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    Structured Review

    Integrated DNA Technologies mers genomic rna
    NHC is effective against multiple genetically distinct Bat-CoV. Top: Antiviral efficacy of NHC in HAE cells against SARS-like (HKU3, SHC014, group 2b) and <t>MERS-like</t> (HKU5, group 2c) bat-CoV. HAE cells were infected at an MOI of 0.5 in the presence of NHC in duplicate. After 48 hours, virus produced was titrated via plaque assay. Each data point represents the titer per culture. Bottom: qRT-PCR for CoV ORF1 and ORFN mRNA in total <t>RNA</t> from cultures in the top panel. Representative data from two separate experiments with two different cell donors are displayed.
    Mers Genomic Rna, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 23 article reviews
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    mers genomic rna - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and multiple endemic, epidemic and bat coronavirus"

    Article Title: An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and multiple endemic, epidemic and bat coronavirus

    Journal: bioRxiv

    doi: 10.1101/2020.03.19.997890

    NHC is effective against multiple genetically distinct Bat-CoV. Top: Antiviral efficacy of NHC in HAE cells against SARS-like (HKU3, SHC014, group 2b) and MERS-like (HKU5, group 2c) bat-CoV. HAE cells were infected at an MOI of 0.5 in the presence of NHC in duplicate. After 48 hours, virus produced was titrated via plaque assay. Each data point represents the titer per culture. Bottom: qRT-PCR for CoV ORF1 and ORFN mRNA in total RNA from cultures in the top panel. Representative data from two separate experiments with two different cell donors are displayed.
    Figure Legend Snippet: NHC is effective against multiple genetically distinct Bat-CoV. Top: Antiviral efficacy of NHC in HAE cells against SARS-like (HKU3, SHC014, group 2b) and MERS-like (HKU5, group 2c) bat-CoV. HAE cells were infected at an MOI of 0.5 in the presence of NHC in duplicate. After 48 hours, virus produced was titrated via plaque assay. Each data point represents the titer per culture. Bottom: qRT-PCR for CoV ORF1 and ORFN mRNA in total RNA from cultures in the top panel. Representative data from two separate experiments with two different cell donors are displayed.

    Techniques Used: Infection, Produced, Plaque Assay, Quantitative RT-PCR

    Remdesivir resistance mutations in the highly conserved RNA-dependent RNA polymerase increase susceptibility to NHC. a , Neighbor-joining trees created with representatives from all four CoV genogroups showing the genetic similarity of CoV nsp12 (RdRp) and CoV spike glycoprotein, which mediates host tropism and entry into cells. Text color of the virus strain label corresponds to virus host species on the left. The heatmap adjacent to each neighbor-joining tree depicts percent amino acid identity (% A.A. similarity) against mouse hepatitis virus (MHV), SARS-CoV or MERS-CoV. b , Core residues of the CoV RdRp are highly conserved among CoV. The variation encompassed in panel a was modeled onto the RdRp structure of the SARS-CoV RdRp. c , Amino acid sequence of CoV in panel a at known resistance alleles to antiviral drug remdesivir (RDV). d , RDV resistant viruses are more susceptible to NHC antiviral activity. Virus titer reduction assay across a dose response of NHC with recombinant MHV bearing resistance mutations to RDV. Asterisks indicate statistically significant differences by Mann-Whitney test.
    Figure Legend Snippet: Remdesivir resistance mutations in the highly conserved RNA-dependent RNA polymerase increase susceptibility to NHC. a , Neighbor-joining trees created with representatives from all four CoV genogroups showing the genetic similarity of CoV nsp12 (RdRp) and CoV spike glycoprotein, which mediates host tropism and entry into cells. Text color of the virus strain label corresponds to virus host species on the left. The heatmap adjacent to each neighbor-joining tree depicts percent amino acid identity (% A.A. similarity) against mouse hepatitis virus (MHV), SARS-CoV or MERS-CoV. b , Core residues of the CoV RdRp are highly conserved among CoV. The variation encompassed in panel a was modeled onto the RdRp structure of the SARS-CoV RdRp. c , Amino acid sequence of CoV in panel a at known resistance alleles to antiviral drug remdesivir (RDV). d , RDV resistant viruses are more susceptible to NHC antiviral activity. Virus titer reduction assay across a dose response of NHC with recombinant MHV bearing resistance mutations to RDV. Asterisks indicate statistically significant differences by Mann-Whitney test.

    Techniques Used: Sequencing, Activity Assay, Recombinant, MANN-WHITNEY

    NHC potently Inhibits MERS-CoV, SARS-CoV and newly emerging SARS-CoV-2 Replication. a , NHC antiviral activity and cytotoxicity in Calu3 cells infected with MERS-CoV. Calu3 cells were infected in triplicate with MERS-CoV nanoluciferase (nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a dose response of drug for 48 hours, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via Cell-Titer-Glo assay. Data is combined from 3 independent experiments. b , NHC antiviral activity and cytotoxicity in Vero cells infected with SARS-CoV-2. Vero cells were infected in duplicate with SARS-CoV-2 clinical isolate virus at an MOI of 0.05 in the presence of a dose response of drug for 48 hours, after which replication was measured through quantitation of cell viability by Cell-Titer-Glo assay. Cytotoxicity was measured as in a . Data is combined from 2 independent experiments. c , NHC inhibits MERS-CoV virus production and RNA synthesis in primary human lung epithelial cell cultures (HAE). HAE cells were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of NHC for 48 hours, after which apical washes were collected for virus titration. qRT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from cultures in c for qRT-PCR analysis. Representative data from three separate experiments with three different cell donors are displayed. PFU, plaque-forming units. d , NHC inhibits SARS-CoV virus production and RNA synthesis in primary human lung epithelial cell cultures (HAE). Studies performed as in c but with SARS-CoV green fluorescent protein (GFP). Representative data from two separate experiments with two different cell donors are displayed.
    Figure Legend Snippet: NHC potently Inhibits MERS-CoV, SARS-CoV and newly emerging SARS-CoV-2 Replication. a , NHC antiviral activity and cytotoxicity in Calu3 cells infected with MERS-CoV. Calu3 cells were infected in triplicate with MERS-CoV nanoluciferase (nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a dose response of drug for 48 hours, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via Cell-Titer-Glo assay. Data is combined from 3 independent experiments. b , NHC antiviral activity and cytotoxicity in Vero cells infected with SARS-CoV-2. Vero cells were infected in duplicate with SARS-CoV-2 clinical isolate virus at an MOI of 0.05 in the presence of a dose response of drug for 48 hours, after which replication was measured through quantitation of cell viability by Cell-Titer-Glo assay. Cytotoxicity was measured as in a . Data is combined from 2 independent experiments. c , NHC inhibits MERS-CoV virus production and RNA synthesis in primary human lung epithelial cell cultures (HAE). HAE cells were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of NHC for 48 hours, after which apical washes were collected for virus titration. qRT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from cultures in c for qRT-PCR analysis. Representative data from three separate experiments with three different cell donors are displayed. PFU, plaque-forming units. d , NHC inhibits SARS-CoV virus production and RNA synthesis in primary human lung epithelial cell cultures (HAE). Studies performed as in c but with SARS-CoV green fluorescent protein (GFP). Representative data from two separate experiments with two different cell donors are displayed.

