitoneally (i.p.) with 0.5 mg anti-CD4 antibody (clone GK1.5) and/or 0.5 mg anti-CD8 antibody (clone 2.43) or 0.5 mg rat IgG at days −2 and 0 p.i. Virus titers in the lungs were measured at the indicated time points. Titers are expressed as FFU/g tissue (n = 4 mice per group per time point) (B–D) To identify SARS-CoV-2 T cell responses, single-cell suspensions were prepared from the BALF of transduced/infected BALB/c mice and stimulated with 2 μM structural protein peptide pools for 5–6 h in the presence of brefeldin A. Flow plots (B, 7 d.p.i), and summary of frequencies and cell numbers of SARS-CoV-2-N pool specific CD4 + T cells (C) and S1 pool specific CD8 + T cells (D) (determined by IFN-γ intracellular staining) are shown (n = 3 to 4 mice per time point). (E) PRNT 50 titers in the sera of transduced/infected C57BL/6 mice at indicated time points p.i. are shown. (F) BALB/c and C57BL/6 mice were immunized with 1 × 10 5 infectious units (IU) of VRP-S intranasally in 50 μL of PBS. Mice were transduced and infected with 1 × 10 5 PFU of SARS-CoV-2 3 weeks after vaccination. Virus titers in the lungs were measured at the indicated time points (n = 4 mice per group per time point). (G) For adoptive transfer of serum, BALB/c mice were immunized with 1 × 10 5 IU of VRP in the footpad in 50 μL of PBS and boosted with the same dose 3 weeks later. Sera were obtained 1–2 weeks after VRP booster. Then, 150 μL of serum was transferred into transduced mice intravenously (i.v.) 1 day before SARS-CoV-2 infection (n = 3 mice per group per time point). ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values ≤ 0.0001.
    Figure Legend Snippet: Requirements for T Cells and Antibodies for SARS-CoV-2 Clearance and Protection from Subsequent Challenge Ad5-hACE2-transduced mice were infected with 1 × 10 5 PFU of SARS-CoV-2. (A) For systemic depletion of CD4 + or CD8 + T cells, mice were injected intraperitoneally (i.p.) with 0.5 mg anti-CD4 antibody (clone GK1.5) and/or 0.5 mg anti-CD8 antibody (clone 2.43) or 0.5 mg rat IgG at days −2 and 0 p.i. Virus titers in the lungs were measured at the indicated time points. Titers are expressed as FFU/g tissue (n = 4 mice per group per time point) (B–D) To identify SARS-CoV-2 T cell responses, single-cell suspensions were prepared from the BALF of transduced/infected BALB/c mice and stimulated with 2 μM structural protein peptide pools for 5–6 h in the presence of brefeldin A. Flow plots (B, 7 d.p.i), and summary of frequencies and cell numbers of SARS-CoV-2-N pool specific CD4 + T cells (C) and S1 pool specific CD8 + T cells (D) (determined by IFN-γ intracellular staining) are shown (n = 3 to 4 mice per time point). (E) PRNT 50 titers in the sera of transduced/infected C57BL/6 mice at indicated time points p.i. are shown. (F) BALB/c and C57BL/6 mice were immunized with 1 × 10 5 infectious units (IU) of VRP-S intranasally in 50 μL of PBS. Mice were transduced and infected with 1 × 10 5 PFU of SARS-CoV-2 3 weeks after vaccination. Virus titers in the lungs were measured at the indicated time points (n = 4 mice per group per time point). (G) For adoptive transfer of serum, BALB/c mice were immunized with 1 × 10 5 IU of VRP in the footpad in 50 μL of PBS and boosted with the same dose 3 weeks later. Sera were obtained 1–2 weeks after VRP booster. Then, 150 μL of serum was transferred into transduced mice intravenously (i.v.) 1 day before SARS-CoV-2 infection (n = 3 mice per group per time point). ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values ≤ 0.0001.

    Techniques Used: Mouse Assay, Infection, Injection, Staining, Plaque Reduction Neutralization Test, Adoptive Transfer Assay

    Convalescent Plasma from COVID-19 Patients and Remdesivir Protect Mice from SARS-CoV-2 Infection (A and B) For plasma adoptive transfer, Ad5-hACE2-transduced mice were injected with 150 μL of plasma i.v. from a healthy donor or COVID-19, MERS, or SARS convalescent patients, at −1 d.p.i. Weight and virus titers in lung tissues were monitored (A) and expressed as FFU/g tissue (n = 4 mice per group per time point). Sections of paraffin embedded lungs from plasma adoptive transferred and infected mice were stained with HE at day 4 p.i. (B). Scale bar, 100 μm. (C and D) For remdesivir treatment, Ad5-hACE2-transduced mice were treated with remdesivir (25 mg/kg, bid s.c.) or vehicle at −1 d.p.i. Weight loss of infected mice and virus titers in the lungs were monitored (C), and hematoxylin/eosin staining of sections of paraffin-embedded lungs is shown at 4 d.p.i. (D) (n = 4 mice per group per time point). Data are representative of two independent experiments. Scale bar, 100 μm. ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values of ≤ 0.0001.
    Figure Legend Snippet: Convalescent Plasma from COVID-19 Patients and Remdesivir Protect Mice from SARS-CoV-2 Infection (A and B) For plasma adoptive transfer, Ad5-hACE2-transduced mice were injected with 150 μL of plasma i.v. from a healthy donor or COVID-19, MERS, or SARS convalescent patients, at −1 d.p.i. Weight and virus titers in lung tissues were monitored (A) and expressed as FFU/g tissue (n = 4 mice per group per time point). Sections of paraffin embedded lungs from plasma adoptive transferred and infected mice were stained with HE at day 4 p.i. (B). Scale bar, 100 μm. (C and D) For remdesivir treatment, Ad5-hACE2-transduced mice were treated with remdesivir (25 mg/kg, bid s.c.) or vehicle at −1 d.p.i. Weight loss of infected mice and virus titers in the lungs were monitored (C), and hematoxylin/eosin staining of sections of paraffin-embedded lungs is shown at 4 d.p.i. (D) (n = 4 mice per group per time point). Data are representative of two independent experiments. Scale bar, 100 μm. ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values of ≤ 0.0001.

    Techniques Used: Mouse Assay, Infection, Adoptive Transfer Assay, Injection, Staining

    Development of Mice Sensitized to SARS-CoV-2 Infection (A and B) To assess hACE2 expression and surface localization, 17CL-1 cells were transduced with Ad5-hACE2 or Ad5-Empty at MOI of 100 at 37°C for 4 h. hACE2 expression was monitored by western blot assay (A) or flow cytometry (B). (C) Ad5-hACE2 transduced 17CL-1 cells were infected with SARS-CoV-2 at MOI of 0.5 at 48 h post transduction, and virus titers were determined by foci forming assay (FFA) at 24, 48, and 72 hours post infection (h.p.i.). (D) Five days after transduction with 2.5 × 10 8 FFU of Ad5-hACE2 or Ad5-Empty in 75 μL of DMEM intranasally, lungs were harvested from BALB/c mice, fixed in zinc formalin, and embedded in paraffin. Sections were stained with an anti-hACE2 antibody (brown color). hACE2 protein (brown color) was detected only in Ad-hACE2-treated mice and was predominantly localized to alveolar epithelial cells. Scale bars, 467 and 94 μm, top and bottom panels, respectively. (E and F) Ad5-hACE2- or Ad5-Empty-transduced BALB/c or C57BL/6 mice were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2 in 50 μL of DMEM. Weight changes in 6-to-8-week old BALB/c (E) and C57BL/6 (F) mice were monitored daily (n = 5 mice per group). To obtain virus kinetics in BALB/c (E) and C57BL/6 (F) mice, lungs were harvested and homogenized at the indicated time points, and virus was titered by plaque assay. Titers are expressed as PFU/g lung tissue (n = 3 mice per group per time point). Data are representative of two independent experiments. (G) 2 d.p.i., lungs were harvested from BALB/c mice, fixed in zinc formalin, and embedded in paraffin. Sections were stained with anti-SARS-CoV-2 N protein. Scale bar, 476 μm. (H) Representative Hematoxylin-eosin (HE) staining of lungs from BALB/c and C57BL/6 mice harvested at the indicated time points p.i. Scale bars, 443 and 88 μm, top and bottom panels, respectively. Asterisk, edema. (I) Summary histology scores determined at the indicated time points (n = 4 to 5 mice per group). PMN, neutrophils. (J) Photographs of lung specimens isolated from infected mice at indicated time points are shown. Arrowheads indicate regions with vascular congestion and hemorrhage.
    Figure Legend Snippet: Development of Mice Sensitized to SARS-CoV-2 Infection (A and B) To assess hACE2 expression and surface localization, 17CL-1 cells were transduced with Ad5-hACE2 or Ad5-Empty at MOI of 100 at 37°C for 4 h. hACE2 expression was monitored by western blot assay (A) or flow cytometry (B). (C) Ad5-hACE2 transduced 17CL-1 cells were infected with SARS-CoV-2 at MOI of 0.5 at 48 h post transduction, and virus titers were determined by foci forming assay (FFA) at 24, 48, and 72 hours post infection (h.p.i.). (D) Five days after transduction with 2.5 × 10 8 FFU of Ad5-hACE2 or Ad5-Empty in 75 μL of DMEM intranasally, lungs were harvested from BALB/c mice, fixed in zinc formalin, and embedded in paraffin. Sections were stained with an anti-hACE2 antibody (brown color). hACE2 protein (brown color) was detected only in Ad-hACE2-treated mice and was predominantly localized to alveolar epithelial cells. Scale bars, 467 and 94 μm, top and bottom panels, respectively. (E and F) Ad5-hACE2- or Ad5-Empty-transduced BALB/c or C57BL/6 mice were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2 in 50 μL of DMEM. Weight changes in 6-to-8-week old BALB/c (E) and C57BL/6 (F) mice were monitored daily (n = 5 mice per group). To obtain virus kinetics in BALB/c (E) and C57BL/6 (F) mice, lungs were harvested and homogenized at the indicated time points, and virus was titered by plaque assay. Titers are expressed as PFU/g lung tissue (n = 3 mice per group per time point). Data are representative of two independent experiments. (G) 2 d.p.i., lungs were harvested from BALB/c mice, fixed in zinc formalin, and embedded in paraffin. Sections were stained with anti-SARS-CoV-2 N protein. Scale bar, 476 μm. (H) Representative Hematoxylin-eosin (HE) staining of lungs from BALB/c and C57BL/6 mice harvested at the indicated time points p.i. Scale bars, 443 and 88 μm, top and bottom panels, respectively. Asterisk, edema. (I) Summary histology scores determined at the indicated time points (n = 4 to 5 mice per group). PMN, neutrophils. (J) Photographs of lung specimens isolated from infected mice at indicated time points are shown. Arrowheads indicate regions with vascular congestion and hemorrhage.

    Techniques Used: Mouse Assay, Infection, Expressing, Transduction, Western Blot, Flow Cytometry, Staining, Plaque Assay, Isolation

    The Role for IFN and STAT1 Signaling in SARS-CoV-2 Infection (A) 5 days after transduction with 2.5 × 10 8 FFU of Ad5-hACE2, C57BL/6 mice were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2. Weight changes were monitored daily (n = 5 mice per group), and virus titers in the lungs were measured at the indicated time points using FFA (n = 3–4 mice per group per time point). Titers are expressed as FFU/g tissue. (B) Sections of paraffin embedded lungs from SARS-CoV-2-infected Ad5-hACE2-transduced, wild-type and genetically modified C57BL/6 mice at 4 d.p.i. were stained with hematoxylin/eosin. Scale bar, 100 μm. (C) Photographs of gross pathological lung specimens isolated from infected C57BL/6 mice at 4 d.p.i. Arrowheads indicate regions with vascular congestion and hemorrhage. (D) Ad5-hACE2-transduced C57BL/6 mice were treated with 80 μg of poly I:C in 50 μL of PBS 6 h before intranasal infection with SARS-CoV-2. Weight changes were monitored daily, and viral titers in lungs were measured at the indicated time points. ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values ≤ 0.0001.
    Figure Legend Snippet: The Role for IFN and STAT1 Signaling in SARS-CoV-2 Infection (A) 5 days after transduction with 2.5 × 10 8 FFU of Ad5-hACE2, C57BL/6 mice were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2. Weight changes were monitored daily (n = 5 mice per group), and virus titers in the lungs were measured at the indicated time points using FFA (n = 3–4 mice per group per time point). Titers are expressed as FFU/g tissue. (B) Sections of paraffin embedded lungs from SARS-CoV-2-infected Ad5-hACE2-transduced, wild-type and genetically modified C57BL/6 mice at 4 d.p.i. were stained with hematoxylin/eosin. Scale bar, 100 μm. (C) Photographs of gross pathological lung specimens isolated from infected C57BL/6 mice at 4 d.p.i. Arrowheads indicate regions with vascular congestion and hemorrhage. (D) Ad5-hACE2-transduced C57BL/6 mice were treated with 80 μg of poly I:C in 50 μL of PBS 6 h before intranasal infection with SARS-CoV-2. Weight changes were monitored daily, and viral titers in lungs were measured at the indicated time points. ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values ≤ 0.0001.

