Journal: Virus Evolution

Article Title: The rise and spread of the SARS-CoV-2 AY.122 lineage in Russia

doi: 10.1093/ve/veac017

Figure Lengend Snippet: The dynamics of the effective reproduction number R e for the main PII of the Delta clade in Moscow inferred by BDSKY (black line; shaded red bars show 50 per cent and 95 per cent posterior credible intervals); and for all (blue line) or for Delta (red line) SARS-CoV-2 cases in Moscow inferred by EpiEstim. The gray line shows the 7-day rolling average of the daily number of new cases in Moscow independent of the genotype.

Article Snippet: For CoRGI samples, whole-genome amplification (WGA) of SARS-CoV-2 virus genome was performed using ARTIC Network protocol V3 ( https://github.com/joshquick/artic-ncov2019/tree/master/primer_schemes/nCoV-2019/V3 ) with modifications by , using NEBNext® ARTIC SARS-CoV-2 Companion Kit (New England Biolabs, USA).

Techniques:

Journal: Scientific Reports

Article Title: Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing

doi: 10.1038/s41598-022-07871-4

Figure Lengend Snippet: Effective SDS and Tween concentration determination for qPCR and virus inactivation. ( A ) Determination of the minimal Tween 20 and Tween 80 concentration necessary for SDS sequestration to enable amplification of 20,000 copies/µL of RNase P DNA. Ct cycle threshold, ND not detected. ( B ) Fluorescence assay of eGFP-expressing recombinant VSV-SARS-CoV-2 (10 7 pfu/mL) infecting Vero-E6 cells after incubation with 0.1% (w/v) SDS for 15–120 s. Only a sample that was not exposed to SDS showed infectivity. 15 s exposure to 0.1% (w/v) SDS proved sufficient to completely inactivate the virus, likely due to dissolution of its membrane and denaturation of the sample proteins.

Article Snippet: RT-LAMP reactions contained 2 µL 5× RT-LAMP reaction buffer (1× reaction buffer: 20 mM Tris–HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 10 mM MgSO 4 , 0.1% [v/v] Tween 20, 0.9% [v/v] Tween 80, 5 µM SYTO 82, pH 8.8), 2 µL 7 mM dNTPs, 1 µL Bst 2.0 WarmStart DNA polymerase (8 U/µL) (NEB) 0.25 µL WarmStart RTx reverse transcriptase (150 U/µL) (NEB), and 1 µL 10× SARS-CoV-2 N1 LAMP primer mix (2 µM F3/B3, 16 µM FIP/BIP, 4 µM LF/LB; Supplementary Table ) and dH 2 O to 9 µL.

Techniques: Concentration Assay, Amplification, Fluorescence, Expressing, Recombinant, Incubation, Infection

Journal: Scientific Reports

Article Title: Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing

doi: 10.1038/s41598-022-07871-4

Figure Lengend Snippet: Comparison of standard qPCR to SDS/Tween and SDS/Tween agarose reactions. ( A ) Comparison of 10 µL qPCR reactions for SARS-CoV-2 N1 gene DNA amplification, containing 3% (v/v) of Tween 80 with (R 2 = 0.9781) and without 0.01% SDS (w/v, final; R 2 = 0.9818) and 1% (w/v) agarose (R 2 = 0.9260). ( B ) Comparison of a 10 µL qPCR reaction for SARS-CoV-2 N1 gene DNA containing 3% (v/v) Tween 80 (R 2 = 0.9908) with a 5 µL qPCR reaction (R 2 = 0.8451) for SARS-CoV-2 N1 DNA detection, containing 3% (v/v) Tween 80, 0.01% SDS (w/v, final) and 1% (w/v) agarose. In all conditions containing SDS, the DNA samples were initially incubated with 0.1% SDS (w/v) in Tris/EDTA buffer to simulate denaturing conditions for clinical samples. Ct cycle threshold.

Article Snippet: RT-LAMP reactions contained 2 µL 5× RT-LAMP reaction buffer (1× reaction buffer: 20 mM Tris–HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 10 mM MgSO 4 , 0.1% [v/v] Tween 20, 0.9% [v/v] Tween 80, 5 µM SYTO 82, pH 8.8), 2 µL 7 mM dNTPs, 1 µL Bst 2.0 WarmStart DNA polymerase (8 U/µL) (NEB) 0.25 µL WarmStart RTx reverse transcriptase (150 U/µL) (NEB), and 1 µL 10× SARS-CoV-2 N1 LAMP primer mix (2 µM F3/B3, 16 µM FIP/BIP, 4 µM LF/LB; Supplementary Table ) and dH 2 O to 9 µL.