    Techniques Used: Activity Assay, Infection, Quantitation Assay, Glo Assay, Titration, Quantitative RT-PCR, Isolation

    NHC antiviral activity is associated with increased viral mutation rates. a , Both remdesivir (RDV) and NHC reduce MERS-CoV infectious virus production in primary human HAE. Cultures were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of vehicle, RDV or NHC for 48 hours, after which apical washes were collected for virus titration. Data is combined from two independent studies. b, A deep sequencing approach called Primer ID to gain accurate sequence data for single RNA genomes of MERS-CoV. c , The total error rate for MERS-CoV RNA isolated from cultures in panel a as determined by Primer ID. Error rate values are # mutations per 10,000 bases. Asterisks indicate significant differences as compared to untreated by 2-way ANOVA with a Dunnett’s multiple comparison test. d , description of potential NHC mutational spectra on both positive and negative sense viral RNA. e , Nucleotide transitions adenine (A) to guanine (G) and uridine (U) to cytosine (C) transitions are enriched in MERS-CoV genomic RNA in an NHC dose dependent manner.
    Figure Legend Snippet: NHC antiviral activity is associated with increased viral mutation rates. a , Both remdesivir (RDV) and NHC reduce MERS-CoV infectious virus production in primary human HAE. Cultures were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of vehicle, RDV or NHC for 48 hours, after which apical washes were collected for virus titration. Data is combined from two independent studies. b, A deep sequencing approach called Primer ID to gain accurate sequence data for single RNA genomes of MERS-CoV. c , The total error rate for MERS-CoV RNA isolated from cultures in panel a as determined by Primer ID. Error rate values are # mutations per 10,000 bases. Asterisks indicate significant differences as compared to untreated by 2-way ANOVA with a Dunnett’s multiple comparison test. d , description of potential NHC mutational spectra on both positive and negative sense viral RNA. e , Nucleotide transitions adenine (A) to guanine (G) and uridine (U) to cytosine (C) transitions are enriched in MERS-CoV genomic RNA in an NHC dose dependent manner.

    Techniques Used: Activity Assay, Mutagenesis, Infection, Titration, Sequencing, Isolation

    Prophylactic and therapeutic EIDD-2801 reduces MERS-CoV replication and pathogenesis coincident with increased viral mutation rates. Equivalent numbers of 10-14 week old male and female C57BL/6 hDPP4 mice were administered vehicle (10% PEG, 2.5% Cremophor RH40 in water) or NHC prodrug EIDD-2801 beginning at −2hr, +12, +24 or +48hr post infection and every 12hr thereafter by oral gavage (n = 10/group). Mice were intranasally infected with 5E+04 PFU mouse-adapted MERS-CoV M35C4 strain. a, Percent starting weight. Asterisks indicate differences by two-way ANOVA with Tukey’s multiple comparison test. b, Lung hemorrhage in mice from panel a scored on a scale of 0-4 where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. c , Virus lung titer in mice from panel a as determined by plaque assay. Asterisks in both panel b and c indicate differences by Kruskal-Wallis with Dunn’s multiple comparison test. d , MERS-CoV genomic RNA in lung tissue by qRT-PCR. Asterisks indicate differences by one-way ANOVA with a Dunnett’s multiple comparison test. e , Pulmonary function by whole body plethysmography was performed daily on four animals per group. Asterisks indicate differences by two-way ANOVA with Tukey’s multiple comparison test. f , Workflow to measure mutation rate in MERS-CoV RNA and host transcript ISG15 by Primer ID in mouse lung tissue. g , Number of template consensus sequences for MERS-CoV nsp10 and ISG15. h , Total error rate in MERS-CoV nsp10 and ISG15. i , The cytosine to uridine transition rate in MERS-CoV nsp10 and ISG15. In panels g-i, asterisks indicate differences by two-way ANOVA with Tukey’s multiple comparison test. j , Codon change frequency in MERS-CoV nsp10. Asterisks indicate differences on Kruskal-Wallis with Dunn’s multiple comparison test.
    Figure Legend Snippet: Prophylactic and therapeutic EIDD-2801 reduces MERS-CoV replication and pathogenesis coincident with increased viral mutation rates. Equivalent numbers of 10-14 week old male and female C57BL/6 hDPP4 mice were administered vehicle (10% PEG, 2.5% Cremophor RH40 in water) or NHC prodrug EIDD-2801 beginning at −2hr, +12, +24 or +48hr post infection and every 12hr thereafter by oral gavage (n = 10/group). Mice were intranasally infected with 5E+04 PFU mouse-adapted MERS-CoV M35C4 strain. a, Percent starting weight. Asterisks indicate differences by two-way ANOVA with Tukey’s multiple comparison test. b, Lung hemorrhage in mice from panel a scored on a scale of 0-4 where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. c , Virus lung titer in mice from panel a as determined by plaque assay. Asterisks in both panel b and c indicate differences by Kruskal-Wallis with Dunn’s multiple comparison test. d , MERS-CoV genomic RNA in lung tissue by qRT-PCR. Asterisks indicate differences by one-way ANOVA with a Dunnett’s multiple comparison test. e , Pulmonary function by whole body plethysmography was performed daily on four animals per group. Asterisks indicate differences by two-way ANOVA with Tukey’s multiple comparison test. f , Workflow to measure mutation rate in MERS-CoV RNA and host transcript ISG15 by Primer ID in mouse lung tissue. g , Number of template consensus sequences for MERS-CoV nsp10 and ISG15. h , Total error rate in MERS-CoV nsp10 and ISG15. i , The cytosine to uridine transition rate in MERS-CoV nsp10 and ISG15. In panels g-i, asterisks indicate differences by two-way ANOVA with Tukey’s multiple comparison test. j , Codon change frequency in MERS-CoV nsp10. Asterisks indicate differences on Kruskal-Wallis with Dunn’s multiple comparison test.