    Techniques Used: Infection, Transduction, Mouse Assay, Genetically Modified, Staining, Isolation

    11) Product Images from "Susceptibility of rabbits to SARS-CoV-2"

    Article Title: Susceptibility of rabbits to SARS-CoV-2

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2020.1868951

    Susceptibility of rabbits to SARS-CoV-2 infection. Infection kinetics of (A) viral RNA and (B) authentic SARS-CoV-2 virus growth curves from rabbits inoculated with 10 6 TCID 50 and followed up for 21 days. (C–E) Viral RNA growth curved in rabbits inoculated with either (C) 10 6 , (D) 10 5 , or (E) 10 4 TCID 50 and followed up for four days post infection. (F) Viral RNA in lung and nasal turbinates of 10 6 TCID 50 infected rabbits, sacrificed after four days. The RNA detection limit was 3.5 × 10 −1 RNA copies/mL, while the live virus detection limit is 12.5 TCID 50 /mL. Error bars depict SEM. n = 3.
    Figure Legend Snippet: Susceptibility of rabbits to SARS-CoV-2 infection. Infection kinetics of (A) viral RNA and (B) authentic SARS-CoV-2 virus growth curves from rabbits inoculated with 10 6 TCID 50 and followed up for 21 days. (C–E) Viral RNA growth curved in rabbits inoculated with either (C) 10 6 , (D) 10 5 , or (E) 10 4 TCID 50 and followed up for four days post infection. (F) Viral RNA in lung and nasal turbinates of 10 6 TCID 50 infected rabbits, sacrificed after four days. The RNA detection limit was 3.5 × 10 −1 RNA copies/mL, while the live virus detection limit is 12.5 TCID 50 /mL. Error bars depict SEM. n = 3.

    Techniques Used: Infection, RNA Detection

    Rabbit ACE2-mediated SARS-CoV-2 infection. SARS-CoV-2 pseudovirus (A) and authentic virus (B) infection of Cos-7 cells transfected with ACE2 of various species. Infectivity was quantified by staining live virus with anti-SARS-CoV nucleocapsid and scanning live virus and pseudovirus infected cells. (C) Confocal imaging of ACE2-mediated live virus infection; cells were stained using anti-human ACE2 in green, anti-SARS-CoV nucleocapsid in red and TO-PRO3 in blue to stain nuclei. Scale indicates 50 µm. n.d. = not detected. Error bars depict SEM.
    Figure Legend Snippet: Rabbit ACE2-mediated SARS-CoV-2 infection. SARS-CoV-2 pseudovirus (A) and authentic virus (B) infection of Cos-7 cells transfected with ACE2 of various species. Infectivity was quantified by staining live virus with anti-SARS-CoV nucleocapsid and scanning live virus and pseudovirus infected cells. (C) Confocal imaging of ACE2-mediated live virus infection; cells were stained using anti-human ACE2 in green, anti-SARS-CoV nucleocapsid in red and TO-PRO3 in blue to stain nuclei. Scale indicates 50 µm. n.d. = not detected. Error bars depict SEM.

    Techniques Used: Infection, Transfection, Staining, Imaging

    Histological analysis of SARS-CoV-2 infected rabbits. Histopathological analysis of rabbits inoculated with 10 6 TCID 50 , sacrificed after four days. (A) Alveolar thickening and inflammatory infiltrates. Scale indicates 100 µm (B) Enlarged, syncytial cells in the alveolar lumina. Scale indicates 20 µm. (C) Lung pathology overview. Arrow indicates thickening and asterisk bronchus-associated lymphoid tissue (BALT). Scale indicates 200 µm. (D) Eosinophilic infiltrates in the nose. Scale indicates 40 µm.
    Figure Legend Snippet: Histological analysis of SARS-CoV-2 infected rabbits. Histopathological analysis of rabbits inoculated with 10 6 TCID 50 , sacrificed after four days. (A) Alveolar thickening and inflammatory infiltrates. Scale indicates 100 µm (B) Enlarged, syncytial cells in the alveolar lumina. Scale indicates 20 µm. (C) Lung pathology overview. Arrow indicates thickening and asterisk bronchus-associated lymphoid tissue (BALT). Scale indicates 200 µm. (D) Eosinophilic infiltrates in the nose. Scale indicates 40 µm.

    Techniques Used: Infection

    12) Product Images from "Mapping and role of T cell response in SARS-CoV-2–infected mice"

    Article Title: Mapping and role of T cell response in SARS-CoV-2–infected mice

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20202187

    Kinetics of virus-specific T cell responses in BALF and lung of SARS-CoV-2–infected BALB/c and C57BL/6 mice. (A–D) Lymphocytes from airway and lung of transduced/infected WT BALB/c mice were harvested at indicated time points after infection and stimulated with 5 µM N351 (A and B) and 1 µM S535 (C and D) for 6 h in the presence of brefeldin A. The frequencies (left) and cell numbers of antigen-specific T cells (right) in BALF (A and C) and lung (B and D) are shown ( n = 3 or 4 mice; data are representative of three independent experiments). (E–H) Lymphocytes from airway and lung of transduced/infected C57BL/6 mice were harvested at indicated time points and stimulated with 5 µM ORF3a 266 (E and F) and 1 µM S538 (G and H) for 6 h in the presence of brefeldin A. The frequencies (left) and cell numbers (right) of antigen-specific T cells are shown ( n = 3 or 4 mice; data are representative of three independent experiments). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide; p.i., post-infection.
    Figure Legend Snippet: Kinetics of virus-specific T cell responses in BALF and lung of SARS-CoV-2–infected BALB/c and C57BL/6 mice. (A–D) Lymphocytes from airway and lung of transduced/infected WT BALB/c mice were harvested at indicated time points after infection and stimulated with 5 µM N351 (A and B) and 1 µM S535 (C and D) for 6 h in the presence of brefeldin A. The frequencies (left) and cell numbers of antigen-specific T cells (right) in BALF (A and C) and lung (B and D) are shown ( n = 3 or 4 mice; data are representative of three independent experiments). (E–H) Lymphocytes from airway and lung of transduced/infected C57BL/6 mice were harvested at indicated time points and stimulated with 5 µM ORF3a 266 (E and F) and 1 µM S538 (G and H) for 6 h in the presence of brefeldin A. The frequencies (left) and cell numbers (right) of antigen-specific T cells are shown ( n = 3 or 4 mice; data are representative of three independent experiments). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide; p.i., post-infection.

    Techniques Used: Infection, Mouse Assay

    Mapping SARS-CoV-2 T cell epitopes in BALB/c mice. (A) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) peptides. Antigen-specific CD4 + T cell responses were determined by intracellular IFN-γ staining. (B) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) peptides. Antigen-specific CD8 + T cell responses were determined. (C) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) and corresponding truncated 13–15-mer peptides for 5–6 h in the presence of brefeldin A. Antigen-specific CD4 + T cell responses were determined. (D) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) and corresponding truncated 8–9-mer peptides for 5–6 h in the presence of brefeldin A. Antigen-specific CD8 + T cell responses were determined. Candidate truncated epitopes are labeled with # ( n = 3; data are representative of at least two independent experiments). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide.
    Figure Legend Snippet: Mapping SARS-CoV-2 T cell epitopes in BALB/c mice. (A) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) peptides. Antigen-specific CD4 + T cell responses were determined by intracellular IFN-γ staining. (B) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) peptides. Antigen-specific CD8 + T cell responses were determined. (C) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) and corresponding truncated 13–15-mer peptides for 5–6 h in the presence of brefeldin A. Antigen-specific CD4 + T cell responses were determined. (D) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) and corresponding truncated 8–9-mer peptides for 5–6 h in the presence of brefeldin A. Antigen-specific CD8 + T cell responses were determined. Candidate truncated epitopes are labeled with # ( n = 3; data are representative of at least two independent experiments). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide.

    Techniques Used: Mouse Assay, Staining, Labeling

    SARS-CoV-2–specific CD4 + T cells and CD8 + T cells were polyfunctional. (A and E) Phenotyping BALF-derived SARS-CoV-2-N351–specific CD4 + T cells (A) and SARS-CoV-2-S535–specific CD8 + T cells (E). (B and F) Cytokine expression of airway-derived N351-specific CD4 + T cells (B) and S535-specific CD8 + T cells (F) are shown ( n = 3 mice; data are representative of two independent experiments). (C and G) Functional avidity curves (left) of airway- and lung-derived N351-specific CD4 + T cells (C) and S535-specific CD8 + T cells (G) and the amount of peptide required for half-maximum response (EC 50 ) are shown (right; n = 3 mice; data are representative of two independent experiments; Student’s t tests; P value of C is 0.0004; P value of G is 0.0051). (D and H) Representative flow histograms (left) and killing rates (right) of in vivo cytotoxicity of N351-specific CD4 + T cells (D) and S535-specific CD8 + T cells (H) in SARS-CoV-2–infected mice and mock-infected mice are shown ( n = 5 mice per group; data are representative of two independent experiments; Student’s t tests; P value of D is 0.0062; P value of H is 0.0002). *, P
    Figure Legend Snippet: SARS-CoV-2–specific CD4 + T cells and CD8 + T cells were polyfunctional. (A and E) Phenotyping BALF-derived SARS-CoV-2-N351–specific CD4 + T cells (A) and SARS-CoV-2-S535–specific CD8 + T cells (E). (B and F) Cytokine expression of airway-derived N351-specific CD4 + T cells (B) and S535-specific CD8 + T cells (F) are shown ( n = 3 mice; data are representative of two independent experiments). (C and G) Functional avidity curves (left) of airway- and lung-derived N351-specific CD4 + T cells (C) and S535-specific CD8 + T cells (G) and the amount of peptide required for half-maximum response (EC 50 ) are shown (right; n = 3 mice; data are representative of two independent experiments; Student’s t tests; P value of C is 0.0004; P value of G is 0.0051). (D and H) Representative flow histograms (left) and killing rates (right) of in vivo cytotoxicity of N351-specific CD4 + T cells (D) and S535-specific CD8 + T cells (H) in SARS-CoV-2–infected mice and mock-infected mice are shown ( n = 5 mice per group; data are representative of two independent experiments; Student’s t tests; P value of D is 0.0062; P value of H is 0.0002). *, P

    Techniques Used: Derivative Assay, Expressing, Mouse Assay, Functional Assay, In Vivo, Infection

    Mapping SARS-CoV-2 T cell epitopes in C57BL/6 mice. (A) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) peptides. Antigen-specific CD4 + T cell responses were determined by intracellular IFN-γ staining. (B) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) peptides. Antigen-specific CD8 + T cell responses were determined. (C) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) and corresponding truncated 13–15-mer peptides. Antigen-specific CD4 + T cell responses were determined. (D) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) and corresponding truncated 8–9-mer peptides. Antigen-specific CD8 + T cell responses were determined. Candidate truncated epitopes are labeled with # ( n = 3; data are representative of at least two independent experiments). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide.
    Figure Legend Snippet: Mapping SARS-CoV-2 T cell epitopes in C57BL/6 mice. (A) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) peptides. Antigen-specific CD4 + T cell responses were determined by intracellular IFN-γ staining. (B) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) peptides. Antigen-specific CD8 + T cell responses were determined. (C) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) and corresponding truncated 13–15-mer peptides. Antigen-specific CD4 + T cell responses were determined. (D) Lymphocytes from vaccinated lungs were stimulated with 5 µM 20-mer (20 amino acids) and corresponding truncated 8–9-mer peptides. Antigen-specific CD8 + T cell responses were determined. Candidate truncated epitopes are labeled with # ( n = 3; data are representative of at least two independent experiments). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide.

