Journal: Microbiology Spectrum

Article Title: Monitoring SARS-CoV-2 Infection Using a Double Reporter-Expressing Virus

doi: 10.1128/spectrum.02379-22

Figure Lengend Snippet: Generation of a bireporter rSARS-CoV-2 expressing mCherry and Nluc (rSARS-CoV-2/mCherry-Nluc). (A) Schematic representation of the rSARS-CoV-2/mCherry-Nluc viral genome: SARS-CoV-2 structural, nonstructural, and accessory open reading frame (ORF) proteins are indicated in white boxes. mCherry (red), Nluc (blue), and the PTV-1 2A autoproteolytic sequence (black) were inserted in front of the viral N protein. NCR, noncoding region. (B) mCherry expression and immunofluorescence assays: Vero E6 cells (1.2 × 10 6 cells/well, 6-well format, triplicates) were mock infected or infected (MOI, 0.01) with rSARS-CoV-2 WT, rSARS-CoV-2/mCherry, rSARS-CoV-2/Nluc, or rSARS-CoV-2/mCherry-Nluc. Cells were fixed in 10% neutral buffered formalin 24 hpi before directly visualizing mCherry expression under a fluorescence microscope or the viral N protein using a specific 1C7C7 MAb. Cell nuclei were strained with DAPI. Representative images are shown. Scale bars = 100 μm. Magnification = ×20. (C) Western blots: Vero E6 cells (1.2 × 10 6 cells/well, 6-well format, triplicates) were mock infected or infected (MOI, 0.01) with rSARS-CoV-2 WT, rSARS-CoV-2/mCherry, rSARS-CoV-2/Nluc, or rSARS-CoV-2/mCherry-Nluc. At 24 hpi, cells were collected and protein expression in cell lysates were evaluated by Western blotting using specific antibodies against SARS-CoV-2 N protein or the mCherry and Nluc reporter proteins. Tubulin was included as a loading control. The molecular mass of proteins is indicated in kilodaltons (kDa) on the left. (D) Deep sequencing analysis of reporter-expressing rSARS-CoV-2: the nonreference allele frequency of rSARS-CoV-2/mCherry (top), rSARS-CoV-2/Nluc (middle), and rSARS-CoV-2/mCherry-Nluc (bottom) was calculated by comparing the short reads to the respective reference SARS-CoV-2 WA-1 viral genome (MN985325.1). Nonreference alleles present in less than 10% of reads are not shown (dotted line), and the nonreference allele frequency that is greater than 10% is indicated.

Article Snippet: Membranes were blocked for 1 h with 5% dried skim milk in 0.1% Tween 20 PBS (T-PBS) and incubated at 4°C overnight with the following specific primary MAbs or polyclonal antibodies (PAbs): N (mouse MAb 1C7C7), mCherry (rabbit Pab; Raybiotech), and Nluc (rabbit Pab, Promega).

Techniques: Expressing, Sequencing, Immunofluorescence, Infection, Fluorescence, Microscopy, Western Blot

Journal: Microbiology Spectrum

Article Title: Monitoring SARS-CoV-2 Infection Using a Double Reporter-Expressing Virus