    Techniques Used: Mutagenesis, Mouse Assay, Infection, Plaque Assay, Quantitative RT-PCR

    2) Product Images from "An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice"

    Article Title: An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice

    Journal: Science Translational Medicine

    doi: 10.1126/scitranslmed.abb5883

    NHC potently inhibits MERS-CoV and newly emerging SARS-CoV-2 replication. (A) Percent inhibition of MERS-CoV replication and NHC cytotoxicity in Calu-3 cells. Calu-3 cells were infected in triplicate with MERS-CoV nanoluciferase (nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via Cell-Titer-Glo assay. Data are combined from 3 independent experiments. (B) NHC antiviral activity and cytotoxicity in Vero E6 cells infected with SARS-CoV-2. Vero E6 cells were infected in duplicate with SARS-CoV-2 clinical isolate 2019-nCoV/USA-WA1/2020 virus at an MOI of 0.05 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of cell viability by Cell-Titer-Glo assay. Cytotoxicity was measured as in A . Data are combined from 2 independent experiments. ( C) SARS-CoV-2 titer reduction (left) and percent inhibition (right) in Calu-3 cells. Cells were infected with at an MOI of 0.1 for 30 min, washed and exposed to a dose response of NHC in triplicate per condition. 72 hours post infection, virus production was measured by plaque assay. (D) SARS-CoV-2 genomic RNA reduction (left) and percent inhibition (right) in Calu-3 cells. Viral RNA was isolated from clarified supernatants from the study in panel C . Genome copy numbers were quantitated by qRT-PCR with primer/probes targeting the N gene. For A-D , the symbol is at the mean and the error bars represent the standard deviation.
    Figure Legend Snippet: NHC potently inhibits MERS-CoV and newly emerging SARS-CoV-2 replication. (A) Percent inhibition of MERS-CoV replication and NHC cytotoxicity in Calu-3 cells. Calu-3 cells were infected in triplicate with MERS-CoV nanoluciferase (nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via Cell-Titer-Glo assay. Data are combined from 3 independent experiments. (B) NHC antiviral activity and cytotoxicity in Vero E6 cells infected with SARS-CoV-2. Vero E6 cells were infected in duplicate with SARS-CoV-2 clinical isolate 2019-nCoV/USA-WA1/2020 virus at an MOI of 0.05 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of cell viability by Cell-Titer-Glo assay. Cytotoxicity was measured as in A . Data are combined from 2 independent experiments. ( C) SARS-CoV-2 titer reduction (left) and percent inhibition (right) in Calu-3 cells. Cells were infected with at an MOI of 0.1 for 30 min, washed and exposed to a dose response of NHC in triplicate per condition. 72 hours post infection, virus production was measured by plaque assay. (D) SARS-CoV-2 genomic RNA reduction (left) and percent inhibition (right) in Calu-3 cells. Viral RNA was isolated from clarified supernatants from the study in panel C . Genome copy numbers were quantitated by qRT-PCR with primer/probes targeting the N gene. For A-D , the symbol is at the mean and the error bars represent the standard deviation.

    Techniques Used: Inhibition, Infection, Quantitation Assay, Glo Assay, Activity Assay, Plaque Assay, Isolation, Quantitative RT-PCR, Standard Deviation

    NHC is highly active against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary human airway epithelial cell cultures. (A) HAE were infected at an MOI of 0.5 with clinical isolate SARS-CoV-2 for 2 hours in the presence of NHC in duplicate after which virus was removed and cultures were washed in incubated in NHC for 48 hours when apical washes were collected for virus titration by plaque assay. The line is at the mean. Each symbol represents the titer from a single well. (B) HAE cells were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in triplicate and treated similarly to A . qRT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from cultures in C for qRT-PCR analysis. Representative data from three separate experiments with three different cell donors are displayed. PFU, plaque-forming units. (C) Studies performed as in A but with SARS-CoV green fluorescent protein (GFP). Representative data from two separate experiments with two different cell donors are displayed. Each symbol represents the data from one HAE culture, the line is at the mean and the error bars represent the standard deviation.
    Figure Legend Snippet: NHC is highly active against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary human airway epithelial cell cultures. (A) HAE were infected at an MOI of 0.5 with clinical isolate SARS-CoV-2 for 2 hours in the presence of NHC in duplicate after which virus was removed and cultures were washed in incubated in NHC for 48 hours when apical washes were collected for virus titration by plaque assay. The line is at the mean. Each symbol represents the titer from a single well. (B) HAE cells were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in triplicate and treated similarly to A . qRT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from cultures in C for qRT-PCR analysis. Representative data from three separate experiments with three different cell donors are displayed. PFU, plaque-forming units. (C) Studies performed as in A but with SARS-CoV green fluorescent protein (GFP). Representative data from two separate experiments with two different cell donors are displayed. Each symbol represents the data from one HAE culture, the line is at the mean and the error bars represent the standard deviation.

    Techniques Used: Infection, Incubation, Titration, Plaque Assay, Quantitative RT-PCR, Isolation, Standard Deviation

    Remdesivir (RDV) resistance mutations in the highly conserved RNA-dependent RNA polymerase increase susceptibility to NHC. (A) Neighbor-joining trees created with representatives from all four CoV genogroups showing the genetic similarity of CoV nsp12 (RdRp) and CoV spike glycoprotein, which mediates host tropism and entry into cells. Text color of the virus strain label corresponds to virus host species on the left. The heatmap adjacent to each neighbor-joining tree depicts percent amino acid identity (% A.A. similarity) against mouse hepatitis virus (MHV), SARS-CoV, or MERS-CoV. (B) The variation encompassed in panel A was modeled onto the RdRp structure of the SARS-CoV RdRp. (C) Amino acid sequence of CoV in panel A at known resistance alleles to antiviral drug RDV. (D) Virus titer reduction assay in DBT cells across a range of NHC with recombinant MHV bearing resistance mutations to RDV. Data shown are combined from three independent experiments performed with biological duplicates or triplicates per condition. Asterisks indicate statistically significant differences by Mann-Whitney test as indicated on the graph.
    Figure Legend Snippet: Remdesivir (RDV) resistance mutations in the highly conserved RNA-dependent RNA polymerase increase susceptibility to NHC. (A) Neighbor-joining trees created with representatives from all four CoV genogroups showing the genetic similarity of CoV nsp12 (RdRp) and CoV spike glycoprotein, which mediates host tropism and entry into cells. Text color of the virus strain label corresponds to virus host species on the left. The heatmap adjacent to each neighbor-joining tree depicts percent amino acid identity (% A.A. similarity) against mouse hepatitis virus (MHV), SARS-CoV, or MERS-CoV. (B) The variation encompassed in panel A was modeled onto the RdRp structure of the SARS-CoV RdRp. (C) Amino acid sequence of CoV in panel A at known resistance alleles to antiviral drug RDV. (D) Virus titer reduction assay in DBT cells across a range of NHC with recombinant MHV bearing resistance mutations to RDV. Data shown are combined from three independent experiments performed with biological duplicates or triplicates per condition. Asterisks indicate statistically significant differences by Mann-Whitney test as indicated on the graph.