    Techniques Used: Mouse Assay, Staining, Labeling

    IFN-I signaling was critical for the generation of robust T cell responses against SARS-CoV-2 infection. (A and B) Frequencies (left) and cell numbers (right) of airway-derived N351-specific CD4 + T cells (A) and S535-specific CD8 + T cells (B) at indicated time points are shown ( n = 3 or 4 mice per time point; data are representative of three independent experiments). (C and D) Airway-derived N351-specific CD4 + T cell responses (C) and S535-specific CD8 + T cell responses (D) in WT and KO BALB/c mice are compared ( n = 3 or 4 mice; data are representative of two independent experiments; Student’s t tests; P values of C are 0.0006 and 0.0013; P values of D are 0.0005 and 0.0029). (E and F) Representative flow plots of N351-specific CD4 + T cells (E) and S535-specific CD8 + T cells (F) are shown (left). Bi-cytokine expression capability (right three panels) is statistically different between WT and KO mice ( n = 3 or 4 mice; data are representative of two indep endent experiments; Student’s t tests; P values of E are 0.0003, 0.0057, and 0.0004; P values of F are
    Figure Legend Snippet: IFN-I signaling was critical for the generation of robust T cell responses against SARS-CoV-2 infection. (A and B) Frequencies (left) and cell numbers (right) of airway-derived N351-specific CD4 + T cells (A) and S535-specific CD8 + T cells (B) at indicated time points are shown ( n = 3 or 4 mice per time point; data are representative of three independent experiments). (C and D) Airway-derived N351-specific CD4 + T cell responses (C) and S535-specific CD8 + T cell responses (D) in WT and KO BALB/c mice are compared ( n = 3 or 4 mice; data are representative of two independent experiments; Student’s t tests; P values of C are 0.0006 and 0.0013; P values of D are 0.0005 and 0.0029). (E and F) Representative flow plots of N351-specific CD4 + T cells (E) and S535-specific CD8 + T cells (F) are shown (left). Bi-cytokine expression capability (right three panels) is statistically different between WT and KO mice ( n = 3 or 4 mice; data are representative of two indep endent experiments; Student’s t tests; P values of E are 0.0003, 0.0057, and 0.0004; P values of F are

    Techniques Used: Infection, Derivative Assay, Mouse Assay, Expressing

    Identification of CD4 + and CD8 + T cell epitopes in SARS-CoV-2–infected WT BALB/c and C57BL/6 mice. (A and B) Confirmation of CD4 + T cell epitopes (A) and CD8 + T cell epitopes (B) in infected BALB/c mice. Flow plots and summary columns are shown ( n = 3; data verified in two independent experiments). (C and D) Confirmation of CD4 + T cell epitopes (C) and CD8 + T cell epitopes (D) in infected C57BL/6 mice. Flow plots and summary columns are shown ( n = 3; data verified in two independent experiments). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide.
    Figure Legend Snippet: Identification of CD4 + and CD8 + T cell epitopes in SARS-CoV-2–infected WT BALB/c and C57BL/6 mice. (A and B) Confirmation of CD4 + T cell epitopes (A) and CD8 + T cell epitopes (B) in infected BALB/c mice. Flow plots and summary columns are shown ( n = 3; data verified in two independent experiments). (C and D) Confirmation of CD4 + T cell epitopes (C) and CD8 + T cell epitopes (D) in infected C57BL/6 mice. Flow plots and summary columns are shown ( n = 3; data verified in two independent experiments). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide.

    Techniques Used: Infection, Mouse Assay

    Kinetics of virus-specific T cell responses in DLNs and spleens of SARS-CoV-2–infected BALB/c and C57BL/6 mice. (A–D) Lymphocytes from DLN and spleen of transduced/infected WT BALB/c mice were harvested at indicated time points after infection and stimulated with 5 µM N351 (A and B) and 1 µM S535 (C and D) for 6 h in the presence of brefeldin A. The frequencies (left) and cell numbers of antigen-specific T cells (right) in DLN (A and C) and spleen (B and D) are shown ( n = 3 mice; data are representative of one experiment). (E–H) Lymphocytes from DLN and spleen of transduced/infected C57BL/6 mice were harvested at indicated time points and stimulated with 5 µM ORF3a 266 (E and F) and 1 µM S538 (G and H) for 6 h in the presence of brefeldin A. The frequencies (left) and cell numbers (right) of antigen-specific T cells are shown ( n = 3; data are representative of one experiment). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide; p.i., post-infection.
    Figure Legend Snippet: Kinetics of virus-specific T cell responses in DLNs and spleens of SARS-CoV-2–infected BALB/c and C57BL/6 mice. (A–D) Lymphocytes from DLN and spleen of transduced/infected WT BALB/c mice were harvested at indicated time points after infection and stimulated with 5 µM N351 (A and B) and 1 µM S535 (C and D) for 6 h in the presence of brefeldin A. The frequencies (left) and cell numbers of antigen-specific T cells (right) in DLN (A and C) and spleen (B and D) are shown ( n = 3 mice; data are representative of one experiment). (E–H) Lymphocytes from DLN and spleen of transduced/infected C57BL/6 mice were harvested at indicated time points and stimulated with 5 µM ORF3a 266 (E and F) and 1 µM S538 (G and H) for 6 h in the presence of brefeldin A. The frequencies (left) and cell numbers (right) of antigen-specific T cells are shown ( n = 3; data are representative of one experiment). All results are expressed as mean ± SEM. Ag, antigen; pep, peptide; p.i., post-infection.

    Techniques Used: Infection, Mouse Assay

    Epitope-specific CD4 + and CD8 + T cells partially protected SARS-CoV-2–infected mice from severe disease. (A) Strategy of VRP vaccination and SARS-CoV-2 challenge. (B and C) Effects of N351-specific CD4 + T cells (B) and S535-specific CD8 + T cells (C) in BALB/c mice. Cell numbers of airway-derived antigen-specific T cells are shown ( n = 3 or 4 mice per group per time point; left). Viral titers in the lungs were measured at the indicated time points ( n = 4 mice per group per time point; middle; data are representative of at least two independent experiments; Student’s t tests; P values of B are 0.0051 and 0.0403; P values of C are 0.0026 and 0.0804). Sections of paraffin-embedded lungs from infected mice at 4 d.p.i. were stained with hematoxylin/eosin ( n = 3 mice per group per time point; right; data are representative of at least two independent experiments). Scale bar, 100 mm. (D) Effects of S538-specific CD8 + T cells in C57BL/6 mice. Cell numbers at indicated time points are shown ( n = 3 mice per group per time point; left). Viral titers in the lungs were measured at the indicated time points ( n = 4 mice per group per time point; middle; data are representative of at least two independent experiments; Student’s t tests; P values of D are 0.0372 and 0.0043). Sections of paraffin-embedded lungs from infected mice at 6 d after infection were stained with hematoxylin/eosin ( n = 3 mice per group per time point; right; data are representative of at least two independent experiments). Scale bar, 100 mm. *, P
    Figure Legend Snippet: Epitope-specific CD4 + and CD8 + T cells partially protected SARS-CoV-2–infected mice from severe disease. (A) Strategy of VRP vaccination and SARS-CoV-2 challenge. (B and C) Effects of N351-specific CD4 + T cells (B) and S535-specific CD8 + T cells (C) in BALB/c mice. Cell numbers of airway-derived antigen-specific T cells are shown ( n = 3 or 4 mice per group per time point; left). Viral titers in the lungs were measured at the indicated time points ( n = 4 mice per group per time point; middle; data are representative of at least two independent experiments; Student’s t tests; P values of B are 0.0051 and 0.0403; P values of C are 0.0026 and 0.0804). Sections of paraffin-embedded lungs from infected mice at 4 d.p.i. were stained with hematoxylin/eosin ( n = 3 mice per group per time point; right; data are representative of at least two independent experiments). Scale bar, 100 mm. (D) Effects of S538-specific CD8 + T cells in C57BL/6 mice. Cell numbers at indicated time points are shown ( n = 3 mice per group per time point; left). Viral titers in the lungs were measured at the indicated time points ( n = 4 mice per group per time point; middle; data are representative of at least two independent experiments; Student’s t tests; P values of D are 0.0372 and 0.0043). Sections of paraffin-embedded lungs from infected mice at 6 d after infection were stained with hematoxylin/eosin ( n = 3 mice per group per time point; right; data are representative of at least two independent experiments). Scale bar, 100 mm. *, P

    Techniques Used: Infection, Mouse Assay, Derivative Assay, Staining

    SARS-CoV-2–specific CD8 + T cells were polyfunctional in infected C57BL/6 mice. (A) Cells from BALF were stained with antibodies against the indicated markers. Histograms shown here were gated on SARS-CoV-2-S538–specific CD8 + T cells. (B) Cytokine expression of airway derived SARS-CoV-2-S538–specific CD8 + T cells are shown ( n = 3 or 4 mice; data are representative of one experiment). (C) Functional avidity curves (left) of airway- and lung-derived SARS-CoV-2-S538–specific CD8 + T cells and the amount of peptide required for half-maximum response (EC 50 ) are shown (right; n = 3; data are representative of two independent experiments; Student’s t tests; P value of C is 0.011). (D) Representative flow histograms (left) and killing rates (right) of in vivo cytotoxicity of SARS-CoV-2-S538–specific CD8 + T cells in SARS-CoV-2–infected mice and mock-infected mice are shown ( n = 5; data are representative of one experiment; Student’s t tests; P value of C is
    Figure Legend Snippet: SARS-CoV-2–specific CD8 + T cells were polyfunctional in infected C57BL/6 mice. (A) Cells from BALF were stained with antibodies against the indicated markers. Histograms shown here were gated on SARS-CoV-2-S538–specific CD8 + T cells. (B) Cytokine expression of airway derived SARS-CoV-2-S538–specific CD8 + T cells are shown ( n = 3 or 4 mice; data are representative of one experiment). (C) Functional avidity curves (left) of airway- and lung-derived SARS-CoV-2-S538–specific CD8 + T cells and the amount of peptide required for half-maximum response (EC 50 ) are shown (right; n = 3; data are representative of two independent experiments; Student’s t tests; P value of C is 0.011). (D) Representative flow histograms (left) and killing rates (right) of in vivo cytotoxicity of SARS-CoV-2-S538–specific CD8 + T cells in SARS-CoV-2–infected mice and mock-infected mice are shown ( n = 5; data are representative of one experiment; Student’s t tests; P value of C is

    Techniques Used: Infection, Mouse Assay, Staining, Expressing, Derivative Assay, Functional Assay, In Vivo

    Cross-reactive T cell responses were found between SARS-CoV-2 and SARS-CoV in SARS-CoV-2–infected mice. (A) Characteristics of conserved T cell epitopes in SARS-CoV-2, SARS-CoV, and MERS-CoV. (B and C) BALB/c mice were transduced and infected with SARS-CoV-2. Lymphocytes derived from airway were prepared at 8 d.p.i. and stimulated with conversed epitopes. CD4 + (B) and CD8 + T cell responses (C) were detected by IFN-γ expression. Flow plots (left) and cross-reactivity rate (right) are shown ( n = 3 or 4 mice per group; data are representative of two independent experiments). (D) Functional avidity curves (left) of S535-specific CD8 + T cells and S521–cross-reactive CD8 + T cells and the amount of peptide required for half-maximum response (EC 50 ) are shown (right; n = 3 mice; data are representative of two independent experiments; Student’s t tests; P value of D is 0.0182). *, P
    Figure Legend Snippet: Cross-reactive T cell responses were found between SARS-CoV-2 and SARS-CoV in SARS-CoV-2–infected mice. (A) Characteristics of conserved T cell epitopes in SARS-CoV-2, SARS-CoV, and MERS-CoV. (B and C) BALB/c mice were transduced and infected with SARS-CoV-2. Lymphocytes derived from airway were prepared at 8 d.p.i. and stimulated with conversed epitopes. CD4 + (B) and CD8 + T cell responses (C) were detected by IFN-γ expression. Flow plots (left) and cross-reactivity rate (right) are shown ( n = 3 or 4 mice per group; data are representative of two independent experiments). (D) Functional avidity curves (left) of S535-specific CD8 + T cells and S521–cross-reactive CD8 + T cells and the amount of peptide required for half-maximum response (EC 50 ) are shown (right; n = 3 mice; data are representative of two independent experiments; Student’s t tests; P value of D is 0.0182). *, P