doi: 10.1128/spectrum.02379-22

Figure Lengend Snippet: In vitro characterization of the bireporter rSARS-CoV-2/mCherry-Nluc virus. (A) Viral growth kinetics: viral titers (PFU/ml) in the cell culture supernatants of Vero E6 cells (1.2 × 10 6 cells/well, 6-well format, triplicates) infected (MOI, 0.01) with rSARS-CoV-2 WT (WT), rSARS-CoV-2/mCherry (mCherry), rSARS-CoV-2/Nluc (Nluc), or rSARS-CoV-2/mCherry-Nluc (mCherry-Nluc) at the indicated time points postinfection were determined by plaque assay. Data represent the mean values and SD of triplicates. LOD, limit of detection. (B) Nluc activity: Nluc activity in the cell culture supernatants obtained from the experiment in panel A is represented in relative light units (RLU). (C) mCherry expression kinetics: Vero E6 cells (1.2 × 10 6 cells/well, 6-well format, triplicates) were infected (MOI, 0.01) with rSARS-CoV-2 WT, rSARS-CoV-2/mCherry, rSARS-CoV-2/Nluc, or rSARS-CoV-2/mCherry-Nluc and mCherry expression was directly visualized under a fluorescence microscope at the indicated times postinfection. Representative images are shown. Scale bars = 300 μm. Magnification = ×10. (D) Plaque phenotype: viral plaques from Vero E6 cells (2 × 10 5 cells/well, 24-well plate format, triplicates) infected with rSARS-CoV-2 WT, rSARS-CoV-2/mCherry, rSARS-CoV-2/Nluc, or rSARS-CoV-2/mCherry-Nluc at 3 dpi were observed under a fluorescence imaging system (first column, red filter), fluorescently stained with an antibody against Nluc (second column, FITC), or immunostaining with an antibody against the viral N protein (third column, N). White arrowheads depict the overlapping signal of mCherry fluorescence (left), Nluc bioluminescence (middle), and immunostaining of the viral N protein (right) in Vero E6 cells infected with rSARS-CoV-2/mCherry, rSARS-CoV-2/Nluc, or rSARS-CoV-2/mCherry-Nluc. *, P < 0.05; **, P < 0.01; ns, not significant.

Article Snippet: Membranes were blocked for 1 h with 5% dried skim milk in 0.1% Tween 20 PBS (T-PBS) and incubated at 4°C overnight with the following specific primary MAbs or polyclonal antibodies (PAbs): N (mouse MAb 1C7C7), mCherry (rabbit Pab; Raybiotech), and Nluc (rabbit Pab, Promega).

Techniques: In Vitro, Cell Culture, Infection, Plaque Assay, Activity Assay, Expressing, Fluorescence, Microscopy, Imaging, Staining, Immunostaining

Journal: Microbiology Spectrum

Article Title: Monitoring SARS-CoV-2 Infection Using a Double Reporter-Expressing Virus

doi: 10.1128/spectrum.02379-22

Figure Lengend Snippet: Bireporter-based microneutralization assay to identify NAbs and antivirals against SARS-CoV-2. (A) A bireporter microneutralization assay to identify NAbs: three-fold serial dilutions of the SARS-CoV-2 1212C2 hMAb (starting concentration of 500 ng) were prepared in postinfection media and incubated with 100–200 PFU/well of rSARS-CoV-2 WT (WT), rSARS-CoV-2/mCherry (mCherry), rSARS-CoV-2/Nluc (Nluc), or rSARS-CoV-2/mCherry-Nluc (mCherry-Nluc) for 1 h at RT. Vero E6 cells (96-well plate format, 4 × 10 4 cells/well, quadruplicates) were infected and incubated with the virus-antibody mixture at 37 °C for 24 h. Viral neutralization was determined by immunostaining using an anti-N protein MAb (1C7C7) for rSARS-CoV-2/WT (left) or by fluorescence expression for rSARS-CoV-2/mCherry and rSARS-CoV-2/mCherry-Nluc (middle), or bioluminescence for rSARS-CoV-2/Nluc and rSARS-CoV-2/mCherry-Nluc (right) using a microplate reader. The 50% neutralization titer (NT 50 ) was calculated using sigmoidal dose-response curves on GraphPad Prism. Viral neutralization was normalized to wells containing infected cells without the 1212C2 hMAb. The dotted line indicates 50% virus inhibition. Data are represented by the mean values and SD of quadruplicates. (B) A bireporter microneutralization assay to assess antivirals: vero E6 cells (96-well plate format, 4 × 10 4 cells/well, quadruplicates) were infected with 100 to 200 PFU of rSARS-CoV-2/WT, or reporter viruses expressing mCherry, Nluc, or mCherry-Nluc. After 1 h viral absorption, cells were incubated in postinfection media containing 3-fold serial dilutions of remdesivir (starting concentration of 100 μM). Viral inhibition was determined by immunostaining using an anti-N protein MAb (1C7C7) for rSARS-CoV-2/WT (left) or by fluorescence expression for rSARS-CoV-2/mCherry and rSARS-CoV-2/mCherry-Nluc (middle), or bioluminescence for rSARS-CoV-2/Nluc and rSARS-CoV-2/mCherry-Nluc (right) using a microplate reader. The 50% effective concentration (EC 50 ) was calculated using sigmoidal dose-response curves on GraphPad Prism. Viral inhibition was normalized to wells containing infected cells without remdesivir. The dotted line indicates the 50% virus inhibition. The data are represented by the mean values and SD of quadruplicates.