    Techniques Used: Sequencing, Recombinant, MANN-WHITNEY

    NHC is effective against multiple genetically distinct Bat-CoV. Top: Antiviral efficacy of NHC in HAE cells against SARS-like (HKU3, SHC014, group 2b) and MERS-like (HKU5, group 2c) bat-CoV. HAE cells were infected at an MOI of 0.5 in the presence of NHC in duplicate. After 48 hours, virus produced was titrated via plaque assay. Each data point represents the titer per culture. Bottom: qRT-PCR for CoV ORF1 and ORFN mRNA in total RNA from cultures in the top panel. Mock, mock-treated. Representative data from two separate experiments with two different cell donors are displayed.
    Figure Legend Snippet: NHC is effective against multiple genetically distinct Bat-CoV. Top: Antiviral efficacy of NHC in HAE cells against SARS-like (HKU3, SHC014, group 2b) and MERS-like (HKU5, group 2c) bat-CoV. HAE cells were infected at an MOI of 0.5 in the presence of NHC in duplicate. After 48 hours, virus produced was titrated via plaque assay. Each data point represents the titer per culture. Bottom: qRT-PCR for CoV ORF1 and ORFN mRNA in total RNA from cultures in the top panel. Mock, mock-treated. Representative data from two separate experiments with two different cell donors are displayed.

    Techniques Used: Infection, Produced, Plaque Assay, Quantitative RT-PCR

    Prophylactic and therapeutic EIDD-2801 reduces MERS-CoV replication and pathogenesis coincident with increased viral mutation rates . Equivalent numbers of 10-14 week old male and female C57BL/6 hDPP4 mice were administered vehicle (10% PEG, 2.5% Cremophor RH40 in water) or NHC prodrug EIDD-2801 beginning at -2 hours, +12, +24 or +48 hours post infection and every 12 hours thereafter by oral gavage (n = 10/group). Mice were intranasally infected with 5E+04 PFU mouse-adapted MERS-CoV M35C4 strain. ( A) Percent starting weight. Asterisks indicate differences between -2 hours and +12 hours group from vehicle by two-way ANOVA with Tukey’s multiple comparison test. ( B) Lung hemorrhage in mice from panel A scored on a scale of 0-4 where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. (C) Virus lung titer in mice from panel A as determined by plaque assay. Asterisks in both panel B and C indicate differences from vehicle by Kruskal-Wallis with Dunn’s multiple comparison test. (D) MERS-CoV genomic RNA in lung tissue by qRT-PCR. Asterisks indicate differences by one-way ANOVA with a Dunnett’s multiple comparison test. (E) Pulmonary function by whole body plethysmography was performed daily on four animals per group. Asterisks indicate differences from vehicle by two-way ANOVA with Tukey’s multiple comparison test. (F) Workflow to measure mutation rate in MERS-CoV RNA and host transcript ISG15 by Primer ID in mouse lung tissue. (G) Number of template consensus sequences (TCS) for MERS-CoV nsp10 and ISG15. (H) Total error rate in MERS-CoV nsp10 and ISG15. (I) The cytosine to uridine transition rate in MERS-CoV nsp10 and ISG15. In panels G-I, asterisks indicate differences from vehicle by two-way ANOVA with Tukey’s multiple comparison test. (J) Codon change frequency in MERS-CoV nsp10. Asterisks indicate differences from vehicle by Kruskal-Wallis with Dunn’s multiple comparison test. For all panels, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range.
    Figure Legend Snippet: Prophylactic and therapeutic EIDD-2801 reduces MERS-CoV replication and pathogenesis coincident with increased viral mutation rates . Equivalent numbers of 10-14 week old male and female C57BL/6 hDPP4 mice were administered vehicle (10% PEG, 2.5% Cremophor RH40 in water) or NHC prodrug EIDD-2801 beginning at -2 hours, +12, +24 or +48 hours post infection and every 12 hours thereafter by oral gavage (n = 10/group). Mice were intranasally infected with 5E+04 PFU mouse-adapted MERS-CoV M35C4 strain. ( A) Percent starting weight. Asterisks indicate differences between -2 hours and +12 hours group from vehicle by two-way ANOVA with Tukey’s multiple comparison test. ( B) Lung hemorrhage in mice from panel A scored on a scale of 0-4 where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. (C) Virus lung titer in mice from panel A as determined by plaque assay. Asterisks in both panel B and C indicate differences from vehicle by Kruskal-Wallis with Dunn’s multiple comparison test. (D) MERS-CoV genomic RNA in lung tissue by qRT-PCR. Asterisks indicate differences by one-way ANOVA with a Dunnett’s multiple comparison test. (E) Pulmonary function by whole body plethysmography was performed daily on four animals per group. Asterisks indicate differences from vehicle by two-way ANOVA with Tukey’s multiple comparison test. (F) Workflow to measure mutation rate in MERS-CoV RNA and host transcript ISG15 by Primer ID in mouse lung tissue. (G) Number of template consensus sequences (TCS) for MERS-CoV nsp10 and ISG15. (H) Total error rate in MERS-CoV nsp10 and ISG15. (I) The cytosine to uridine transition rate in MERS-CoV nsp10 and ISG15. In panels G-I, asterisks indicate differences from vehicle by two-way ANOVA with Tukey’s multiple comparison test. (J) Codon change frequency in MERS-CoV nsp10. Asterisks indicate differences from vehicle by Kruskal-Wallis with Dunn’s multiple comparison test. For all panels, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range.