Techniques: Amplification, Incubation

Journal: Scientific Reports

Article Title: Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing

doi: 10.1038/s41598-022-07871-4

Figure Lengend Snippet: Single-tube sample-to-assay RT-qPCR of SARS-CoV-2 positive-patient RNA sample and inactivated viral particles. ( A ) Comparison of five-fold diluted purified and unpurified SARS-CoV-2 patient RNA samples (nasal swabs stored in VTM + 0.5% [w/v] SDS) in triplicate 10 µL RT-qPCR reactions containing SARS-CoV-2 N1 primers and probe, 3% (v/v) Tween 80, and 0.5% (w/v) agarose. Samples were diluted five-fold to achieve a 0.1% (w/v) SDS working concentration for unpurified samples in VTM. Data are presented as ± SEM. ( B ) Comparison of 5 µL and 10 µL RT-qPCR reactions containing 3% (v/v) Tween 80, and 0.5% (w/v) agarose with a purified SARS-CoV-2 patient RNA sample. Triplicate reactions were performed at each volume, comparing the patient sample with and without spiking into saliva. Reactions were performed with SARS-CoV-2 N1 and beta-actin (ACTB) primer–probe sets. Data are presented as ± SEM. ( C ) RT-qPCR results of SARS-CoV-2 synthetic RNA (n = 11) in water. RT-qPCR was performed for 45 cycles using SARS-CoV-2 N1- and N2-specific primers and probes on a Quantabio Q thermal cycler. Samples were tested blind with concentrations and storage media revealed thereafter (ND = not detected). ( D ) Scatter plot of Ct value (y-axis) versus log[copies] of SARS-CoV-2 viral particles (x-axis) of inactivated SARS-CoV-2 viral particle samples (n = 49) in PBS, saliva, and nasal media. RT-qPCR was performed for 45 cycles with SARS-CoV-2 N1-specific primers and fluorescent probe on a Bio-Rad CFX Connect thermal cycler. Ct cycle threshold.

Article Snippet: RT-LAMP reactions contained 2 µL 5× RT-LAMP reaction buffer (1× reaction buffer: 20 mM Tris–HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 10 mM MgSO 4 , 0.1% [v/v] Tween 20, 0.9% [v/v] Tween 80, 5 µM SYTO 82, pH 8.8), 2 µL 7 mM dNTPs, 1 µL Bst 2.0 WarmStart DNA polymerase (8 U/µL) (NEB) 0.25 µL WarmStart RTx reverse transcriptase (150 U/µL) (NEB), and 1 µL 10× SARS-CoV-2 N1 LAMP primer mix (2 µM F3/B3, 16 µM FIP/BIP, 4 µM LF/LB; Supplementary Table ) and dH 2 O to 9 µL.

Techniques: Quantitative RT-PCR, Purification, Concentration Assay

Journal: Scientific Reports

Article Title: Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing

doi: 10.1038/s41598-022-07871-4

Figure Lengend Snippet: RT-LAMP amplification with SDS and Tween 80. ( A ) Real-time fluorescence of SARS-CoV-2N gene amplification by RT-LAMP was performed at 65 °C for 50 min. Indicated concentrations (copies) of SARS-CoV-2 viral particles were assessed including a no-reverse-transcriptase (NRT) and no-template control (NTC; 0 copies), with all SARS-CoV-2 samples providing a positive result after ~ 30 min. ( B ) Amplification products (1 µL) from the real-time fluorescence RT-LAMP reaction were fractionated using agarose gel electrophoresis. The expected laddering pattern of LAMP amplification products was observed at all SARS-CoV-2 viral particle concentrations and not observed in NRT and NTC controls. The uncropped gel can be found in Supplementary Fig. . ( C ) Fluorescence of RT-LAMP reactions of SARS-CoV-2 viral particles containing 20 µM SYTO 82 observed after 30 and 50 min of incubation at 65 °C. Reactions were analyzed with white, green, and UV (365 nm) handheld LED flashlights at each time point, with readily observable fluorescence of amplification reactions starting from 100 to 10 copies (labeled) after 30 min with each illumination. After 50 min, fluorescence in the 100-copy and 10-copy tubes increased with no observable amplification in the 0-copy control. RFU relative fluorescence units.