Article Snippet: Membranes were blocked for 1 h with 5% dried skim milk in 0.1% Tween 20 PBS (T-PBS) and incubated at 4°C overnight with the following specific primary MAbs or polyclonal antibodies (PAbs): N (mouse MAb 1C7C7), mCherry (rabbit Pab; Raybiotech), and Nluc (rabbit Pab, Promega).

Techniques: Microneutralization Assay, Concentration Assay, Incubation, Infection, Neutralization, Immunostaining, Fluorescence, Expressing, Inhibition

Journal: Microbiology Spectrum

Article Title: Monitoring SARS-CoV-2 Infection Using a Double Reporter-Expressing Virus

doi: 10.1128/spectrum.02379-22

Figure Lengend Snippet: Virulence of rSARS-CoV-2/mCherry-Nluc in K18 hACE2 transgenic mice: 4- to 6-week-old female K18 hACE2 transgenic mice ( n = 4) were mock infected or intranasally inoculated with 10 5 PFU/mouse of rSARS-CoV-2 WT (WT), rSARS-CoV-2/mCherry (mCherry), rSARS-CoV-2/Nluc (Nluc), or the bireporter rSARS-CoV-2/mCherry-Nluc (mCherry-Nluc). A group of 4- to 6-week-old female K18 hACE2 transgenic mice ( n = 4) were also coinfected with rSARS-CoV-2/mCherry and rSARS-CoV-2/Nluc (mCherry + Nluc). Body weight loss (A) and survival (B) of mice were monitored for 12 days after viral infection.

Article Snippet: Membranes were blocked for 1 h with 5% dried skim milk in 0.1% Tween 20 PBS (T-PBS) and incubated at 4°C overnight with the following specific primary MAbs or polyclonal antibodies (PAbs): N (mouse MAb 1C7C7), mCherry (rabbit Pab; Raybiotech), and Nluc (rabbit Pab, Promega).

Techniques: Transgenic Assay, Infection

Journal: Microbiology Spectrum

Article Title: Monitoring SARS-CoV-2 Infection Using a Double Reporter-Expressing Virus

doi: 10.1128/spectrum.02379-22

Figure Lengend Snippet: In vivo kinetics of rSARS-CoV-2/mCherry-Nluc in K18 hACE2 transgenic mice: 4- to 6-week-old female K18 hACE2 transgenic mice ( n = 4) were mock infected or infected intranasally with 10 5 PFU/mouse of rSARS-CoV-2 WT, rSARS-CoV-2/mCherry, rSARS-CoV-2/Nluc, rSARS-CoV-2/mCherry + rSARS-CoV-2/Nluc, or with rSARS-CoV-2/mCherry-Nluc (mCherry-Nluc). Nluc activity in the whole mouse at the indicated dpi was evaluated with an Ami HT in vivo imaging system. Representative images of the same mouse at 1, 2, 4, and 6 dpi are shown (A). Means and SD of the radiance (number of photons per second per square centimeter per steradian [p/second/cm 2 /sr]) and bioluminescence (total flux [log 10 photons per second (p/s)]) over each mouse are shown (B). ***, P < 0.001; ns, not significant.