    Techniques Used: Mutagenesis, Mouse Assay, Infection, Plaque Assay, Quantitative RT-PCR

    NHC antiviral activity is associated with increased viral mutation rates . ( A ) HAE cultures were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of vehicle, RDV, or NHC for 48 hours, after which apical washes were collected for virus titration. Data are combined from two independent studies. The boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. (B) Schematic of Primer ID deep sequencing for single RNA genomes of MERS-CoV. (C) The total error rate for MERS-CoV RNA isolated from cultures in panel A as determined by Primer ID. Error rate values are # mutations per 10,000 bases. Asterisks indicate significant differences as compared to untreated group by two-way ANOVA with a Dunnett’s multiple comparison test. (D) Description of potential NHC mutational spectra on both positive and negative sense viral RNA. (E) Nucleotide transitions in cDNA derived from MERS-CoV genomic RNA.
    Figure Legend Snippet: NHC antiviral activity is associated with increased viral mutation rates . ( A ) HAE cultures were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of vehicle, RDV, or NHC for 48 hours, after which apical washes were collected for virus titration. Data are combined from two independent studies. The boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. (B) Schematic of Primer ID deep sequencing for single RNA genomes of MERS-CoV. (C) The total error rate for MERS-CoV RNA isolated from cultures in panel A as determined by Primer ID. Error rate values are # mutations per 10,000 bases. Asterisks indicate significant differences as compared to untreated group by two-way ANOVA with a Dunnett’s multiple comparison test. (D) Description of potential NHC mutational spectra on both positive and negative sense viral RNA. (E) Nucleotide transitions in cDNA derived from MERS-CoV genomic RNA.

    Techniques Used: Activity Assay, Mutagenesis, Infection, Titration, Sequencing, Isolation, Derivative Assay

    3) Product Images from "Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction"

    Article Title: Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction

    Journal: bioRxiv

    doi: 10.1101/2020.06.18.159434

    Effect of sample volume on SARS-CoV-2 detection. γ-irradiated SARS-CoV-2 (1.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined with TBE buffer 1:1 at a final working concentration of 1X. The sample was distributed into either 50 mL conical or 1.5 mL microfuge tubes, at either 10% (5 mL in 50 mL conical, 150 μL in 1.5 ml microfuge), 5% (2.5 mL in 50 ml conical, 75 μL in 1.5 ml microfuge), or 1% (0.5 mL in 50 mL conical, 15 μL in 1.5 mL microfuge) the vessel storage capacity. Samples were incubated in a hot water bath at 95°C for 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.
    Figure Legend Snippet: Effect of sample volume on SARS-CoV-2 detection. γ-irradiated SARS-CoV-2 (1.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined with TBE buffer 1:1 at a final working concentration of 1X. The sample was distributed into either 50 mL conical or 1.5 mL microfuge tubes, at either 10% (5 mL in 50 mL conical, 150 μL in 1.5 ml microfuge), 5% (2.5 mL in 50 ml conical, 75 μL in 1.5 ml microfuge), or 1% (0.5 mL in 50 mL conical, 15 μL in 1.5 mL microfuge) the vessel storage capacity. Samples were incubated in a hot water bath at 95°C for 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.

    Techniques Used: Irradiation, Concentration Assay, Incubation, Purification, Positive Control, Negative Control, Quantitative RT-PCR

    RNA stabilizing additive optimization. γ-irradiated SARS-CoV-2 (1.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined with TBE buffer, at a final working concentration of 1X. Samples (0.5 mL in 50 mL conical tubes) were incubated in a hot water bath at 95°C for 30 min. Following heat treatment, virus-spiked saliva was combined with various RNA stabilizing agents, including RNaseI (1 U/μL), carrier RNA (0.05 μg/mL), glycogen (1 μg/μL), TCEP/EDTA (1X), Proteinase K (5 μg/μL), RNase-free BSA (1.25 mg/mL), RNAlater (1:1 ratio in place of TBE), or PBS/DTT (6.5 mM DTT in PBS, diluted 1:1 in place of TBE). All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples with or without additives, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.
    Figure Legend Snippet: RNA stabilizing additive optimization. γ-irradiated SARS-CoV-2 (1.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined with TBE buffer, at a final working concentration of 1X. Samples (0.5 mL in 50 mL conical tubes) were incubated in a hot water bath at 95°C for 30 min. Following heat treatment, virus-spiked saliva was combined with various RNA stabilizing agents, including RNaseI (1 U/μL), carrier RNA (0.05 μg/mL), glycogen (1 μg/μL), TCEP/EDTA (1X), Proteinase K (5 μg/μL), RNase-free BSA (1.25 mg/mL), RNAlater (1:1 ratio in place of TBE), or PBS/DTT (6.5 mM DTT in PBS, diluted 1:1 in place of TBE). All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples with or without additives, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.

    Techniques Used: Irradiation, Concentration Assay, Incubation, Purification, Positive Control, Negative Control, Quantitative RT-PCR

    The effect of heat on SARS-CoV-2 detection. γ-irradiated SARS-CoV-2 (from BEI, used at 1.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative). Samples diluted 1:1 with 2X Tris-borate-EDTA (TBE) buffer (0.5 mL in 50 mL conical tubes) were incubated at 25°C (ambient temperature), or in a hot water bath at 65°C, 75°C, or 95°C, for 1, 5, 15, or 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.
    Figure Legend Snippet: The effect of heat on SARS-CoV-2 detection. γ-irradiated SARS-CoV-2 (from BEI, used at 1.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative). Samples diluted 1:1 with 2X Tris-borate-EDTA (TBE) buffer (0.5 mL in 50 mL conical tubes) were incubated at 25°C (ambient temperature), or in a hot water bath at 65°C, 75°C, or 95°C, for 1, 5, 15, or 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.

    Techniques Used: Irradiation, Incubation, Purification, Positive Control, Negative Control, Quantitative RT-PCR

    Specificity of SARS-CoV-2 detection system. Commercially available saliva (Lee Biosciences and Innovative Research) were combined in equal proportions, diluted 1:1 with 2X TBE buffer, and spiked 1.0×10 5 viral copies/mL of SARS-CoV-2 (γ-irradiated virus or synthetic N-transcript RNA), human coronaviruses (229E, OC43), SARS and MERS synthetic RNA, and human RNA (purified from HEK 293 cells). Samples were heat treated at 95°C for 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.
    Figure Legend Snippet: Specificity of SARS-CoV-2 detection system. Commercially available saliva (Lee Biosciences and Innovative Research) were combined in equal proportions, diluted 1:1 with 2X TBE buffer, and spiked 1.0×10 5 viral copies/mL of SARS-CoV-2 (γ-irradiated virus or synthetic N-transcript RNA), human coronaviruses (229E, OC43), SARS and MERS synthetic RNA, and human RNA (purified from HEK 293 cells). Samples were heat treated at 95°C for 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.