Article Snippet: RT-LAMP reactions contained 2 µL 5× RT-LAMP reaction buffer (1× reaction buffer: 20 mM Tris–HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 10 mM MgSO 4 , 0.1% [v/v] Tween 20, 0.9% [v/v] Tween 80, 5 µM SYTO 82, pH 8.8), 2 µL 7 mM dNTPs, 1 µL Bst 2.0 WarmStart DNA polymerase (8 U/µL) (NEB) 0.25 µL WarmStart RTx reverse transcriptase (150 U/µL) (NEB), and 1 µL 10× SARS-CoV-2 N1 LAMP primer mix (2 µM F3/B3, 16 µM FIP/BIP, 4 µM LF/LB; Supplementary Table ) and dH 2 O to 9 µL.

Techniques: Amplification, Fluorescence, Agarose Gel Electrophoresis, Incubation, Labeling

Journal: Biosensors & Bioelectronics

Article Title: Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip

doi: 10.1016/j.bios.2022.114192

Figure Lengend Snippet: Flow profile assay of SARS-CoV-2 on a paper-based microfluidic chip. (A) Paper-based microfluidic chip design containing green edge and three red squares for recognizing the chip area in automated flow distance measurement. (B) The chip holder and the chip lock. (C) A paper-based microfluidic chip was placed into a chip holder to flatten the chip. (D) 4 μL of sample was loaded directly onto the square inlet (top) of each channel and dried for 10 min. (E) A smartphone camera was held just above the chip to view the chip area and start recording the video. (F) 4 μL of Ab-particles were loaded, and the liquid flow on the paper microfluidic chip was recorded with the smartphone camera.

Article Snippet: Two types of antibodies were used in this study: rabbit monoclonal antibody to SARS-CoV-2 (2019-nCoV) nucleocapsid (Sino Biological US Inc., PA, USA) and rabbit polyclonal antibody to SARS-CoV/SARS-CoV-2 nucleocapsid (Sino Biological US Inc., PA, USA).

Techniques:

Journal: Biosensors & Bioelectronics

Article Title: Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip

doi: 10.1016/j.bios.2022.114192

Figure Lengend Snippet: Video processing algorithm. Python code was developed to automatically obtain the flow distance over time. (A) The red squares were detected, and each frame is rotated for orientation correction. (B) The green edge was recognized and cropped. (C) The cropped area was analyzed to generate an intensity histogram plot. Using appropriate thresholding, the liquid flow was recognized on paper. (D) The flow on each channel was read separately by recognizing it as the black pixels increasing along the vertical centerline. (E) Flow distance vs. time profile of mAb-particles on the preloaded 1000 fg/μL SARS-CoV-2 spiked sample. (F) Flow distance vs. time profile of pAb-particles on the preloaded 1000 fg/μL SARS-CoV-2 spiked sample.

Article Snippet: Two types of antibodies were used in this study: rabbit monoclonal antibody to SARS-CoV-2 (2019-nCoV) nucleocapsid (Sino Biological US Inc., PA, USA) and rabbit polyclonal antibody to SARS-CoV/SARS-CoV-2 nucleocapsid (Sino Biological US Inc., PA, USA).

Techniques:

Journal: Biosensors & Bioelectronics

Article Title: Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip

doi: 10.1016/j.bios.2022.114192

Figure Lengend Snippet: Assay LOD and Specificity. NC indicates negative control and * shows p < 0.05 between sample and NC using one-tailed student's t-test with unequal variance. Error bars represent standard error. (A) Flow distances at 30 s on the paper microfluidic chips preloaded with SARS-CoV-2 spiked in 1% v/v human pooled saliva, using polyclonal antibody conjugated particles at 0.04 μg/μL (n = 3). (B) Flow distances at 30 s on the paper microfluidic chips preloaded with SARS-CoV-2 spiked in simulated saline gargle samples (∼15% v/v saliva and 0.9% saline), using polyclonal antibody conjugated particles at 0.04 μg/μL, with the addition of 0.5% w/v Tween 20 (n = 3). (C) Specificity test results with 1 pg/μL SARS-CoV-2 and influenza A/H1N1 (Ct values of 25–28) spiked in 1% v/v and 10% v/v saliva in 0.9% saline using the pAb-particles, shown together with the no target control samples (1% or 10% saliva in 0.9% saline) (n = 3).