Article Snippet: Membranes were blocked for 1 h with 5% dried skim milk in 0.1% Tween 20 PBS (T-PBS) and incubated at 4°C overnight with the following specific primary MAbs or polyclonal antibodies (PAbs): N (mouse MAb 1C7C7), mCherry (rabbit Pab; Raybiotech), and Nluc (rabbit Pab, Promega).

Techniques: In Vivo, Transgenic Assay, Infection, Activity Assay, In Vivo Imaging

Journal: Microbiology Spectrum

Article Title: Monitoring SARS-CoV-2 Infection Using a Double Reporter-Expressing Virus

doi: 10.1128/spectrum.02379-22

Figure Lengend Snippet: In vivo bioluminescence and ex vivo fluorescence in K18 hACE2 transgenic mice infected with rSARS-CoV-2/mCherry-Nluc. (A) In vivo Nluc expression: Nluc activity in live mice ( n = 4) mock infected or infected (10 5 PFU/mouse) with rSARS-CoV-2 WT, rSARS-CoV-2/mCherry, rSARS-CoV-2/Nluc, rSARS-CoV-2/mCherry + rSARS-CoV-2/Nluc, or the bireporter rSARS-CoV-2/mCherry-Nluc (mCherry-Nluc) were determined on 2 and 4 dpi using the Ami HT IVIS. A representative image of a mouse per time point is shown. (B) Quantification of Nluc signal: means and SD of the radiance (number of photons per second per square centimeter per steradian [p/second/cm 2 /sr]) and bioluminescence (total flux [log 10 photons per second (p/s)]) of mock and infected mice is shown. (C) Ex vivo mCherry expression: excised lungs from mock-infected or infected mice from panel A were monitored for mCherry fluorescent expression (FL, top) and bright field (BF, bottom) at 2 and 4 dpi. Representative lung images from the same mouse used in panel A are shown. (D) Quantification of mCherry expression: the mean values of mCherry signal around the regions of interest were normalized to the autofluorescence of mock-infected lungs at each time point and the fold changes in fluorescence were calculated. (E) Gross pathology score: pathology lesions, consolidation, congestion, and atelectasis, of excised lungs were measured using NIH ImageJ and are represented as percentages of total lung surface area affected. *, P < 0.05; ***, P < 0.001; ns, not significant.

Article Snippet: Membranes were blocked for 1 h with 5% dried skim milk in 0.1% Tween 20 PBS (T-PBS) and incubated at 4°C overnight with the following specific primary MAbs or polyclonal antibodies (PAbs): N (mouse MAb 1C7C7), mCherry (rabbit Pab; Raybiotech), and Nluc (rabbit Pab, Promega).

Techniques: In Vivo, Ex Vivo, Fluorescence, Transgenic Assay, Infection, Expressing, Activity Assay

Journal: Microbiology Spectrum

Article Title: Monitoring SARS-CoV-2 Infection Using a Double Reporter-Expressing Virus

doi: 10.1128/spectrum.02379-22

Figure Lengend Snippet: Nluc activity and viral titers in tissue homogenates from infected K18 hACE2 transgenic mice: the nasal turbinate (left), lungs (middle), and brain (right) of four-to-6-weeks-old female K18 hACE2 transgenic mice ( n = 4) mock infected or infected intranasally with 10 5 PFU/mouse of rSARS-CoV-2 WT, rSARS-CoV-2/mCherry, rSARS-CoV-2/Nluc, rSARS-CoV-2/mCherry + rSARS-CoV-2/Nluc, or the bireporter rSARS-CoV-2/mCherry-Nluc were collected after imaging on an Ami HT IVIS on 2 and 4 dpi. After homogenization, Nluc activity (A) and viral titers (B) in tissue homogenates were determined on a microplate reader or by plaque assay, respectively. The results are the mean values and SD. LOD, limit of detection. **, P < 0.01; ***, P < 0.001; ns, not significant; nd, not detected.

Article Snippet: Membranes were blocked for 1 h with 5% dried skim milk in 0.1% Tween 20 PBS (T-PBS) and incubated at 4°C overnight with the following specific primary MAbs or polyclonal antibodies (PAbs): N (mouse MAb 1C7C7), mCherry (rabbit Pab; Raybiotech), and Nluc (rabbit Pab, Promega).

