sars-cov-2 (2019-ncov) nucleocapsid antibody, rabbit mab  (Sino Biological)

Journal: EBioMedicine

Article Title: Q493K and Q498H substitutions in Spike promote adaptation of SARS-CoV-2 in mice

doi: 10.1016/j.ebiom.2021.103381

Figure Lengend Snippet: Generation of mouse-adapted SARS-CoV-2 WBP-1. Wild type (WT) Wuhan-Hu-1 was passaged in old and young mice to obtain WBP-1 that was adapted to cause respiratory disease in mice. (a) Hematoxylin and eosin (H&E) staining of lung sections from mice infected with virus obtained through different passages (P2, P5, P8, P11) of SARS-CoV-2. Scale bars, 100 μm. (b) Viral copies were detected through qRT-PCR at three days post infection (dpi) in the lungs. (c) Five clonal isolates were obtained from the P11 virus pool by three rounds of plaque purification in Vero cells. The strain #2 that showed a high mortality rate in mice was defined as WBP-1. (d) Location of the mutations and deletion in the genome of WBP-1. WBP-1 obtained five synonymous (purple triangles), five nonsynonymous (red triangles), and a 740 bp deletion (black line). (e) Mutational frequency during SARS-CoV-2 experimental passage in pooled mouse groups. Single nucleotide variants are represented along the genome for the mice of passage 5, passage 8, passage 11 and WBP-1 virus.

Article Snippet: For immunohistochemistry, fixed tissues were paraffin-embedded, sectioned, and stained with rabbit-anti ACE2 (Abcam, ab108252, 1:200), SARS-CoV-2 nucleocapsid antibody (Sino Biological, China, 40588-R001, 1:200).

Techniques: Staining, Infection, Quantitative RT-PCR, Purification

Journal: EBioMedicine

Article Title: Q493K and Q498H substitutions in Spike promote adaptation of SARS-CoV-2 in mice

doi: 10.1016/j.ebiom.2021.103381

Figure Lengend Snippet: Characterization of mouse-adapted SARS-CoV-2 WBP-1 in mice. (a, b) Groups of mice ( n = 5) were intranasally infected with 10-fold serial dilutions of WBP-1 virus. The infected mice were observed for weight loss and survival for 10 dpi. (c- f) Mice ( n = 6) were intranasally infected with 2 LD 50 of the WBP-1 virus and mice in the control group were mock-infected with DMEM. (c) Viral RNA copies were determined through qRT-PCR at 3 and 5 dpi in the heart, liver, lung, kidney, brain, intestine, trachea, turbinate, and spleen of WBP-1. (d) Viral titers in the lung were determined through plaque assays. (e) Cytokine (IL1β, TNFα, MCP1, and IL10) mRNA levels in the lung were evaluated at 3 and 5 dpi. Statistical significance was analyzed by unpaired Student's t tests. * p < 0.05; ** p < 0.01; *** p < 0.001 **** p < 0.0001. (f) Hematoxylin and eosin (H&E) staining of lung sections from mice infected Mouse-adapted WBP-1 virus (Exfoliated necrotic cells, black arrow; Mononuclear lymphocytes, green arrow; Inflammatory exudate, red arrow; Vascular thrombosis, blue arrow). Scale bars (black), 100 μm. Scale bars (green), 50 μm. (g) Immunohistochemical detection of viral antigen using anti-Spike protein Rabbit monoclonal antibody in bronchi and alveoli. Scale bars (black), 100 μm. Scale bars (green), 50 μm.

Article Snippet: For immunohistochemistry, fixed tissues were paraffin-embedded, sectioned, and stained with rabbit-anti ACE2 (Abcam, ab108252, 1:200), SARS-CoV-2 nucleocapsid antibody (Sino Biological, China, 40588-R001, 1:200).