Article Snippet: Two types of antibodies were used in this study: rabbit monoclonal antibody to SARS-CoV-2 (2019-nCoV) nucleocapsid (Sino Biological US Inc., PA, USA) and rabbit polyclonal antibody to SARS-CoV/SARS-CoV-2 nucleocapsid (Sino Biological US Inc., PA, USA).

Techniques: Negative Control, One-tailed Test

Journal: Biosensors & Bioelectronics

Article Title: Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip

doi: 10.1016/j.bios.2022.114192

Figure Lengend Snippet: Turbidity assessment of clinical saline gargle samples. Error bars represent standard error. (A) Photographs of negative and positive clinical saline gargle samples, obtained from human subjects. The normalized turbidity was determined by comparing the pixel intensities of the sample tubes against the black background. Red boxes indicate samples that were determined to be turbid using the procedure described in part B. (B) Using the normalized (to empty tube) turbidity, all clinical samples were classified into two categories, turbid and clear, using the threshold value of 1.41. Note: while all samples were classified in this manner, some could not undergo all subsequent testing due to low sample volume. (C) Surface tension measurements showed a decreasing trend with increased turbidity. (D) Total protein concentration of samples according to the Bradford assay. Turbid and clear samples showed no difference in total protein concentration, but SARS-CoV-2 positive samples had a higher (not significant) total protein concentration than negative samples. (n = 5 for negative clear, n = 3 for negative turbid, n = 6 for positive clear, and n = 4 for positive turbid). (E) Samples with a last oral intake (LOI) of 10–30 min prior to sample acquisition (n = 6) had higher turbidity than samples with a longer time since LOI (60+ min; n = 10), and the difference was statistically significant ( p < 0.05). Average values are shown in the bar chart. (F) The time to constant velocity (n = 2) and surface tension of no toothpaste vs. toothpaste-added (10 mg/mL) NC samples, along with photos of the samples. Surface tension was measured at 0, 2, 4, 6, 8, and 10 s and the stabilized final value was chosen (hence no error bar). The accuracy of surface tension measurement is less than 1 mN/mm.

Article Snippet: Two types of antibodies were used in this study: rabbit monoclonal antibody to SARS-CoV-2 (2019-nCoV) nucleocapsid (Sino Biological US Inc., PA, USA) and rabbit polyclonal antibody to SARS-CoV/SARS-CoV-2 nucleocapsid (Sino Biological US Inc., PA, USA).

Techniques: Protein Concentration, Bradford Assay

Journal: Cell

Article Title: Vaccine protection against the SARS-CoV-2 Omicron variant in macaques

doi: 10.1016/j.cell.2022.03.024

Figure Lengend Snippet: Humoral immune responses following vaccination Antibody responses at weeks 0 (baseline), 8 (post-prime), 14 (pre-boost), and 18 (post-boost) following vaccination with BNTx3, BNTx2/Ad26, Ad26/BNT, Ad26x2, or sham (N = 30; N = 6/group). (A) Neutralizing antibody (NAb) titers by a luciferase-based pseudovirus neutralization assay. (B) Receptor-binding domain (RBD)-specific binding antibody titers by ELISA. Responses were measured against the SARS-CoV-2 WA1/2020 (black), B.1.617.2 (Delta; blue), B.1.351 (Beta; red), and B.1.1.529 (Omicron; green) variants. Dotted lines represent limits of quantitation. Medians (red bars) are shown. Omicron-specific NAbs in the vaccinated groups were compared with the sham controls by two-sided Mann-Whitney tests. ∗ p < 0.05.

Article Snippet: SARS-CoV-2 (Omicron) RBD , Sino Biological , Cat # 40592-V08H121.

Techniques: Luciferase, Neutralization, Binding Assay, Enzyme-linked Immunosorbent Assay, Quantitation Assay, MANN-WHITNEY

Journal: Cell

Article Title: Vaccine protection against the SARS-CoV-2 Omicron variant in macaques

doi: 10.1016/j.cell.2022.03.024

Figure Lengend Snippet: Cellular immune responses following vaccination T cell responses at weeks 14 (pre-boost) and 18 (post-boost) following vaccination with BNTx3, BNTx2/Ad26, Ad26/BNT, Ad26x2, or sham (N = 30; N = 6/group). (A and B) Pooled peptide spike-specific IFN-γ (A) CD8+ T cell responses and (B) CD4+ T cell responses by intracellular cytokine staining assays. Responses were measured against the SARS-CoV-2 WA1/2020 (black), B.1.617.2 (Delta; blue), and B.1.1.529 (Omicron; green) variants. Dotted lines represent limits of quantitation. Medians (red bars) are shown. Omicron-specific CD8+ and CD4+ T cell responses in the vaccinated groups were compared with the sham controls by two-sided Mann-Whitney tests. ∗ p < 0.05.