Techniques: Activity Assay, Infection, Transgenic Assay, Imaging, Homogenization, Plaque Assay

Journal: Microbiology Spectrum

Article Title: Monitoring SARS-CoV-2 Infection Using a Double Reporter-Expressing Virus

doi: 10.1128/spectrum.02379-22

Figure Lengend Snippet: In vivo bioluminescence and ex vivo fluorescence in golden Syrian hamsters infected with rSARS-CoV-2/mCherry-Nluc. (A) In vivo Nluc expression: Nluc activity in 4- to 6-week-old female golden Syrian hamsters ( n = 4) mock infected or infected with 10 5 PFU/hamster of rSARS-CoV-2/mCherry-Nluc were determined on 2, 4, and 6 dpi using the Ami HT IVIS. Contact animals were exposed to infected animals 1 dpi. A representative image of a hamster per time points and experimental condition is shown. (B) Quantification of Nluc signal: means and SD of the radiance (number of photons per second per square centimeter per steradian [p/second/cm 2 /sr]) and bioluminescence (total flux [log 10 photons per second (p/s)]) were quantified from whole hamsters after IVIS imaging. (C) Ex vivo mCherry and Nluc expression: excised lungs from mock-infected or infected golden Syrian hamsters from panel A were monitored for mCherry fluorescence (FL, top), Nluc signal (Nluc, middle), and bright field (BF, bottom) at 2, 4, and 6 dpi. Representative lung images from the same hamster used in panel A are shown. (D) Quantification of mCherry expression: the mean values of mCherry signal around the regions of interest were normalized to the autofluorescence of mock-infected lungs at each time point and the fold changes in fluorescence were calculated. (E) Gross pathology score: pathological lesions, consolidation, congestion, and atelectasis, of excised lungs were measured using NIH ImageJ and are represented as percentages of total lung surface area affected. *, P < 0.05; ***, P < 0.001; ns, not significant.

Article Snippet: Membranes were blocked for 1 h with 5% dried skim milk in 0.1% Tween 20 PBS (T-PBS) and incubated at 4°C overnight with the following specific primary MAbs or polyclonal antibodies (PAbs): N (mouse MAb 1C7C7), mCherry (rabbit Pab; Raybiotech), and Nluc (rabbit Pab, Promega).

Techniques: In Vivo, Ex Vivo, Fluorescence, Infection, Expressing, Activity Assay, Imaging

Journal: Microbiology Spectrum

Article Title: Monitoring SARS-CoV-2 Infection Using a Double Reporter-Expressing Virus

doi: 10.1128/spectrum.02379-22

Figure Lengend Snippet: Nluc activity and viral titers in golden Syrian hamster tissue homogenates infected with rSARS-CoV-2/mCherry-Nluc: the nasal turbinate (left) and lungs (right) of 4- to 6-week-old female golden Syrian hamsters ( n = 4) mock infected or infected intranasally with 10 5 PFU/hamster of rSARS-CoV-2/mCherry-Nluc were collected after imaging on an Ami HT IVIS at 2 and 4 dpi. In addition, after 24 hpi, contact golden Syrian hamsters ( n = 4, contact) were added to the cages of infected animals. After homogenization, Nluc activity (A) and viral titers (B) in tissue homogenates were determined on a microplate reader or by plaque assay, respectively. Results are the means and SD. LOD, limit of detection. *, P < 0.05; ***, P < 0.001; ns, not significant; nd, not detected.

Article Snippet: Membranes were blocked for 1 h with 5% dried skim milk in 0.1% Tween 20 PBS (T-PBS) and incubated at 4°C overnight with the following specific primary MAbs or polyclonal antibodies (PAbs): N (mouse MAb 1C7C7), mCherry (rabbit Pab; Raybiotech), and Nluc (rabbit Pab, Promega).

Techniques: Activity Assay, Infection, Imaging, Homogenization, Plaque Assay