    Techniques Used: Irradiation, Purification, Positive Control, Negative Control, Quantitative RT-PCR

    LOD of direct saliva-to-RT-qPCR SARS-CoV-2 detection using CDC-approved primers and probes. Heat-inactivated (a, b, c) and γ-irradiated (d, e, f) SARS-CoV-2 was spiked into fresh human saliva (SARS-CoV-2 negative) in 1X Tris-Borate-EDTA buffer (TBE) at 1.0×10 2 , 5.0×10 2 , 1.0×10 3 , 2.5×10 3 , 5.0×10 3 , 1.0×10 4 , and 5.0×10 4 viral copies/mL. Samples were incubated at 95°C for 30 min. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL) and a negative control (neg; water) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 N1 gene (a, d) and N2 gene (b, e), and the human RP gene (c, f). Undetermined Ct values are plotted at 0.
    Figure Legend Snippet: LOD of direct saliva-to-RT-qPCR SARS-CoV-2 detection using CDC-approved primers and probes. Heat-inactivated (a, b, c) and γ-irradiated (d, e, f) SARS-CoV-2 was spiked into fresh human saliva (SARS-CoV-2 negative) in 1X Tris-Borate-EDTA buffer (TBE) at 1.0×10 2 , 5.0×10 2 , 1.0×10 3 , 2.5×10 3 , 5.0×10 3 , 1.0×10 4 , and 5.0×10 4 viral copies/mL. Samples were incubated at 95°C for 30 min. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL) and a negative control (neg; water) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 N1 gene (a, d) and N2 gene (b, e), and the human RP gene (c, f). Undetermined Ct values are plotted at 0.

    Techniques Used: Quantitative RT-PCR, Irradiation, Incubation, Positive Control, Negative Control

    ( A ) The effect of collection buffer on SARS-CoV-2 detection. γ-irradiated SARS-CoV-2 (from BEI, at 1.0×10 3 or 1.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined at a 1:1 ratio with Tris-Borate-EDTA (TBE), Tris-EDTA (TE), Phosphate Buffered Saline (PBS), DNA/RNA shield (Zymo Research), or SDNA-1000 (Spectrum Solutions) such that the final concentration of each buffer was 1X. Samples (0.5 mL in 50 mL conical tubes) were incubated in a hot water bath at 95°C for 30 min. ( B ) Detergent optimization. γ-irradiated SARS-CoV-2 (1.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined 1:1 with TBE buffer at a final working concentration of 1X. Samples were treated with detergents (Triton X-100, 1%, 0.5%, 0.25%; Tween 20, 1%, 0.5%, 0.25%; NP-40, 2%, 1%, 0.5%) after heating at 95°C for 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.
    Figure Legend Snippet: ( A ) The effect of collection buffer on SARS-CoV-2 detection. γ-irradiated SARS-CoV-2 (from BEI, at 1.0×10 3 or 1.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined at a 1:1 ratio with Tris-Borate-EDTA (TBE), Tris-EDTA (TE), Phosphate Buffered Saline (PBS), DNA/RNA shield (Zymo Research), or SDNA-1000 (Spectrum Solutions) such that the final concentration of each buffer was 1X. Samples (0.5 mL in 50 mL conical tubes) were incubated in a hot water bath at 95°C for 30 min. ( B ) Detergent optimization. γ-irradiated SARS-CoV-2 (1.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined 1:1 with TBE buffer at a final working concentration of 1X. Samples were treated with detergents (Triton X-100, 1%, 0.5%, 0.25%; Tween 20, 1%, 0.5%, 0.25%; NP-40, 2%, 1%, 0.5%) after heating at 95°C for 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.

    Techniques Used: Irradiation, Concentration Assay, Incubation, Purification, Positive Control, Negative Control, Quantitative RT-PCR

    Limit of Detection (LOD) reproducibility. γ-irradiated SARS-CoV-2 was spiked into human saliva (SARS-CoV-2 negative), sourced fresh from two healthy donors, and purchased from two companies, in 1X TBE buffer at 1.0×10 3 viral copies/mL. Samples were incubated at 95°C for 30 min, then Tween 20 was added to a final concentration of 0.5%. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples were directly analyzed by RT-qPCR (direct saliva). All samples, including a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were analyzed by RT-qPCR, in replicates of 5, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.
    Figure Legend Snippet: Limit of Detection (LOD) reproducibility. γ-irradiated SARS-CoV-2 was spiked into human saliva (SARS-CoV-2 negative), sourced fresh from two healthy donors, and purchased from two companies, in 1X TBE buffer at 1.0×10 3 viral copies/mL. Samples were incubated at 95°C for 30 min, then Tween 20 was added to a final concentration of 0.5%. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples were directly analyzed by RT-qPCR (direct saliva). All samples, including a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were analyzed by RT-qPCR, in replicates of 5, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.

    Techniques Used: Irradiation, Incubation, Concentration Assay, Purification, Quantitative RT-PCR, Positive Control, Negative Control

    Effect of centrifugation on SARS-CoV-2 detection. Heat-inactivated SARS-CoV-2 (1.0×10 2 , 5.0×10 2 , 1.0×10 3 , 5.0×10 3 , 1.0×10 4 , and 5.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined with TBE buffer 1:1 at a final working concentration of 1X. Samples were heat treated at 95°C for 30 min, then treated with or without centrifugation at 3000 rpm for 2 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, centrifugation supernatants, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL) and a negative control (neg; water) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.
    Figure Legend Snippet: Effect of centrifugation on SARS-CoV-2 detection. Heat-inactivated SARS-CoV-2 (1.0×10 2 , 5.0×10 2 , 1.0×10 3 , 5.0×10 3 , 1.0×10 4 , and 5.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined with TBE buffer 1:1 at a final working concentration of 1X. Samples were heat treated at 95°C for 30 min, then treated with or without centrifugation at 3000 rpm for 2 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, centrifugation supernatants, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL) and a negative control (neg; water) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.

    Techniques Used: Centrifugation, Concentration Assay, Purification, Positive Control, Negative Control, Quantitative RT-PCR

    Limit of Detection (LOD) for assessment of SARS-CoV-2 from saliva, comparing a process utilizing RNA isolation/purification to one that bypasses RNA isolation/purification. γ-irradiated SARS-CoV-2 was spiked into fresh human saliva (SARS-CoV-2 negative), with or without TBE buffer(1X) at 1.0×10 2 , 5.0×10 2 , 1.0×10 3 , 2.5×10 3 , 5.0×10 3 , 1.0×10 4 , 5.0×10 4 , 1.0×10 5 , and 5.0×10 5 viral copies/mL. Samples were incubated at 95°C for 30 min, then combined with or without Tween 20 (0.5%). All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples were either processed for RNA extraction followed by RT-qPCR (purified RNA), or directly analyzed by RT-qPCR (direct saliva). All samples, including a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0. The limit of detection (LOD) is indicated by the dotted vertical line.
    Figure Legend Snippet: Limit of Detection (LOD) for assessment of SARS-CoV-2 from saliva, comparing a process utilizing RNA isolation/purification to one that bypasses RNA isolation/purification. γ-irradiated SARS-CoV-2 was spiked into fresh human saliva (SARS-CoV-2 negative), with or without TBE buffer(1X) at 1.0×10 2 , 5.0×10 2 , 1.0×10 3 , 2.5×10 3 , 5.0×10 3 , 1.0×10 4 , 5.0×10 4 , 1.0×10 5 , and 5.0×10 5 viral copies/mL. Samples were incubated at 95°C for 30 min, then combined with or without Tween 20 (0.5%). All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples were either processed for RNA extraction followed by RT-qPCR (purified RNA), or directly analyzed by RT-qPCR (direct saliva). All samples, including a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0. The limit of detection (LOD) is indicated by the dotted vertical line.