    Techniques Used: Infection, Mouse Assay, Derivative Assay, Expressing, Functional Assay

    13) Product Images from "Published anti-SARS-CoV-2 in vitro hits share common mechanisms of action that synergize with antivirals"

    Article Title: Published anti-SARS-CoV-2 in vitro hits share common mechanisms of action that synergize with antivirals

    Journal: Briefings in Bioinformatics

    doi: 10.1093/bib/bbab249

    Key pathways regulated by anti-SARS-CoV-2 active compounds indicate patients severity and compound efficacy in vitro . A, Expression change of genes involved in cholesterol homeostasis or microtubule cytoskeleton organization induced by anti-SARS-CoV-2 compounds or three randomly selected compounds namely heliotrine, telenzepine, and dipivefrine. B, The expression change of the selected ChoMCyto genes in different COVID-19 patient groups (Sequence Read Archive ID: SRP267176). The bar plot shows the overall ChoMCyto scores. C, ChoMCyto scores correlate with patients’ SOFA (Sequence Read Archive ID: SRP279280). The P -value is one-tailed. D, Drugs targeting cytoskeleton, and their effects on cholesterol homeostasis, microtubule cytoskeleton organization and ChoMCyto genes. Dashed rectangle incorporates anti-SARS-CoV-2 active compounds. E, The dose–response (blue) and dose–viability (red) curves of monensin, with IC50, CC50 and selectivity index labeled. F, mRNA fold changes (compared with TATA-box-binding protein (TBP)) of NPC1, INSIG1 and HMGCS1 induced by cepharanthine, lomitapide and monensin at 0.5 μM in human lung primary small airway cells. Error bars denote standard deviations. For reference, the fold change of 1.0 is shown with grey dashed lines. In both heatmaps and their labels, red indicates upregulation, and blue means downregulation. * P
    Figure Legend Snippet: Key pathways regulated by anti-SARS-CoV-2 active compounds indicate patients severity and compound efficacy in vitro . A, Expression change of genes involved in cholesterol homeostasis or microtubule cytoskeleton organization induced by anti-SARS-CoV-2 compounds or three randomly selected compounds namely heliotrine, telenzepine, and dipivefrine. B, The expression change of the selected ChoMCyto genes in different COVID-19 patient groups (Sequence Read Archive ID: SRP267176). The bar plot shows the overall ChoMCyto scores. C, ChoMCyto scores correlate with patients’ SOFA (Sequence Read Archive ID: SRP279280). The P -value is one-tailed. D, Drugs targeting cytoskeleton, and their effects on cholesterol homeostasis, microtubule cytoskeleton organization and ChoMCyto genes. Dashed rectangle incorporates anti-SARS-CoV-2 active compounds. E, The dose–response (blue) and dose–viability (red) curves of monensin, with IC50, CC50 and selectivity index labeled. F, mRNA fold changes (compared with TATA-box-binding protein (TBP)) of NPC1, INSIG1 and HMGCS1 induced by cepharanthine, lomitapide and monensin at 0.5 μM in human lung primary small airway cells. Error bars denote standard deviations. For reference, the fold change of 1.0 is shown with grey dashed lines. In both heatmaps and their labels, red indicates upregulation, and blue means downregulation. * P

    Techniques Used: In Vitro, Expressing, Sequencing, One-tailed Test, Labeling, Binding Assay

    Anti-SARS-CoV-2 compound signature gene expression change. A, The workflow of this research. B, Example genes were induced (SQSTM1 and NPC1) or suppressed (MYBL2 and CCNA2) by anti-SARS-CoV-2 compounds. The y -axis indicates the LINCS z -score of a specific compound and a higher score means higher expression change. P -values were derived from Wilcox rank sum tests, and further corrected across all LINCS 978 genes. C, A boxplot showing the comparison of the effects on the 63 signature genes between active compounds and other compounds. The y -axis indicates an overall effect of a specific compound on the 63 genes, and a higher score means a better alignment with the up/downregulation pattern. On the x -axis, the number of gene expression profiles in each group is labeled under the group name. P -values were derived from Wilcox rank sum tests. D, A boxplot comparison between anti-SARS-CoV-2 CRISPR screening gene hits and non-hits. A higher RGES score on y -axis indicates a query host cell shRNA knockdown-induced gene expression profile more closely resembles the summarized gene expression signature of anti-SARS-CoV-2 active compounds. x -axis denotes whether a query host gene knock-out makes the cells resistant to SARS-CoV-2 infection. In the box plot, the central line represents the median value, and the bounds represent the 25th and 75th percentiles. The whiskers are 25th/75th quartiles plus 1.5 times the interquartile range.
    Figure Legend Snippet: Anti-SARS-CoV-2 compound signature gene expression change. A, The workflow of this research. B, Example genes were induced (SQSTM1 and NPC1) or suppressed (MYBL2 and CCNA2) by anti-SARS-CoV-2 compounds. The y -axis indicates the LINCS z -score of a specific compound and a higher score means higher expression change. P -values were derived from Wilcox rank sum tests, and further corrected across all LINCS 978 genes. C, A boxplot showing the comparison of the effects on the 63 signature genes between active compounds and other compounds. The y -axis indicates an overall effect of a specific compound on the 63 genes, and a higher score means a better alignment with the up/downregulation pattern. On the x -axis, the number of gene expression profiles in each group is labeled under the group name. P -values were derived from Wilcox rank sum tests. D, A boxplot comparison between anti-SARS-CoV-2 CRISPR screening gene hits and non-hits. A higher RGES score on y -axis indicates a query host cell shRNA knockdown-induced gene expression profile more closely resembles the summarized gene expression signature of anti-SARS-CoV-2 active compounds. x -axis denotes whether a query host gene knock-out makes the cells resistant to SARS-CoV-2 infection. In the box plot, the central line represents the median value, and the bounds represent the 25th and 75th percentiles. The whiskers are 25th/75th quartiles plus 1.5 times the interquartile range.

    Techniques Used: Expressing, Derivative Assay, Labeling, CRISPR, shRNA, Knock-Out, Infection

    The drug effect on ChoMCyto genes correlates with antiviral synergism. A, heatmaps of synergism effects (top), ChoMCyto score (middle) and ChoMCyto genes expression change (bottom). Heatmaps share drug columns. In the top panel, the row ‘Synergism’ summarizes the average synergistic effect of antiviral drugs with each host-targeting compound in the x -axis. White: missing values, purple: synergism, orange: antagonism. The middle row illustrates the ChoMCyto scores multiplied by −1, for a better color agreement with ‘Synergism’. In the bottom heatmap and gene labels, red indicates upregulation, and blue means downregulation. B, ChoMCyto scores and average synergism effects of repurposed anti-SARS-CoV-2 candidates in a scatter plot. Each dot represents a compound. Spearman R correlation and the P -value (one-tailed) are labeled.
    Figure Legend Snippet: The drug effect on ChoMCyto genes correlates with antiviral synergism. A, heatmaps of synergism effects (top), ChoMCyto score (middle) and ChoMCyto genes expression change (bottom). Heatmaps share drug columns. In the top panel, the row ‘Synergism’ summarizes the average synergistic effect of antiviral drugs with each host-targeting compound in the x -axis. White: missing values, purple: synergism, orange: antagonism. The middle row illustrates the ChoMCyto scores multiplied by −1, for a better color agreement with ‘Synergism’. In the bottom heatmap and gene labels, red indicates upregulation, and blue means downregulation. B, ChoMCyto scores and average synergism effects of repurposed anti-SARS-CoV-2 candidates in a scatter plot. Each dot represents a compound. Spearman R correlation and the P -value (one-tailed) are labeled.

    Techniques Used: Expressing, One-tailed Test, Labeling

    14) Product Images from "High prevalence of SARS-CoV-2 antibodies in care homes affected by COVID-19: Prospective cohort study, England"

    Article Title: High prevalence of SARS-CoV-2 antibodies in care homes affected by COVID-19: Prospective cohort study, England

    Journal: EClinicalMedicine

    doi: 10.1016/j.eclinm.2020.100597

    Flow diagram of residents and staff in 6 London care homes experiencing a COVID-19 outbreak during the pandemic who consented to follow-up testing including blood sampling for SARS-CoV-2 antibodies four to six weeks later. ‘Ever-symptomatic’ indicates that symptoms were experienced at some point during the follow-up period. *Three individuals in this group became SARS-CoV-2 PCR positive at follow-up RT-PCR testing conducted simultaneously with SARS-CoV-2 antibody testing.
    Figure Legend Snippet: Flow diagram of residents and staff in 6 London care homes experiencing a COVID-19 outbreak during the pandemic who consented to follow-up testing including blood sampling for SARS-CoV-2 antibodies four to six weeks later. ‘Ever-symptomatic’ indicates that symptoms were experienced at some point during the follow-up period. *Three individuals in this group became SARS-CoV-2 PCR positive at follow-up RT-PCR testing conducted simultaneously with SARS-CoV-2 antibody testing.

    Techniques Used: Sampling, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    15) Product Images from "Development of immunohistochemistry and in situ hybridisation for the detection of SARS-CoV and SARS-CoV-2 in formalin-fixed paraffin-embedded specimens"

    Article Title: Development of immunohistochemistry and in situ hybridisation for the detection of SARS-CoV and SARS-CoV-2 in formalin-fixed paraffin-embedded specimens

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-78949-0

    Immunohistochemistry labelling of FFPE cells expressing SARS-CoV, SARS-CoV-2 and MERS spike proteins. Immunodetection performed using SARS-CoV spike rabbit monoclonal antibody on producer cells for SARS-CoV (a) , SARS-CoV-2 (b) and MERS-CoV pseudotype virus (c) and non-transfected cells (d) . Scale bars, 20 µm.
    Figure Legend Snippet: Immunohistochemistry labelling of FFPE cells expressing SARS-CoV, SARS-CoV-2 and MERS spike proteins. Immunodetection performed using SARS-CoV spike rabbit monoclonal antibody on producer cells for SARS-CoV (a) , SARS-CoV-2 (b) and MERS-CoV pseudotype virus (c) and non-transfected cells (d) . Scale bars, 20 µm.

    Techniques Used: Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Expressing, Immunodetection, Transfection

    Immunohistochemistry and in situ hybridisation detection of SARS-CoV-2 and RNA on infected ferret tissues. Detection of spike protein (a) , nucleoprotein (b) and dsRNA antigens (c) and spike RNA (d) labelling. Tissue shrinkage artefact with ISH pre-treatment (d) . Scale bars, 20 µm.
    Figure Legend Snippet: Immunohistochemistry and in situ hybridisation detection of SARS-CoV-2 and RNA on infected ferret tissues. Detection of spike protein (a) , nucleoprotein (b) and dsRNA antigens (c) and spike RNA (d) labelling. Tissue shrinkage artefact with ISH pre-treatment (d) . Scale bars, 20 µm.