Article Snippet: SARS-CoV-2 (Omicron) RBD , Sino Biological , Cat # 40592-V08H121.

Techniques: Staining, Quantitation Assay, MANN-WHITNEY

Journal: Cell

Article Title: Vaccine protection against the SARS-CoV-2 Omicron variant in macaques

doi: 10.1016/j.cell.2022.03.024

Figure Lengend Snippet: Viral loads following SARS-CoV-2 Omicron challenge (A) Log subgenomic RNA (sgRNA) copies/mL in bronchoalveolar lavage (BAL) following SARS-CoV-2 Omicron challenge. (B) Log subgenomic RNA (sgRNA) copies/swab in nasal swabs (NS) following SARS-CoV-2 Omicron challenge. Medians (red lines) are shown.

Article Snippet: SARS-CoV-2 (Omicron) RBD , Sino Biological , Cat # 40592-V08H121.

Techniques:

Journal: Cell

Article Title: Vaccine protection against the SARS-CoV-2 Omicron variant in macaques

doi: 10.1016/j.cell.2022.03.024

Figure Lengend Snippet: Comparison of peak and day 4 viral loads (A) Log subgenomic RNA (sgRNA) copies/mL in bronchoalveolar lavage (BAL) at peak and on day 4 following SARS-CoV-2 Omicron challenge. (B) Log subgenomic RNA (sgRNA) copies/swab in nasal swabs (NS) at peak and on day 4 following SARS-CoV-2 Omicron challenge. Dotted lines represent limits of quantitation. Medians (red bars) are shown. Vaccinated groups were compared with the sham controls by two-sided Mann-Whitney tests. ∗ p < 0.05.

Article Snippet: SARS-CoV-2 (Omicron) RBD , Sino Biological , Cat # 40592-V08H121.

Techniques: Quantitation Assay, MANN-WHITNEY

Journal: Cell

Article Title: Vaccine protection against the SARS-CoV-2 Omicron variant in macaques

doi: 10.1016/j.cell.2022.03.024

Figure Lengend Snippet: TCID50 titers, related to Figure 3 Log TCID50/mL in bronchoalveolar lavage (BAL) and nasal swabs on day 2 following SARS-CoV-2 Omicron challenge (top). Log TCID50/mL is also shown in nasal swabs on day 7 following SARS-CoV-2 Omicron challenge in the 4 vaccinated animals in the BNTx3 and BNTx2/Ad26 groups and in the 6 sham controls with persistently positive sgRNA levels on day 7 (bottom). Dotted lines represent limits of quantitation. Medians (red bars) are shown. Vaccinated groups were compared with the sham controls by two-sided Mann-Whitney tests. ∗ p < 0.05.

Article Snippet: SARS-CoV-2 (Omicron) RBD , Sino Biological , Cat # 40592-V08H121.

Techniques: Quantitation Assay, MANN-WHITNEY

Journal: Cell

Article Title: Vaccine protection against the SARS-CoV-2 Omicron variant in macaques

doi: 10.1016/j.cell.2022.03.024

Figure Lengend Snippet: Histopathology and immunohistochemistry of Omicron infection, related to Figure 1 (A–K) (A–C) Pharynx and (D–K) lungs from macaques on day 2 following Omicron infection demonstrated lymphoid hyperplasia of the pharynx (A and B), SARS-N positive ciliated epithelial cells in the pharynx (C), foamy macrophages and degenerating neutrophils in bronchiole lumen (D), cellular necrotic debris adhering to bronchiolar ciliated epithelium (E), alveolar syncytia (F), SARS-N-positive ciliated epithelial cells in the pulmonary interstitium (G), neutrophilic bronchitis (H), hyaline membranes (I), endothelialitis (J), and type II pneumocyte hyperplasia (K). Scoring involved assessment of the following lesions: interstitial inflammation and septal thickening, interstitial infiltrate (eosinophils), interstitial infiltrate (neutrophils), hyaline membranes, interstitial fibrosis, alveolar infiltrate (macrophages), bronchoalveolar infiltrate (neutrophils), epithelial syncytia, type II pneumocyte hyperplasia, bronchi infiltrate (macrophages), bronchi infiltrate (neutrophils), bronchi (hyperplasia of bronchus-associated lymphoid tissue), bronchiolar or peribronchiolar infiltrate (mononuclear cells), perivascular infiltrate (mononuclear cells), and endothelialitis. Each feature assessed was assigned a score of: 0, no substantial findings; 1, minimal; 2, mild; 3, moderate; 4, moderate to severe; 5, marked or severe. Scores were added for all lesions across all lung lobes for each macaque, for a maximum possible score of 600 for each macaque. (L) Summary of lung pathology scores from SARS-CoV-2 WA1/2020- and Omicron-infected macaques. Medians (red bars) are shown. Dotted line represents no pathology. Lung pathology scores were compared in macaques infected with Omicron versus WA1/2020 by two-sided Mann-Whitney tests. ∗ p < 0.05.