    Techniques Used: Isolation, Purification, Irradiation, Incubation, RNA Extraction, Quantitative RT-PCR, Positive Control, Negative Control

    Schematic of SARS-CoV-2 testing. A) The current, widely-utilized diagnostic process involves nasopharyngeal (NP) swabs and viral transport media (VTM), followed by RNA extraction and isolation, with RT-qPCR analysis of the samples. NP swabs, VTM, and RNA purification kits have been in short supply at various times. B) In April of 2020, saliva was emergency use authorized (EUA) as a diagnostic sample, using RNA extraction and isolation, followed by RT-qPCR. C) Other groups have reported direct testing of NP swabs in VTM by RT-qPCR. D) The University of Illinois at Urbana-Champaign (UIUC) protocol involves saliva collection in standard 50 mL conical tubes, heating (95°C for 30 min), followed by addition of buffer and analysis by RT-qPCR.
    Figure Legend Snippet: Schematic of SARS-CoV-2 testing. A) The current, widely-utilized diagnostic process involves nasopharyngeal (NP) swabs and viral transport media (VTM), followed by RNA extraction and isolation, with RT-qPCR analysis of the samples. NP swabs, VTM, and RNA purification kits have been in short supply at various times. B) In April of 2020, saliva was emergency use authorized (EUA) as a diagnostic sample, using RNA extraction and isolation, followed by RT-qPCR. C) Other groups have reported direct testing of NP swabs in VTM by RT-qPCR. D) The University of Illinois at Urbana-Champaign (UIUC) protocol involves saliva collection in standard 50 mL conical tubes, heating (95°C for 30 min), followed by addition of buffer and analysis by RT-qPCR.

    Techniques Used: Diagnostic Assay, RNA Extraction, Isolation, Quantitative RT-PCR, Purification

    Workflow of TBE and Tween addition in relation to heat. γ-irradiated SARS-CoV-2 (1.0×10 5 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined with TBE buffer (1:10, final concentration 1X) and Tween 20 (1:20, final concentration 0.5%) alone or in combination, before or after heat treatment at 95°C for 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.
    Figure Legend Snippet: Workflow of TBE and Tween addition in relation to heat. γ-irradiated SARS-CoV-2 (1.0×10 5 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined with TBE buffer (1:10, final concentration 1X) and Tween 20 (1:20, final concentration 0.5%) alone or in combination, before or after heat treatment at 95°C for 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.

    Techniques Used: Irradiation, Concentration Assay, Purification, Positive Control, Negative Control, Quantitative RT-PCR

    Saliva collection buffer titration. γ-irradiated SARS-CoV-2 (1.0×10 3 (a) or 1.0×10 4 (b) viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined with Tris-Borate-EDTA buffer (TBE), Tris-EDTA buffer (TE), or Phosphate Buffered Saline (PBS), at a final working concentration of 2X, 1.5X, 1X, or 0.5X. Samples (0.5 mL in 50 mL conical tubes) were incubated in a hot water bath at 95°C for 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.
    Figure Legend Snippet: Saliva collection buffer titration. γ-irradiated SARS-CoV-2 (1.0×10 3 (a) or 1.0×10 4 (b) viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined with Tris-Borate-EDTA buffer (TBE), Tris-EDTA buffer (TE), or Phosphate Buffered Saline (PBS), at a final working concentration of 2X, 1.5X, 1X, or 0.5X. Samples (0.5 mL in 50 mL conical tubes) were incubated in a hot water bath at 95°C for 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples, a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.

    Techniques Used: Titration, Irradiation, Concentration Assay, Incubation, Purification, Positive Control, Negative Control, Quantitative RT-PCR

    Limit of detection optimization. Heat-inactivated SARS-CoV-2 was spiked into fresh human saliva (SARS-CoV-2 negative) in 0.5X TE or water at 5.0×10 2 , 2.5×10 3 , 5.0×10 3 , 2.5×10 4 , 5.0×10 4 , and 2.5×10 5 viral copies/mL. Samples were incubated at 95°C for 30 min. All samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked samples were either processed for RNA extraction using a commercially available kit (MagMAX), or directly analyzed by RT-qPCR (direct saliva). All samples, including a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0. The limit of detection (LOD) is indicated by the vertical dotted line.
    Figure Legend Snippet: Limit of detection optimization. Heat-inactivated SARS-CoV-2 was spiked into fresh human saliva (SARS-CoV-2 negative) in 0.5X TE or water at 5.0×10 2 , 2.5×10 3 , 5.0×10 3 , 2.5×10 4 , 5.0×10 4 , and 2.5×10 5 viral copies/mL. Samples were incubated at 95°C for 30 min. All samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked samples were either processed for RNA extraction using a commercially available kit (MagMAX), or directly analyzed by RT-qPCR (direct saliva). All samples, including a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0. The limit of detection (LOD) is indicated by the vertical dotted line.

    Techniques Used: Incubation, Purification, RNA Extraction, Quantitative RT-PCR, Positive Control, Negative Control

    Stability of saliva samples. γ-irradiated SARS-CoV-2 (1.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined with TBE buffer 1:1 to a final working concentration of 1X. Samples (0.5 mL in 50 mL conical tubes) were stored at 25°C (ambient temperature), 4°C, −20°C, or −80°C for 1, 2, 4, 8, 12, and 24 hours. Following storage, samples were incubated in a hot water bath at 95°C for 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples stored under different conditions, a freshly prepared virus-spiked saliva sample (0 hr), a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.
    Figure Legend Snippet: Stability of saliva samples. γ-irradiated SARS-CoV-2 (1.0×10 4 viral copies/mL) was spiked into fresh human saliva (SARS-CoV-2 negative) and combined with TBE buffer 1:1 to a final working concentration of 1X. Samples (0.5 mL in 50 mL conical tubes) were stored at 25°C (ambient temperature), 4°C, −20°C, or −80°C for 1, 2, 4, 8, 12, and 24 hours. Following storage, samples were incubated in a hot water bath at 95°C for 30 min. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Virus-spiked saliva samples stored under different conditions, a freshly prepared virus-spiked saliva sample (0 hr), a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL, no MS2) and a negative control (neg; water, no MS2) were directly analyzed by RT-qPCR, in triplicate, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). Undetermined Ct values are plotted at 0.