    Techniques Used: Immunohistochemistry, In Situ, Hybridization, Infection, In Situ Hybridization

    Immunohistochemical labelling of FFPE SARS-CoV and SARS-CoV-2 infected cells and uninfected cells. Immunodetection performed using SARS-CoV spike rabbit monoclonal antibody (a–c) , SARS-CoV nucleoprotein rabbit polyclonal antibody (d–f) and double-stranded RNA (dsRNA) rabbit monoclonal antibody (g–i) . Scale bars, 20 µm.
    Figure Legend Snippet: Immunohistochemical labelling of FFPE SARS-CoV and SARS-CoV-2 infected cells and uninfected cells. Immunodetection performed using SARS-CoV spike rabbit monoclonal antibody (a–c) , SARS-CoV nucleoprotein rabbit polyclonal antibody (d–f) and double-stranded RNA (dsRNA) rabbit monoclonal antibody (g–i) . Scale bars, 20 µm.

    Techniques Used: Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Infection, Immunodetection

    In situ hybridisation (ISH) of FFPE cells infected with SARS-CoV and SARS-CoV-2 using RNAScope ® . ISH performed using RNA probes designed specific to SARS-CoV-2 spike RNA. SARS-CoV (a) and SARS-CoV-2 infected cells (b) , uninfected cells (c) . Scale bars, 20 µm.
    Figure Legend Snippet: In situ hybridisation (ISH) of FFPE cells infected with SARS-CoV and SARS-CoV-2 using RNAScope ® . ISH performed using RNA probes designed specific to SARS-CoV-2 spike RNA. SARS-CoV (a) and SARS-CoV-2 infected cells (b) , uninfected cells (c) . Scale bars, 20 µm.

    Techniques Used: In Situ, Hybridization, In Situ Hybridization, Formalin-fixed Paraffin-Embedded, Infection

    16) Product Images from "SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site"

    Article Title: SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site

    Journal: eLife

    doi: 10.7554/eLife.64508

    hAO cultures grown at 2D air–liquid interface are well-differentiated and express ACE2 and TMPRSS2. ( A, B, and C ) Immunofluorescent or immunohistochemistry staining of differentiated airway cultures. Anti-AcTub (green) and anti-FOXJ1 (white) stains ciliated cells ( A ), anti-SCGB1A1 (magenta) stains club cells ( B ), and anti-MUC5AC (yellow) stains goblet cells ( C ). Nuclei are stained with hoechst (blue). ( D and E ) hAO cultures also expressed the SARS-CoV-2 entry receptor ACE2 ( D ) and TMPRSS2 ( E ). Hematoxylin was used as a counterstain in ( D ) and ( E ). Scale bars indicate 20 μm. Representative images are shown from a bronchiolar culture. ACE2, angiotensin-converting enzyme 2; hAO, human airway organoid; TMPRSS2, transmembrane protease serine 2.
    Figure Legend Snippet: hAO cultures grown at 2D air–liquid interface are well-differentiated and express ACE2 and TMPRSS2. ( A, B, and C ) Immunofluorescent or immunohistochemistry staining of differentiated airway cultures. Anti-AcTub (green) and anti-FOXJ1 (white) stains ciliated cells ( A ), anti-SCGB1A1 (magenta) stains club cells ( B ), and anti-MUC5AC (yellow) stains goblet cells ( C ). Nuclei are stained with hoechst (blue). ( D and E ) hAO cultures also expressed the SARS-CoV-2 entry receptor ACE2 ( D ) and TMPRSS2 ( E ). Hematoxylin was used as a counterstain in ( D ) and ( E ). Scale bars indicate 20 μm. Representative images are shown from a bronchiolar culture. ACE2, angiotensin-converting enzyme 2; hAO, human airway organoid; TMPRSS2, transmembrane protease serine 2.

    Techniques Used: Immunohistochemistry, Staining

    SARS-CoV-2 entry and replication are dependent on serine proteases in human airway organoids. ( A and B ) Differentiated bronchiolar ( A ) or bronchial ( B ) hAO cultures were infected at an MOI of 2. Sixteen hours ( A ) or 24 hr ( B ) postinfection they were fixed and stained for viral nucleoprotein (red). Nuclei were stained with hoechst (blue) and actin was stained using phalloidin (white). AcTub stains ciliated cells (green). Scale bars indicate 200 μm in ( A ) and 50 μm in ( B ). Representative images are shown from two independent experiments. ( C–E ) Replication kinetics of SARS-CoV-2 in bronchiolar hAO cultures pretreated with camostat or carrier (DMSO). ( C and D ) TCID50 equivalents (eq.) per mL are shown in culture medium ( C ) and lysed organoids ( D ). Circles indicate DMSO-treated organoids, whereas squares indicate camostat-treated organoids. ( E ) Live virus titers (TCID50/mL) in lysed organoids. Dotted line indicates limit of detection. ( F ) Replication kinetics of SARS-CoV-2 in 2D tracheal air–liquid interface airway cultures pretreated with camostat or carrier (DMSO). TCID50 eq./mL in apical washes are shown. Two-way ANOVA was performed for statistical analysis. Error bars indicate SEM. *p
    Figure Legend Snippet: SARS-CoV-2 entry and replication are dependent on serine proteases in human airway organoids. ( A and B ) Differentiated bronchiolar ( A ) or bronchial ( B ) hAO cultures were infected at an MOI of 2. Sixteen hours ( A ) or 24 hr ( B ) postinfection they were fixed and stained for viral nucleoprotein (red). Nuclei were stained with hoechst (blue) and actin was stained using phalloidin (white). AcTub stains ciliated cells (green). Scale bars indicate 200 μm in ( A ) and 50 μm in ( B ). Representative images are shown from two independent experiments. ( C–E ) Replication kinetics of SARS-CoV-2 in bronchiolar hAO cultures pretreated with camostat or carrier (DMSO). ( C and D ) TCID50 equivalents (eq.) per mL are shown in culture medium ( C ) and lysed organoids ( D ). Circles indicate DMSO-treated organoids, whereas squares indicate camostat-treated organoids. ( E ) Live virus titers (TCID50/mL) in lysed organoids. Dotted line indicates limit of detection. ( F ) Replication kinetics of SARS-CoV-2 in 2D tracheal air–liquid interface airway cultures pretreated with camostat or carrier (DMSO). TCID50 eq./mL in apical washes are shown. Two-way ANOVA was performed for statistical analysis. Error bars indicate SEM. *p

    Techniques Used: Infection, Staining

    SARS-CoV PP infectivity into Calu-3 cells is not altered by the insertion of the multibasic cleavage site. Titrations of SARS-CoV PPs and SARS-PRRA PPs on Calu-3 cells. Error bars indicate SEM. A representative experiment in triplicate from three independent experiments is shown. PP, pseudoparticles.
    Figure Legend Snippet: SARS-CoV PP infectivity into Calu-3 cells is not altered by the insertion of the multibasic cleavage site. Titrations of SARS-CoV PPs and SARS-PRRA PPs on Calu-3 cells. Error bars indicate SEM. A representative experiment in triplicate from three independent experiments is shown. PP, pseudoparticles.

    Techniques Used: Infection

    The SARS-CoV-2 multibasic cleavage site increases serine protease usage. ( A and B ) SARS-CoV PP and SARS-CoV-2 PP entry route on VeroE6 cells pretreated with a concentration range of camostat ( A ) or E64D ( B ) to inhibit serine proteases and cathepsins, respectively. ( C and D ) SARS-CoV PP and SARS-CoV-2 PP entry route on VeroE6-TMPRSS2 cells pretreated with a concentration range of camostat ( C ) or E64D ( D ) to inhibit serine proteases and cathepsins, respectively. T-test was performed for statistical analysis at the highest concentration. *p
    Figure Legend Snippet: The SARS-CoV-2 multibasic cleavage site increases serine protease usage. ( A and B ) SARS-CoV PP and SARS-CoV-2 PP entry route on VeroE6 cells pretreated with a concentration range of camostat ( A ) or E64D ( B ) to inhibit serine proteases and cathepsins, respectively. ( C and D ) SARS-CoV PP and SARS-CoV-2 PP entry route on VeroE6-TMPRSS2 cells pretreated with a concentration range of camostat ( C ) or E64D ( D ) to inhibit serine proteases and cathepsins, respectively. T-test was performed for statistical analysis at the highest concentration. *p

    Techniques Used: Concentration Assay

    A GFP complementation based assay for assessing coronavirus fusogenicity. ( A ) HEK-293T cells expressing an empty vector or S protein together with GFP-11-tagged beta-actin and a BFP containing a nuclear localization signal were added to cells stably expressing GFP1-10. Fusion of these two cell types allowed GFP complementation in cells expressing a nuclear BFP, facilitating easy quantification of nuclei per syncytial cell. Unfused cells only expressed BFP in the nucleus. Fusion with VeroE6 GFP1-10 cells 18 hr after addition of the fusogenic HEK-293T is shown as an example. ( B–D ) Full well scans of the complemented GFP signal 18 hr after addition of the fusogenic HEK-293T cells to Calu-3 GFP1-10 ( B ), VeroE6 GFP1-10 ( C ), and VeroE6-TMPRSS2 GFP1-10 ( D ) cells are shown. Dashed areas are enlarged next to each well. Scale bars indicate 50 μm. ( E and F ) Fusogenicity of SARS-CoV-2 S and SARS-CoV S was assessed after 18 hr by measuring the sum of all GFP+ pixels per well in VeroE6 cells ( E ) and VeroE6 TMPRSS2 cells ( F ). Statistical analysis was performed by one-way ANOVA on SARS-CoV-2 S-mediated fusion compared with SARS-CoV S. *p
    Figure Legend Snippet: A GFP complementation based assay for assessing coronavirus fusogenicity. ( A ) HEK-293T cells expressing an empty vector or S protein together with GFP-11-tagged beta-actin and a BFP containing a nuclear localization signal were added to cells stably expressing GFP1-10. Fusion of these two cell types allowed GFP complementation in cells expressing a nuclear BFP, facilitating easy quantification of nuclei per syncytial cell. Unfused cells only expressed BFP in the nucleus. Fusion with VeroE6 GFP1-10 cells 18 hr after addition of the fusogenic HEK-293T is shown as an example. ( B–D ) Full well scans of the complemented GFP signal 18 hr after addition of the fusogenic HEK-293T cells to Calu-3 GFP1-10 ( B ), VeroE6 GFP1-10 ( C ), and VeroE6-TMPRSS2 GFP1-10 ( D ) cells are shown. Dashed areas are enlarged next to each well. Scale bars indicate 50 μm. ( E and F ) Fusogenicity of SARS-CoV-2 S and SARS-CoV S was assessed after 18 hr by measuring the sum of all GFP+ pixels per well in VeroE6 cells ( E ) and VeroE6 TMPRSS2 cells ( F ). Statistical analysis was performed by one-way ANOVA on SARS-CoV-2 S-mediated fusion compared with SARS-CoV S. *p

    Techniques Used: Expressing, Plasmid Preparation, Stable Transfection

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    Article Snippet: .. Slides were incubated with the anti-necleoprotein (SARS-CoV-2) polyclonal antibody (from rabbit, Cat. 40143-T62, Sino Biological, Chesterbrook, PA, USA) in PBS buffer (1:1000 dilution) supplemented with 0.1% BSA. ..

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    Article Title: Published anti-SARS-CoV-2 in vitro hits share common mechanisms of action that synergize with antivirals
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    Staining:

    Article Title: Hepatic Failure in COVID-19: Is Iron Overload the Dangerous Trigger?
    Article Snippet: .. Samples were immersed in 10 mM sodium citrate pH 6.0, microwaved for antigen retrieval, and stained on the BenchMark ULTRA system fully automated instrument (Roche, Tucson, AZ 85755, USA) with antibodies directed against CD4 (Ventana SP35), CD8 (Ventana SP57), CD68 (Ventana KP-1), CD34 (Ventana QBEnd/10), vWF (Leica 36B11), aSMA (Ventana 760-2833), Coronavirus SARS-CoV Nucleoprotein (Sino Biological 40143-T62), and SARS Nucleocapsid protein (Novusbio NB100-56576). ..