Techniques: Infection, Quantitative RT-PCR, Staining, Immunohistochemical staining

Journal: EBioMedicine

Article Title: Q493K and Q498H substitutions in Spike promote adaptation of SARS-CoV-2 in mice

doi: 10.1016/j.ebiom.2021.103381

Figure Lengend Snippet: WBP-1 virus using mouse ACE2 to gain access to cells was determined by immunofluorescence. Hela cells with transfection of empty vector (Flag), hACE2, or mACE2 plasmids were infected with WT Wuhan-Hu-1 or mouse-adapted WBP-1. ACE2 expression was detcted using rabbit anti-ACE2 IgG followed by CoraLite594-conjugated goat anti-rabbit IgG (H+L). SARS-CoV-2 nucleocapsid expression was detected using mouse anti-NP IgG followed by CoraLite488-conjugated Affinipure goat anti-mouse IgG(H+L). Nuclei were stained with DAPI. Scale bars, 200 μm.

Article Snippet: For immunohistochemistry, fixed tissues were paraffin-embedded, sectioned, and stained with rabbit-anti ACE2 (Abcam, ab108252, 1:200), SARS-CoV-2 nucleocapsid antibody (Sino Biological, China, 40588-R001, 1:200).

Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Infection, Expressing, Staining

Journal: EBioMedicine

Article Title: Q493K and Q498H substitutions in Spike promote adaptation of SARS-CoV-2 in mice

doi: 10.1016/j.ebiom.2021.103381

Figure Lengend Snippet: Mouse-adapted WBP-1 virus RBD enhance interactive affinities with mACE2. (a-d) Modelling of SARS-CoV-2 RBD–ACE2 interface. (a) Wuhan-Hu-1 RBD interacts with of human ACE2. (b) WBP-1 RBD (Q493K/Q498H-RBD) interacts human ACE2. (c) Modelling of Wuhan-Hu-1 RBD and mouse ACE2. (d) Modelling of WBP-1 RBD (Q493K/Q498H-RBD) shows interaction with mouse ACE2. (e-l) The binding affinities of the RBD of WBP-1 and mACE2 or hACE2 were determined through BLI experiments. The sensors were dipped in mACE2-hFC and functionalized sensorgrams captured upon incubation of Q493K-RBD (e), Q498H-RBD (f), Q493K/Q498H-RBD (g), and WT-RBD (h) at 6.25 (black), 12.5 (blue), 25 (green), and 50 nM (red). The sensors were dipped in hACE2-hFC and functionalized sensorgrams captured upon incubation of Q493K-RBD (i), Q498H-RBD (j), Q493K/Q498H-RBD (k), and WT-RBD (l) at 50 (black), 100 (blue), 200 (green), and 400 nM (red).

Article Snippet: For immunohistochemistry, fixed tissues were paraffin-embedded, sectioned, and stained with rabbit-anti ACE2 (Abcam, ab108252, 1:200), SARS-CoV-2 nucleocapsid antibody (Sino Biological, China, 40588-R001, 1:200).

Techniques: Binding Assay, Incubation

Journal: EBioMedicine

Article Title: Q493K and Q498H substitutions in Spike promote adaptation of SARS-CoV-2 in mice

doi: 10.1016/j.ebiom.2021.103381

Figure Lengend Snippet: R848 inhibits SARS-CoV-2 in vitro and in vivo . (a) Caco2 cells were treated with R848 (0-20 μM) for 24 h before infection with viruses at a multiplicity of infection (MOI) of 1. Infected cells were further incubated in the R848. Samples were collected at 48 and 72 h post infection (hpi) and viral titers were determined using plaque assays. (b-e) ( n = 10) Mice were intraperitoneally injected with R848 after immediately intranasal infection with the WBP-1 virus. Infected mice were further treated with the R848 or PBS once a day for four consecutive days. Mice were monitored for weight loss (b) and survival (c) for 10 days. (d) Viral loads in mice lung, trachea and turbinate were determined at 5 dpi. (e) Hematoxylin and eosin (H&E) staining of lung sections from PBS or R848 treated mice. Scale bars (black), 100 μm. Scale bars (green), 50 μm. (a) and (d), Log transformed data analyzed by Two-way ANOVA followed by Sidak's multiple comparisons. The line represents the mean and error bars represent standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001 **** p < 0.0001.

Article Snippet: For immunohistochemistry, fixed tissues were paraffin-embedded, sectioned, and stained with rabbit-anti ACE2 (Abcam, ab108252, 1:200), SARS-CoV-2 nucleocapsid antibody (Sino Biological, China, 40588-R001, 1:200).

Techniques: In Vitro, In Vivo, Infection, Incubation, Injection, Staining, Transformation Assay, Standard Deviation