Article Snippet: SARS-CoV-2 (Omicron) RBD , Sino Biological , Cat # 40592-V08H121.

Techniques: Histopathology, Immunohistochemistry, Infection, MANN-WHITNEY

Journal: Cell

Article Title: Vaccine protection against the SARS-CoV-2 Omicron variant in macaques

doi: 10.1016/j.cell.2022.03.024

Figure Lengend Snippet:

Article Snippet: SARS-CoV-2 (Omicron) RBD , Sino Biological , Cat # 40592-V08H121.

Techniques: Infection, Recombinant, Binding Assay, Blocking Assay, Software

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Codon-optimized full-length Spike protein gene (S F ), S1 subunit gene and the receptor-binding domain (RBD) plus envelope protein genes of SARS-CoV-2 with and without 21 amino acids honeybee melittin signal peptide [(msp) NH 2 -MKFLVNVALVFMVVYISYIYA-COOH] gene in the purple box, and 49 amino acids VSV G protein transmembrane domain and cytoplasmic tail [(Gtc) NH 2 -SSIASFFFIIGLIIGLFL VLRVGIYLCIKLKHTKKRQIYTDIEMNRLGK-COOH] gene in the red box were inserted into the G and L gene junction of rVSV Ind and rVSV NJ . In addition, 25- nucleotides-long VSV intergenic junctions (5´-CATATGAAAAAAACTAACAGATATC-3´), in the green box, were inserted between genes to provide transcription termination, polyadenylation and the transcription reinitiation sequences. Recombinant viruses were rescued by VSV reverse genetics . pT7: Bacteriophage T7 promoter for DNA-dependent RNA polymerase. N : VSV Nucleocapsid Protein gene. P : VSV Phosphoprotein gene. M : VSV Matrix protein gene. G : VSV Glycoprotein gene. L : VSV Large protein, RNA-dependent RNA polymerase gene. l : Leader region in the 3´-end of the VSV genome. t : Trailer region in the 5´-end of the VSV genome. HDV: Hepatitis delta virus ribozyme encoding sequences. T7δ: Bacteriophage T7 transcriptional terminator sequences. nt: nucleotides. aa: amino acids.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Binding Assay, Recombinant

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: To check the expression of SARS-CoV-2 RBD, S1, S F , and E proteins from the rVSV Ind -SARS-CoV-2 infected cells, BHK-21 cells were infected with the virus at an MOI of 6. After six hours incubation at 37°C, cell lysates were prepared and protein expression was determined by Western blot analysis. Cell lysates were loaded in 5 μg quantity for SDS-PAGE. RBD, S1, and S F proteins were detected by rabbit antibody against SARS-CoV-2 RBD. S2 protein was detected by rabbit antibody against SARS-CoV-2 S2. E protein was detected by rabbit antibody against SARS-CoV-2 E peptides. (A) Expression of RBD, S1, and S F with and without msp and Gtc. (B) Expression of S2 with and without Gtc. (C) Expression of E protein. (D) Expression of VSV Ind N, P, M, and G proteins. Purple box: honeybee msp, red box: VSV Gtc.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Expressing, Infection, Incubation, Western Blot, SDS Page