    Article Title: Published anti-SARS-CoV-2 in vitro hits share common mechanisms of action that synergize with antivirals
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    Infection:

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    Microscopy:

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    Article Snippet: .. Then the cells were infected with SARS-CoV-2 (strain: βCoV/Korea/KCDC03/2020, NCCP43326) at a MOI of 0.01, followed with incubation at 37°C for 24 h. After fixation at room temperature for 30 min and permeabilization with 0.25% tritonX-100 for 10 min, the primary antibody, anti-SARS-CoV-2 Nucleoprotein (Sino Biological, 40143-T62), was attached at 37°C for 1.5 h. Then the secondary antibody was attached using goat-anti-rabbit-IgG-alexa-488 + Hoechst 33342, and nucleus staining was conducted at 37°C for 1 h. Then the assay plate was submitted to the Operetta microscope (Perkin Elmer) for imaging. ..

    Imaging:

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    Immunohistochemistry:

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    Article Snippet: .. For IHC, paraffin sections were rehydrated and the antigens retrieved with 10 mM citrate buffer (pH 6.0) at 100°C for 5 min. After blocking the peroxidase activity and background (IHC/ICC kit, BioVision, Milpitas, CA, USA), the serial sections were incubated with primary antibody anti-SARS-CoV nucleoprotein (Sino Biological, cat. No. 40143-T62, PA, USA) and stained following the manufacturer’s protocol (BioVision). ..

    Blocking Assay:

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    Activity Assay:

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    Sino Biological sars cov nucleoprotein np antibody rabbit pab
    In vivo <t>anti-SARS-CoV-2</t> efficacy of GS-441524 in mouse AAV-hACE2 model. AAV-hACE2 transduced mice were infected with SARA-CoV-2. The mice were administrated with either vehicle or GS-441524 25 mg/kg/day at −1 days pi (post innoculation) and the treatment was continued for a total of 8 days. Body weights were monitored every day ( A ). Lung tissues of 3 mice in each group were harvested and the viral titers were analyzed by qRT-PCR at 2 Dpi ( B ). ( C ) Representative Hematoxylin-eosin (HE) staining of lungs from hACE2 transduced mice Scale bars, 500mm (top) and 111 mm (bottom). *p values ≤ 0.05; **p values ≤ 0.005; ***p values ≤ 0.0005.
    Sars Cov Nucleoprotein Np Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov nucleoprotein np antibody rabbit pab/product/Sino Biological
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov nucleoprotein np antibody rabbit pab - by Bioz Stars, 2021-09
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    94
    Sino Biological rabbit anti sars cov np
    Mutations in the multibasic cleavage site and the adjacent serine residue (S686) abrogate S1/S2 cleavage. ( A ) Analysis of S1/S2 cleavage by S1 immunoblot of <t>SARS-CoV-2</t> S (WT), multibasic cleavage site (MBCS) mutant and S686G mutant pseudoviruses. ( B ) Quantification of S1 cleavage from four independent pseudovirus productions. ( C ) Analysis of S1/S2 cleavage by multiplex S1 (red) and S2 (green) immunoblot of SARS-CoV-2 S (WT) and S686G mutant pseudoviruses. S0 indicates uncleaved spike; S1 indicates the S1 domain of cleaved spike; VSV-N indicates VSV nucleoprotein (production control). Numbers indicate the molecular weight (kDa) of bands of the protein standard. ( D ) Quantification of S2 cleavage from four independent pseudovirus productions. Error bars indicate SD. EV = empty vector. WT = wild type. kDa = kilo dalton.
    Rabbit Anti Sars Cov Np, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti sars cov np/product/Sino Biological
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    In vivo anti-SARS-CoV-2 efficacy of GS-441524 in mouse AAV-hACE2 model. AAV-hACE2 transduced mice were infected with SARA-CoV-2. The mice were administrated with either vehicle or GS-441524 25 mg/kg/day at −1 days pi (post innoculation) and the treatment was continued for a total of 8 days. Body weights were monitored every day ( A ). Lung tissues of 3 mice in each group were harvested and the viral titers were analyzed by qRT-PCR at 2 Dpi ( B ). ( C ) Representative Hematoxylin-eosin (HE) staining of lungs from hACE2 transduced mice Scale bars, 500mm (top) and 111 mm (bottom). *p values ≤ 0.05; **p values ≤ 0.005; ***p values ≤ 0.0005.

    Journal: bioRxiv

    Article Title: Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mice Models

    doi: 10.1101/2020.10.26.353300

    Figure Lengend Snippet: In vivo anti-SARS-CoV-2 efficacy of GS-441524 in mouse AAV-hACE2 model. AAV-hACE2 transduced mice were infected with SARA-CoV-2. The mice were administrated with either vehicle or GS-441524 25 mg/kg/day at −1 days pi (post innoculation) and the treatment was continued for a total of 8 days. Body weights were monitored every day ( A ). Lung tissues of 3 mice in each group were harvested and the viral titers were analyzed by qRT-PCR at 2 Dpi ( B ). ( C ) Representative Hematoxylin-eosin (HE) staining of lungs from hACE2 transduced mice Scale bars, 500mm (top) and 111 mm (bottom). *p values ≤ 0.05; **p values ≤ 0.005; ***p values ≤ 0.0005.

    Article Snippet: Cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, Sino Biological), followed by an HRP-labeled goat anti-rabbit secondary antibody (Cat. No.: 109-035-088, Jackson ImmunoResearch Laboratories).

    Techniques: In Vivo, Mouse Assay, Infection, Quantitative RT-PCR, Staining

    The prodrug remdesivir and parent nucleoside GS-441524 potently inhibit SARS-CoV-2 replication in vitro . Vero-E6 ( A ), calu-3 ( B ) and caco-2 ( C ) were infected with SARS-CoV-2 at an MOI of 0.05 and treated with different drugs (GS-441524 and Remdesivir) at different doses (0, 0.01, 0.1, 1, 5, 10, 50 μM) for 48 h. The viral yield in the cell supernatant was then quantified by qRT-PCR. Data represented are the mean value of % inhibition of SARS-CoV-2 on cells. At the same time, the cytotoxicity at different concentrations of drugs was tested. Vero-E6 cells were infected with SARS-CoV-2 at an MOI of 0.05 and treated with different doses (0, 5, 10 μM) of the indicated compounds for 48 h. The viral RNA in the cell supernatant ( D ) and intracellular ( E ) was then quantified by qRT-PCR.

    Journal: bioRxiv

    Article Title: Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mice Models

    doi: 10.1101/2020.10.26.353300

    Figure Lengend Snippet: The prodrug remdesivir and parent nucleoside GS-441524 potently inhibit SARS-CoV-2 replication in vitro . Vero-E6 ( A ), calu-3 ( B ) and caco-2 ( C ) were infected with SARS-CoV-2 at an MOI of 0.05 and treated with different drugs (GS-441524 and Remdesivir) at different doses (0, 0.01, 0.1, 1, 5, 10, 50 μM) for 48 h. The viral yield in the cell supernatant was then quantified by qRT-PCR. Data represented are the mean value of % inhibition of SARS-CoV-2 on cells. At the same time, the cytotoxicity at different concentrations of drugs was tested. Vero-E6 cells were infected with SARS-CoV-2 at an MOI of 0.05 and treated with different doses (0, 5, 10 μM) of the indicated compounds for 48 h. The viral RNA in the cell supernatant ( D ) and intracellular ( E ) was then quantified by qRT-PCR.

    Article Snippet: Cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, Sino Biological), followed by an HRP-labeled goat anti-rabbit secondary antibody (Cat. No.: 109-035-088, Jackson ImmunoResearch Laboratories).

    Techniques: In Vitro, Infection, Quantitative RT-PCR, Inhibition

    Sensitivity and specificity of CALIBURN for SARS-CoV-2 detection. (A) Types of specimens. (B) Sensitivity of CALIBURN. Positive groups are defined as those with all three CALIBURN replicates reporting a positive signal. Presumptive positive groups are defined as those with one or two CALIBURN replicates reporting a positive signal. False negative groups are defined as those with no CALIBURN replicates reporting a positive signal. (C) Composition of specimen types in the positive, presumptive positive, and false negative groups. (D) Specificity of CALIBURN. The RT-RPA primers and crRNA probes for SARS-CoV-2 are identical to those in Figure 1 .

    Journal: ACS Chemical Biology

    Article Title: Development of a Broadly Applicable Cas12a-Linked Beam Unlocking Reaction for Sensitive and Specific Detection of Respiratory Pathogens Including SARS-CoV-2

    doi: 10.1021/acschembio.0c00840

    Figure Lengend Snippet: Sensitivity and specificity of CALIBURN for SARS-CoV-2 detection. (A) Types of specimens. (B) Sensitivity of CALIBURN. Positive groups are defined as those with all three CALIBURN replicates reporting a positive signal. Presumptive positive groups are defined as those with one or two CALIBURN replicates reporting a positive signal. False negative groups are defined as those with no CALIBURN replicates reporting a positive signal. (C) Composition of specimen types in the positive, presumptive positive, and false negative groups. (D) Specificity of CALIBURN. The RT-RPA primers and crRNA probes for SARS-CoV-2 are identical to those in Figure 1 .

    Article Snippet: Cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, Sino Biological, Inc. Beijing), followed by an HRP-labeled goat anti-rabbit secondary antibody (Cat. No.: 109-035-088, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA).

    Techniques: Recombinase Polymerase Amplification

    CALIBURN detection of multiple pathogens in SARS-CoV-2 mouse model. (A) Flowchart showing experimental design. (B) Focus forming assay to determine SARS-CoV-2 titers at different time points. LOD, limit of detection. (C) RT-PCR quantification of SARS-CoV-2 viral loads. (D,E) CALIBURN detection of SARS-CoV-2 (D) and Ad5 transcript (E). For the data in C–E, the results from three biological replicates of infection are shown as mean ± SD ( n = 3). Each data point represents the mean value of three replicates of detection. The RT-RPA primers and crRNA probes for SARS-CoV-2 are identical to those in Figure 1 .

    Journal: ACS Chemical Biology

    Article Title: Development of a Broadly Applicable Cas12a-Linked Beam Unlocking Reaction for Sensitive and Specific Detection of Respiratory Pathogens Including SARS-CoV-2

    doi: 10.1021/acschembio.0c00840

    Figure Lengend Snippet: CALIBURN detection of multiple pathogens in SARS-CoV-2 mouse model. (A) Flowchart showing experimental design. (B) Focus forming assay to determine SARS-CoV-2 titers at different time points. LOD, limit of detection. (C) RT-PCR quantification of SARS-CoV-2 viral loads. (D,E) CALIBURN detection of SARS-CoV-2 (D) and Ad5 transcript (E). For the data in C–E, the results from three biological replicates of infection are shown as mean ± SD ( n = 3). Each data point represents the mean value of three replicates of detection. The RT-RPA primers and crRNA probes for SARS-CoV-2 are identical to those in Figure 1 .

    Article Snippet: Cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, Sino Biological, Inc. Beijing), followed by an HRP-labeled goat anti-rabbit secondary antibody (Cat. No.: 109-035-088, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA).

    Techniques: Focus Forming Assay, Reverse Transcription Polymerase Chain Reaction, Infection, Recombinase Polymerase Amplification

    Specific detection of SARS-CoV-2, IAV, and IBV. (A) Schematic illustration of CALIBURN and coupled RT-RPA reaction. F, fluorophore. Q, quencher. (B) Investigation of the specificity of CALIBURN on each pathogen. For each virus, 5 μL of the extracted nucleic acids is added to each reaction without dilution. The viral copies are in the range of 10 6 –10 7 per reaction for different viruses. The RT-RPA primers are SARSCoV2-S-FWD-1/SARSCoV2-S-REV-1 for SARS-CoV-2, IAV-M-FWD/IAV-M-REV for IAV, and IBV-HA-FWD/IBV-HA-REV for IBV, respectively ( Table S1 ). The crRNA probes are SARSCoV2-S-crRNA2 for SARS-CoV-2, IAV-M-crRNA3 for IAV, and IBV-HA-crRNA4 for IBV, respectively ( Table S2 ). The data from three biological replicates are shown as mean ± SD.

    Journal: ACS Chemical Biology

    Article Title: Development of a Broadly Applicable Cas12a-Linked Beam Unlocking Reaction for Sensitive and Specific Detection of Respiratory Pathogens Including SARS-CoV-2

    doi: 10.1021/acschembio.0c00840

    Figure Lengend Snippet: Specific detection of SARS-CoV-2, IAV, and IBV. (A) Schematic illustration of CALIBURN and coupled RT-RPA reaction. F, fluorophore. Q, quencher. (B) Investigation of the specificity of CALIBURN on each pathogen. For each virus, 5 μL of the extracted nucleic acids is added to each reaction without dilution. The viral copies are in the range of 10 6 –10 7 per reaction for different viruses. The RT-RPA primers are SARSCoV2-S-FWD-1/SARSCoV2-S-REV-1 for SARS-CoV-2, IAV-M-FWD/IAV-M-REV for IAV, and IBV-HA-FWD/IBV-HA-REV for IBV, respectively ( Table S1 ). The crRNA probes are SARSCoV2-S-crRNA2 for SARS-CoV-2, IAV-M-crRNA3 for IAV, and IBV-HA-crRNA4 for IBV, respectively ( Table S2 ). The data from three biological replicates are shown as mean ± SD.

    Article Snippet: Cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, Sino Biological, Inc. Beijing), followed by an HRP-labeled goat anti-rabbit secondary antibody (Cat. No.: 109-035-088, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA).

    Techniques: Recombinase Polymerase Amplification

    LODs of different methods on detecting laboratory strains of SARS-CoV-2, IAV, and IBV. (A) RT-PCR. Positive signal is determined by valid C t values. (B) CALIBURN. Viral RNA of indicated copies is added to 5 μL RT-RPA reactions and the reaction product is directly transferred to Cas12a reaction with a final volume of 20 μL. The threshold of positive signal is set as the mean plus 3-fold standard deviation (mean+3σ) of the mock sample. (C) Fluorescent RT-RPA alone without CALIBURN reaction for detection of SARS-CoV-2. Arrows denote LOD, as defined by the lowest input viral copies resulting in positive signal readouts with more than 95% confidence. The data are shown as mean ± SD ( n = 3). The RT-RPA primers and crRNA probes are identical to those in Figure 1 .

    Journal: ACS Chemical Biology

    Article Title: Development of a Broadly Applicable Cas12a-Linked Beam Unlocking Reaction for Sensitive and Specific Detection of Respiratory Pathogens Including SARS-CoV-2

    doi: 10.1021/acschembio.0c00840

    Figure Lengend Snippet: LODs of different methods on detecting laboratory strains of SARS-CoV-2, IAV, and IBV. (A) RT-PCR. Positive signal is determined by valid C t values. (B) CALIBURN. Viral RNA of indicated copies is added to 5 μL RT-RPA reactions and the reaction product is directly transferred to Cas12a reaction with a final volume of 20 μL. The threshold of positive signal is set as the mean plus 3-fold standard deviation (mean+3σ) of the mock sample. (C) Fluorescent RT-RPA alone without CALIBURN reaction for detection of SARS-CoV-2. Arrows denote LOD, as defined by the lowest input viral copies resulting in positive signal readouts with more than 95% confidence. The data are shown as mean ± SD ( n = 3). The RT-RPA primers and crRNA probes are identical to those in Figure 1 .

    Article Snippet: Cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, Sino Biological, Inc. Beijing), followed by an HRP-labeled goat anti-rabbit secondary antibody (Cat. No.: 109-035-088, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Recombinase Polymerase Amplification, Standard Deviation

    Anti-SARS-CoV-2 efficacy of GS-441524 in an AAV-hACE2 mouse model. AAV-hACE2 transduced mice were infected with SARS-CoV-2. Mice were administrated either vehicle or GS-441524 (25 mg/kg/day) at −1 dpi and were treated for a total of 8 d. (A) Changes in body weight for either vehicle (black) or GS-441524-treated (red) mice. (B) Viral titers from lung tissue of three mice per group were harvested at 2 dpi and analyzed by FFA. *** p -value ≤ 0.0005. (C) Representative H E staining of lungs from hACE2 transduced mice. (D) Scale bars, 500 μm (top) and 100 μm (bottom).

    Journal: Journal of Medicinal Chemistry

    Article Title: Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mouse Models

    doi: 10.1021/acs.jmedchem.0c01929

    Figure Lengend Snippet: Anti-SARS-CoV-2 efficacy of GS-441524 in an AAV-hACE2 mouse model. AAV-hACE2 transduced mice were infected with SARS-CoV-2. Mice were administrated either vehicle or GS-441524 (25 mg/kg/day) at −1 dpi and were treated for a total of 8 d. (A) Changes in body weight for either vehicle (black) or GS-441524-treated (red) mice. (B) Viral titers from lung tissue of three mice per group were harvested at 2 dpi and analyzed by FFA. *** p -value ≤ 0.0005. (C) Representative H E staining of lungs from hACE2 transduced mice. (D) Scale bars, 500 μm (top) and 100 μm (bottom).

    Article Snippet: The cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No. 40143-T62, Sino Biological), followed by an HRP-labeled goat antirabbit secondary antibody (Cat. No. 109-035-088, Jackson ImmunoResearch Laboratories).

    Techniques: Mouse Assay, Infection, Staining

    Remdesivir and GS-441524 potently inhibit SARS-CoV-2 replication in vitro. Vero E6 (A), Calu-3 (B), and Caco-2 (C) were infected with SARS-CoV-2 at an MOI of 0.05 and treated with dilutions of either GS-441524 or remdesivir (0, 0.01, 0.1, 1, 5, 10, 50 μM) for 48 h. Viral yield in the cell supernatant was then quantified by qRT-PCR. Data represented are the mean value of % inhibition of SARS-CoV-2 in cells. Cytotoxicity of GS-441524 (green dots) and remdesivir (orange dots) was determined using a CCK-8 test. Vero E6 cells were infected with SARS-CoV-2 at an MOI of 0.05 and treated with dilutions of the indicated compounds for 48 h. Viral RNA in the cell supernatant (D) and pellet (E) was then quantified by qRT-PCR.

    Journal: Journal of Medicinal Chemistry

    Article Title: Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mouse Models

    doi: 10.1021/acs.jmedchem.0c01929

    Figure Lengend Snippet: Remdesivir and GS-441524 potently inhibit SARS-CoV-2 replication in vitro. Vero E6 (A), Calu-3 (B), and Caco-2 (C) were infected with SARS-CoV-2 at an MOI of 0.05 and treated with dilutions of either GS-441524 or remdesivir (0, 0.01, 0.1, 1, 5, 10, 50 μM) for 48 h. Viral yield in the cell supernatant was then quantified by qRT-PCR. Data represented are the mean value of % inhibition of SARS-CoV-2 in cells. Cytotoxicity of GS-441524 (green dots) and remdesivir (orange dots) was determined using a CCK-8 test. Vero E6 cells were infected with SARS-CoV-2 at an MOI of 0.05 and treated with dilutions of the indicated compounds for 48 h. Viral RNA in the cell supernatant (D) and pellet (E) was then quantified by qRT-PCR.

    Article Snippet: The cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No. 40143-T62, Sino Biological), followed by an HRP-labeled goat antirabbit secondary antibody (Cat. No. 109-035-088, Jackson ImmunoResearch Laboratories).

    Techniques: In Vitro, Infection, Quantitative RT-PCR, Inhibition, CCK-8 Assay

    Mutations in the multibasic cleavage site and the adjacent serine residue (S686) abrogate S1/S2 cleavage. ( A ) Analysis of S1/S2 cleavage by S1 immunoblot of SARS-CoV-2 S (WT), multibasic cleavage site (MBCS) mutant and S686G mutant pseudoviruses. ( B ) Quantification of S1 cleavage from four independent pseudovirus productions. ( C ) Analysis of S1/S2 cleavage by multiplex S1 (red) and S2 (green) immunoblot of SARS-CoV-2 S (WT) and S686G mutant pseudoviruses. S0 indicates uncleaved spike; S1 indicates the S1 domain of cleaved spike; VSV-N indicates VSV nucleoprotein (production control). Numbers indicate the molecular weight (kDa) of bands of the protein standard. ( D ) Quantification of S2 cleavage from four independent pseudovirus productions. Error bars indicate SD. EV = empty vector. WT = wild type. kDa = kilo dalton.

    Journal: bioRxiv

    Article Title: Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

    doi: 10.1101/2021.01.22.427802

    Figure Lengend Snippet: Mutations in the multibasic cleavage site and the adjacent serine residue (S686) abrogate S1/S2 cleavage. ( A ) Analysis of S1/S2 cleavage by S1 immunoblot of SARS-CoV-2 S (WT), multibasic cleavage site (MBCS) mutant and S686G mutant pseudoviruses. ( B ) Quantification of S1 cleavage from four independent pseudovirus productions. ( C ) Analysis of S1/S2 cleavage by multiplex S1 (red) and S2 (green) immunoblot of SARS-CoV-2 S (WT) and S686G mutant pseudoviruses. S0 indicates uncleaved spike; S1 indicates the S1 domain of cleaved spike; VSV-N indicates VSV nucleoprotein (production control). Numbers indicate the molecular weight (kDa) of bands of the protein standard. ( D ) Quantification of S2 cleavage from four independent pseudovirus productions. Error bars indicate SD. EV = empty vector. WT = wild type. kDa = kilo dalton.

    Article Snippet: Spike was stained using polyclonal rabbit-anti-SARS-CoV S1 (1:1000, Sino Biological), mouse-anti-SARS-CoV-2 S2 (1:1000, Genetex), SARS-CoV-2 nucleoprotein was stained using rabbit-anti-SARS-CoV NP (1:1000, Sino Biological) and VSV nucleoprotein was stained using mouse-anti-VSV-N (1:1000, Absolute Antibody) followed by infrared-labelled secondary antibodies (1:20,000; Licor).

    Techniques: Mutagenesis, Multiplex Assay, Molecular Weight, Plasmid Preparation

    A 2D air-liquid interface human airway organoid model for SARS-CoV-2 propagation. ( A ) Human airway organoids were dissociated and plated onto 12 mm transwell inserts. After an 8-12 week differentiation period at air-liquid interface cultures contained ciliated, non-ciliated and basal cells as shown on a hematoxylin-eosin stain. ( B ) Air-exposed cells, but not basal cells, expressed the priming protease TMPRSS2 as shown by immunohistochemistry. ( C ) Immunofluorescent staining indicated that in these cultures, ciliated cells (acetylated tubulin+ or AcTUB+ cells) were infected by SARS-CoV-2. ( D and E ) At 5 days post-infection, whole-well confocal imaging indicated the infection was widespread ( D ) and cytopathic effects, including cilia damage ( D and E ) and syncytial cells ( E ) were visible. Scale bars indicate 20μm in A, B, C; 2mm in D; and 100μm in E.

    Journal: bioRxiv

    Article Title: Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

    doi: 10.1101/2021.01.22.427802

    Figure Lengend Snippet: A 2D air-liquid interface human airway organoid model for SARS-CoV-2 propagation. ( A ) Human airway organoids were dissociated and plated onto 12 mm transwell inserts. After an 8-12 week differentiation period at air-liquid interface cultures contained ciliated, non-ciliated and basal cells as shown on a hematoxylin-eosin stain. ( B ) Air-exposed cells, but not basal cells, expressed the priming protease TMPRSS2 as shown by immunohistochemistry. ( C ) Immunofluorescent staining indicated that in these cultures, ciliated cells (acetylated tubulin+ or AcTUB+ cells) were infected by SARS-CoV-2. ( D and E ) At 5 days post-infection, whole-well confocal imaging indicated the infection was widespread ( D ) and cytopathic effects, including cilia damage ( D and E ) and syncytial cells ( E ) were visible. Scale bars indicate 20μm in A, B, C; 2mm in D; and 100μm in E.

    Article Snippet: Spike was stained using polyclonal rabbit-anti-SARS-CoV S1 (1:1000, Sino Biological), mouse-anti-SARS-CoV-2 S2 (1:1000, Genetex), SARS-CoV-2 nucleoprotein was stained using rabbit-anti-SARS-CoV NP (1:1000, Sino Biological) and VSV nucleoprotein was stained using mouse-anti-VSV-N (1:1000, Absolute Antibody) followed by infrared-labelled secondary antibodies (1:20,000; Licor).