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Incorporation of SARS-CoV-2 S F , S1, S2, and RBD with or without VSV Gtc into rVSV Ind particles was examined by infecting BHK-21 cells with rVSV Ind -SARS-CoV-2 at an MOI of 3. The rVSV Ind -SARS-CoV-2 infected cells were incubated at 31°C for 6 hrs. Infected cell lysates were prepared in lysis buffer (lanes 1, 2, and 5). Culture media from the infected cells was centrifuged at 500 x g for 10 minutes and supernatant was filtered through a 0.45 μm filter to remove cell debris. The filtered culture media was loaded onto 1 ml of 25% sucrose cushion and ultra-centrifuged at 150,900 x g for 3 hrs. Supernatant on top of the 25% sucrose cushion was collected to check the soluble proteins in the media (lanes 3 and 6). Pelleted samples were checked for proteins incorporated into VSV particles (lanes 4 and 7). We detected RBD, S1, and S F proteins by Western blot using an antibody against the SARS-CoV-2 RBD protein. S2 and S F proteins were detected by rabbit antibody against SARS-CoV-2 S2. (A) Detection of S F and S1 proteins in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-S F -Gtc or rVSV Ind -S F . (B) Detection of S F and S2 proteins in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-S F -Gtc or rVSV Ind -S F . (C) Detection of VSV Ind proteins in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-S F -Gtc or rVSV Ind -S F . (D) Detection of S1 protein in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-S1-Gtc or rVSV Ind -S1. (E) Detection of RBD proteins in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-RBD-Gtc+E-Gtc or rVSV Ind -msp-RBD+E. (F) Detection of VSV Ind proteins in cell lysate, concentrated culture media, and virus pellet from the cells infected with rVSV Ind -msp-RBD-Gtc+E-Gtc or rVSV Ind -msp-RBD+E. Purple box: honeybee msp, red box: VSV Gtc.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Infection, Incubation, Lysis, Western Blot

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: To examine immune responses in mice, it was first necessary to purify rVSV-SARS-CoV-2 viral particles by anion-exchange chromatography. One μg of the purified rVSV-SARS-CoV-2 was analyzed by SDS-PAGE and the presence of RBD, S1, S2, and S F was determined by Western blot analysis. (A) Detection of RBD, S1, and S F on VSV particles. (B) Detection of S2 and S F on VSV particles. (C) Detection of VSV Ind and VSV NJ proteins. (D) Depicted model of pseudotype recombinant VSV virions with three different forms of SARS-CoV-2 Spike proteins. rVSV pseudotypes are formed when rVSV-SARS-CoV-2 Spike proteins are expressed with the msp at the NH 2 -terminus and VSV Gtc at the COOH-terminus. Purple box: honeybee msp, red box: VSV Gtc.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Chromatography, Purification, SDS Page, Western Blot, Recombinant