    Techniques: Staining, Immunohistochemistry, Infection, Imaging

    SARS-CoV-2 propagation in Calu-3 cells efficiently prevents SARS-CoV-2 cell culture adaptation. ( A ) Deep-sequencing analysis of Calu-3 passage 2 virus from a VeroE6 passage 1. ( B ) Deep-sequencing analysis of Calu-3 passage 3 virus from the Calu-3 passage 2 in A. ( C ) Deep-sequencing analysis of Calu-3 passage 3 virus grown from a VeroE6 passage 2 stock ( Figure 1A ). Deep-sequencing analysis of Calu-3 passage 5 virus from a Calu-3 passage 3 stock in C. In each graph the amino acid sequence logo of the multibasic cleavage site is shown.

    Journal: bioRxiv

    Article Title: Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

    doi: 10.1101/2021.01.22.427802

    Figure Lengend Snippet: SARS-CoV-2 propagation in Calu-3 cells efficiently prevents SARS-CoV-2 cell culture adaptation. ( A ) Deep-sequencing analysis of Calu-3 passage 2 virus from a VeroE6 passage 1. ( B ) Deep-sequencing analysis of Calu-3 passage 3 virus from the Calu-3 passage 2 in A. ( C ) Deep-sequencing analysis of Calu-3 passage 3 virus grown from a VeroE6 passage 2 stock ( Figure 1A ). Deep-sequencing analysis of Calu-3 passage 5 virus from a Calu-3 passage 3 stock in C. In each graph the amino acid sequence logo of the multibasic cleavage site is shown.

    Article Snippet: Spike was stained using polyclonal rabbit-anti-SARS-CoV S1 (1:1000, Sino Biological), mouse-anti-SARS-CoV-2 S2 (1:1000, Genetex), SARS-CoV-2 nucleoprotein was stained using rabbit-anti-SARS-CoV NP (1:1000, Sino Biological) and VSV nucleoprotein was stained using mouse-anti-VSV-N (1:1000, Absolute Antibody) followed by infrared-labelled secondary antibodies (1:20,000; Licor).

    Techniques: Cell Culture, Sequencing

    Schematic workflow for the production of SARS-CoV-2 stocks on 2D air-liquid interface differentiated airway organoids. Step 1. 3D self-renewing airway organoids are grown from human lung tissue. Next, these are dissociated to single cells and differentiated at air-liquid interface for 4-12 weeks. Step 2. Differentiated cultures are infected at a multiplicity of infection of 0.05 and washed daily for 5 days. The washes from day 2-5 are collected and stored at 4°C. Step 3. Virus collections are cleared by centrifugation and filtered to remove debris larger than 0.45 μm. Next, the medium is exchanged three times using Amicon columns to remove cytokines and debris smaller than 100 kDa. Purified virus preparations are then stored at −80°C in aliquots. Step 4. Stocks can be characterized using plaque assays, Sanger sequencing and deep-sequencing. Created with BioRender.com .

    Journal: bioRxiv

    Article Title: Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

    doi: 10.1101/2021.01.22.427802

    Figure Lengend Snippet: Schematic workflow for the production of SARS-CoV-2 stocks on 2D air-liquid interface differentiated airway organoids. Step 1. 3D self-renewing airway organoids are grown from human lung tissue. Next, these are dissociated to single cells and differentiated at air-liquid interface for 4-12 weeks. Step 2. Differentiated cultures are infected at a multiplicity of infection of 0.05 and washed daily for 5 days. The washes from day 2-5 are collected and stored at 4°C. Step 3. Virus collections are cleared by centrifugation and filtered to remove debris larger than 0.45 μm. Next, the medium is exchanged three times using Amicon columns to remove cytokines and debris smaller than 100 kDa. Purified virus preparations are then stored at −80°C in aliquots. Step 4. Stocks can be characterized using plaque assays, Sanger sequencing and deep-sequencing. Created with BioRender.com .

    Article Snippet: Spike was stained using polyclonal rabbit-anti-SARS-CoV S1 (1:1000, Sino Biological), mouse-anti-SARS-CoV-2 S2 (1:1000, Genetex), SARS-CoV-2 nucleoprotein was stained using rabbit-anti-SARS-CoV NP (1:1000, Sino Biological) and VSV nucleoprotein was stained using mouse-anti-VSV-N (1:1000, Absolute Antibody) followed by infrared-labelled secondary antibodies (1:20,000; Licor).

    Techniques: Infection, Centrifugation, Purification, Sequencing

    The SARS-CoV-2 multibasic cleavage site and the adjacent serine residue (S686) enhance infectivity and serine protease mediated entry on Calu-3 and VeroE6-TMPRSS2 cells. ( A-B ) SARS-CoV-2 (WT), multibasic cleavage site (MBCS) mutant and S686G pseudovirus infectious titers on ( A ) VeroE6 and ( B ) Calu-3 cells. ( C ) Fold change in SARS-CoV-2, MBCS mutant and S686G pseudovirus infectious titers on Calu-3 cells over infectious titers on VeroE6 cells. ( D ) SARS-CoV-2, MBCS mutant and S686G pseudovirus infectious titers on VeroE6-TMPRSS2 cells. ( E ) Fold change in SARS-CoV-2, MBCS mutant and S686G pseudovirus infectious titers on VeroE6-TMPRSS2 cells over infectious titers on VeroE6 cells. One-way ANOVA was performed for statistical analysis comparing all groups with WT. ( F-I ) SARS-CoV-2, MBCS mutant and S686G pseudovirus entry into ( F and G ) VeroE6 cells or ( H and I ) VeroE6-TMPRSS2 cells pre-treated with a concentration range of either ( F and H ) camostat mesylate or ( G and I ) E64D. Two-way ANOVA, followed by a bonferroni post hoc test was performed for statistical analysis comparing all groups to WT. WT pseudovirus entry into VeroE6 cells treated with 10μM E64D was significantly different from del-RRAR, R682A, R685A and S686G pseudovirus entry. * indicates statistical significance (p

    Journal: bioRxiv

    Article Title: Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

    doi: 10.1101/2021.01.22.427802

    Figure Lengend Snippet: The SARS-CoV-2 multibasic cleavage site and the adjacent serine residue (S686) enhance infectivity and serine protease mediated entry on Calu-3 and VeroE6-TMPRSS2 cells. ( A-B ) SARS-CoV-2 (WT), multibasic cleavage site (MBCS) mutant and S686G pseudovirus infectious titers on ( A ) VeroE6 and ( B ) Calu-3 cells. ( C ) Fold change in SARS-CoV-2, MBCS mutant and S686G pseudovirus infectious titers on Calu-3 cells over infectious titers on VeroE6 cells. ( D ) SARS-CoV-2, MBCS mutant and S686G pseudovirus infectious titers on VeroE6-TMPRSS2 cells. ( E ) Fold change in SARS-CoV-2, MBCS mutant and S686G pseudovirus infectious titers on VeroE6-TMPRSS2 cells over infectious titers on VeroE6 cells. One-way ANOVA was performed for statistical analysis comparing all groups with WT. ( F-I ) SARS-CoV-2, MBCS mutant and S686G pseudovirus entry into ( F and G ) VeroE6 cells or ( H and I ) VeroE6-TMPRSS2 cells pre-treated with a concentration range of either ( F and H ) camostat mesylate or ( G and I ) E64D. Two-way ANOVA, followed by a bonferroni post hoc test was performed for statistical analysis comparing all groups to WT. WT pseudovirus entry into VeroE6 cells treated with 10μM E64D was significantly different from del-RRAR, R682A, R685A and S686G pseudovirus entry. * indicates statistical significance (p

    Article Snippet: Spike was stained using polyclonal rabbit-anti-SARS-CoV S1 (1:1000, Sino Biological), mouse-anti-SARS-CoV-2 S2 (1:1000, Genetex), SARS-CoV-2 nucleoprotein was stained using rabbit-anti-SARS-CoV NP (1:1000, Sino Biological) and VSV nucleoprotein was stained using mouse-anti-VSV-N (1:1000, Absolute Antibody) followed by infrared-labelled secondary antibodies (1:20,000; Licor).

    Techniques: Infection, Mutagenesis, Concentration Assay

    Multibasic cleavage site mutations and the adjacent serine residue (S686) impair spike protein fusogenicity. ( A-C ) Fusogenicity of wild type SARS-CoV-2 spike and spike mutants was assessed after 18 hours by measuring the sum of all GFP+ pixels per well in a GFP-complementation fusion assay on VeroE6-GFP1-10 ( A ), VeroE6-TMPRSS2-GFP1-10 ( B ), and Calu-3-GFP1-10 ( C ) cells. The experiment was performed in triplicate. A representative experiment from two independent experiments is shown. Statistical analysis was performed by one-way ANOVA. * indicates a significant difference compared to WT (P

    Journal: bioRxiv

    Article Title: Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

    doi: 10.1101/2021.01.22.427802

    Figure Lengend Snippet: Multibasic cleavage site mutations and the adjacent serine residue (S686) impair spike protein fusogenicity. ( A-C ) Fusogenicity of wild type SARS-CoV-2 spike and spike mutants was assessed after 18 hours by measuring the sum of all GFP+ pixels per well in a GFP-complementation fusion assay on VeroE6-GFP1-10 ( A ), VeroE6-TMPRSS2-GFP1-10 ( B ), and Calu-3-GFP1-10 ( C ) cells. The experiment was performed in triplicate. A representative experiment from two independent experiments is shown. Statistical analysis was performed by one-way ANOVA. * indicates a significant difference compared to WT (P

    Article Snippet: Spike was stained using polyclonal rabbit-anti-SARS-CoV S1 (1:1000, Sino Biological), mouse-anti-SARS-CoV-2 S2 (1:1000, Genetex), SARS-CoV-2 nucleoprotein was stained using rabbit-anti-SARS-CoV NP (1:1000, Sino Biological) and VSV nucleoprotein was stained using mouse-anti-VSV-N (1:1000, Absolute Antibody) followed by infrared-labelled secondary antibodies (1:20,000; Licor).

    Techniques: Single Vesicle Fusion Assay

    SARS-CoV-2 rapidly acquires multibasic cleavage site mutations when propagated on VeroE6 cells. ( A-C ) Deep-sequencing analysis of VeroE6 passage 2 ( A ), passage 3 ( B ) and passage 4 ( C ) virus stocks. In each graph the amino acid sequence logo of the multibasic cleavage site is shown. ( D-F ) Sanger sequencing chromatograms of VeroE6 passage 2 ( D ), passage 3 ( E ), and passage 4 ( F ) viruses. Multibasic cleavage site mutations identified by deep-sequencing are indicated with arrows. Translated sequences are indicated below Sanger reads. ( G ) Plaque size analysis of VeroE6 passage 2-4 virus stocks on VeroE6 cells. Red arrow heads indicate small plaques.

    Journal: bioRxiv

    Article Title: Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

    doi: 10.1101/2021.01.22.427802

    Figure Lengend Snippet: SARS-CoV-2 rapidly acquires multibasic cleavage site mutations when propagated on VeroE6 cells. ( A-C ) Deep-sequencing analysis of VeroE6 passage 2 ( A ), passage 3 ( B ) and passage 4 ( C ) virus stocks. In each graph the amino acid sequence logo of the multibasic cleavage site is shown. ( D-F ) Sanger sequencing chromatograms of VeroE6 passage 2 ( D ), passage 3 ( E ), and passage 4 ( F ) viruses. Multibasic cleavage site mutations identified by deep-sequencing are indicated with arrows. Translated sequences are indicated below Sanger reads. ( G ) Plaque size analysis of VeroE6 passage 2-4 virus stocks on VeroE6 cells. Red arrow heads indicate small plaques.

    Article Snippet: Spike was stained using polyclonal rabbit-anti-SARS-CoV S1 (1:1000, Sino Biological), mouse-anti-SARS-CoV-2 S2 (1:1000, Genetex), SARS-CoV-2 nucleoprotein was stained using rabbit-anti-SARS-CoV NP (1:1000, Sino Biological) and VSV nucleoprotein was stained using mouse-anti-VSV-N (1:1000, Absolute Antibody) followed by infrared-labelled secondary antibodies (1:20,000; Licor).

    Techniques: Sequencing