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Mice were prime immunized with rVSV Ind -SARS-CoV-2 and boost immunized with rVSV NJ -SARS-CoV-2 two weeks after prime-immunization. Serum was collected to determine SARS-CoV-2 S1 protein-specific antibody levels by ELISA on day 13, one day before boost-immunization, and on day 27, two weeks after boost-immunization. (A) Prime-boost vaccination schedule. (B) Spike(ΔTM)-specific IgG titer after the prime-boost vaccination with doses of 5X10 7 PFU/mouse ( C) Spike(ΔTM)-specific IgG titer after the prime-boost vaccination with doses of 5X10 8 PFU/mouse. Statistical significance was determined by two-way ANOVA with Tukey’s correction (*, p < 0.05; **, p < 0.005; ***, p< 0.001; ns, not significant). The data were presented as means with error bars of standard deviation (n = 5 mice per group). Purple box: honeybee msp, red box: VSV Gtc. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Six-week-old female hACE2 transgenic mice were prime vaccinated with rVSV Ind -msp-S F -Gtc and boost immunized with rVSV Ind -msp-S F -Gtc or rVSV NJ -msp-S F -Gtc two weeks after the prime-vaccination. Serum was collected on day 13, one day before the boost-vaccination and on day 27, two weeks after the boost-vaccination. SARS-CoV-2 neutralization was determined by FRNT 50 assay. Statistical significance was determined by two-way ANOVA with Tukey’s correction (*, p < 0.05; **, p<0.005; ***, p< 0.001; ns, not significant). The data were presented as means with error bars of standard deviation (n = 5 mice per group). Purple box: honeybee msp, red box: VSV Gtc. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Neutralization, Standard Deviation, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Six-week-old female hACE2 transgenic mice were prime vaccinated with rVSV Ind -msp-S F -Gtc and boost vaccinated with rVSV Ind -msp-S F -Gtc or rVSV NJ -msp-S F -Gtc two weeks after the prime-vaccination. Serum was collected to determine the SARS-CoV-2 Spike(ΔTM) protein-specific antibody level by ELISA on day 13, one day before the boost-vaccination and on day 27, two weeks after the boost-vaccination. Statistical significance was determined by two-way ANOVA with Tukey’s correction (*, p < 0.05; **, p<0.005, ***, p<0.001, ****, p< 0.0001; ns, not significant). The data were presented as means with error bars of standard deviation (n = 5 mice per group). Purple box: honeybee msp, red box: VSV Gtc. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Mice were primed with rVSV Ind -SARS-CoV-2 followed with rVSV NJ -SARS-CoV-2 two weeks after prime-immunization. Two weeks after the boost-immunization, splenocytes were prepared and stimulated with a PepTivator SARS-CoV-2 Prot_S [ (A) ], or an irrelevant (control) peptide derived from the HIV Gag (B) . IFN-γ spot-forming units (SFUs) were enumerated by ELISPOT. Statistical significance was determined by two-way ANOVA with Tukey’s correction (*, p < 0.05; **, p < 0.005; ns, not significant). Data are presented as mean SFU numbers with error bars representing standard deviation (n = 5 mice per group). Purple box: honeybee msp, red box: VSV Gtc. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Derivative Assay, Enzyme-linked Immunospot, Standard Deviation, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Six-week-old female hACE2 transgenic mice (n = 5 per group) were prime-vaccinated with rVSV Ind -msp-S F -Gtc and boost vaccinated with rVSV Ind -msp-S F -Gtc or rVSV NJ -msp-S F -Gtc two weeks after prime-vaccination. Four weeks after boost-vaccination , mice were challenged intranasally with 1x10 5 PFU of SARS-CoV-2. The survival and body weight of each mouse was monitored daily. (A) Average bodyweights of mice in each vaccinated group. (B) Individual body weights for mice vaccinated with rVSV-Mock and challenged with SARS-CoV-2. (C) Mouse survival after SARS-CoV-2 challenge. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Human ACE2 transgenic mice were vaccinated and challenged with SARS-CoV-2 as described in . Right lobes of mice lungs were aseptically removed from the mice on day 3, day 7, and day 15 after SARS-CoV-2 challenge. Infectious SARS-CoV-2 was quantified by plaque assay on Vero E6 cells. Statistical significance was determined by two-way ANOVA with Tukey’s correction (****, p< 0.0001). VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Plaque Assay, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Human ACE2 transgenic mice were vaccinated and challenged with SARS-CoV-2 as described in . Left lobes of mice lungs were fixed in 10% buffered formalin on day 3 and day 7 after the SARS-CoV-2 challenge. Lung tissues were processed and embedded in low-melting paraffin, sectioned to a thickness of 3 μm, and stained with hematoxylin and eosin. Stained tissues were examined under a light microscope (Olympus CS41, Japan) with 100X magnification. Note: a, alveolus; b, bronchiole; v, blood vessels. (A) Lung tissue 3 days after the SARS-CoV-2 challenge. (B) Lung tissue 7 days after SARS-CoV-2 challenge. Arrows show infiltration of inflammatory cells (lymphocytes and macrophages). G1: empty vector infected mice, G2: 5X10 8 of rVSV Ind -msp-S F -Gtc/ rVSV NJ -msp-S F -Gtc vaccinated mice, G3: 5X10 8 of rVSV Ind -msp-S F -Gtc/ rVSV Ind -msp-S F -Gtc vaccinated mice, G4: 5X10 7 of rVSV Ind -msp-S F -Gtc/ rVSV NJ -msp-S F -Gtc vaccinated mice, G5: uninfected mice.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Staining, Light Microscopy, Plasmid Preparation, Infection

Journal: Journal of Biomolecular Techniques : JBT

Article Title: RT qLAMP—Direct Detection of SARS-CoV-2 in Raw Sewage

doi: 10.7171/jbt.21-32-03-016

Figure Lengend Snippet: Run B: September 23, 2020. Run using NEB E2019 reaction mix. Left to right, a) E-gene amplification, b) E-gene melt, c) E-gene standard curve, d) N-gene amplification, e) N-gene melt, and f) N-gene standard curve.

Article Snippet: It is important to note that although the NEB E2019 kit is supplied with E1-gene and N2-gene primers (both sets differ from the E- and N-gene primers used in our work; ), except for run 16 (October 1, 2020; d-f ), we did not use the NEB primers.

Techniques: Amplification