    Techniques Used: Irradiation, Concentration Assay, Incubation, Purification, Positive Control, Negative Control, Quantitative RT-PCR

    Assessment of clinical samples. Saliva samples from 9 SARS-CoV-2 positive and 91 SARS-CoV-2 negative patients (as judged by NP swabs in VTM with RNA extraction) had TE buffer added to them (at a 1:1 ratio) and were frozen for over a week. Upon thawing, 10X TBE buffer was added to the samples at a final concentration of 1X, heated at 95°C for 30 min, cooled to room temp, and Tween 20 was added to a final concentration of 0.5%. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Saliva samples were directly analyzed by RT-qPCR. All samples, including a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL) and a negative control (neg; water) were analyzed by RT-qPCR, in singlet, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). This figure shows one of the two replicates. Undetermined Ct values are plotted at 0.
    Figure Legend Snippet: Assessment of clinical samples. Saliva samples from 9 SARS-CoV-2 positive and 91 SARS-CoV-2 negative patients (as judged by NP swabs in VTM with RNA extraction) had TE buffer added to them (at a 1:1 ratio) and were frozen for over a week. Upon thawing, 10X TBE buffer was added to the samples at a final concentration of 1X, heated at 95°C for 30 min, cooled to room temp, and Tween 20 was added to a final concentration of 0.5%. All saliva samples were spiked with purified MS2 bacteriophage (1:40 MS2:sample) as an internal control. Saliva samples were directly analyzed by RT-qPCR. All samples, including a positive control (pos; SARS-CoV-2 positive control, 5.0×10 3 copies/mL) and a negative control (neg; water) were analyzed by RT-qPCR, in singlet, for SARS-CoV-2 ORF1ab (green triangle), N-gene (red square), and S-gene (blue circle), and MS2 (open circle). This figure shows one of the two replicates. Undetermined Ct values are plotted at 0.

    Techniques Used: RNA Extraction, Concentration Assay, Purification, Quantitative RT-PCR, Positive Control, Negative Control

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    Integrated DNA Technologies mers genomic rna
    NHC is effective against multiple genetically distinct Bat-CoV. Top: Antiviral efficacy of NHC in HAE cells against SARS-like (HKU3, SHC014, group 2b) and <t>MERS-like</t> (HKU5, group 2c) bat-CoV. HAE cells were infected at an MOI of 0.5 in the presence of NHC in duplicate. After 48 hours, virus produced was titrated via plaque assay. Each data point represents the titer per culture. Bottom: qRT-PCR for CoV ORF1 and ORFN mRNA in total <t>RNA</t> from cultures in the top panel. Representative data from two separate experiments with two different cell donors are displayed.
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    NHC is effective against multiple genetically distinct Bat-CoV. Top: Antiviral efficacy of NHC in HAE cells against SARS-like (HKU3, SHC014, group 2b) and MERS-like (HKU5, group 2c) bat-CoV. HAE cells were infected at an MOI of 0.5 in the presence of NHC in duplicate. After 48 hours, virus produced was titrated via plaque assay. Each data point represents the titer per culture. Bottom: qRT-PCR for CoV ORF1 and ORFN mRNA in total RNA from cultures in the top panel. Representative data from two separate experiments with two different cell donors are displayed.

    Journal: bioRxiv

    Article Title: An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and multiple endemic, epidemic and bat coronavirus

    doi: 10.1101/2020.03.19.997890

    Figure Lengend Snippet: NHC is effective against multiple genetically distinct Bat-CoV. Top: Antiviral efficacy of NHC in HAE cells against SARS-like (HKU3, SHC014, group 2b) and MERS-like (HKU5, group 2c) bat-CoV. HAE cells were infected at an MOI of 0.5 in the presence of NHC in duplicate. After 48 hours, virus produced was titrated via plaque assay. Each data point represents the titer per culture. Bottom: qRT-PCR for CoV ORF1 and ORFN mRNA in total RNA from cultures in the top panel. Representative data from two separate experiments with two different cell donors are displayed.

    Article Snippet: Previously published TaqMan primers were synthesized by Integrated DNA Technologies (IDT) to quantify MERS genomic RNA (targeting orf1a.

    Techniques: Infection, Produced, Plaque Assay, Quantitative RT-PCR

    Remdesivir resistance mutations in the highly conserved RNA-dependent RNA polymerase increase susceptibility to NHC. a , Neighbor-joining trees created with representatives from all four CoV genogroups showing the genetic similarity of CoV nsp12 (RdRp) and CoV spike glycoprotein, which mediates host tropism and entry into cells. Text color of the virus strain label corresponds to virus host species on the left. The heatmap adjacent to each neighbor-joining tree depicts percent amino acid identity (% A.A. similarity) against mouse hepatitis virus (MHV), SARS-CoV or MERS-CoV. b , Core residues of the CoV RdRp are highly conserved among CoV. The variation encompassed in panel a was modeled onto the RdRp structure of the SARS-CoV RdRp. c , Amino acid sequence of CoV in panel a at known resistance alleles to antiviral drug remdesivir (RDV). d , RDV resistant viruses are more susceptible to NHC antiviral activity. Virus titer reduction assay across a dose response of NHC with recombinant MHV bearing resistance mutations to RDV. Asterisks indicate statistically significant differences by Mann-Whitney test.

    Journal: bioRxiv

    Article Title: An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and multiple endemic, epidemic and bat coronavirus

    doi: 10.1101/2020.03.19.997890

    Figure Lengend Snippet: Remdesivir resistance mutations in the highly conserved RNA-dependent RNA polymerase increase susceptibility to NHC. a , Neighbor-joining trees created with representatives from all four CoV genogroups showing the genetic similarity of CoV nsp12 (RdRp) and CoV spike glycoprotein, which mediates host tropism and entry into cells. Text color of the virus strain label corresponds to virus host species on the left. The heatmap adjacent to each neighbor-joining tree depicts percent amino acid identity (% A.A. similarity) against mouse hepatitis virus (MHV), SARS-CoV or MERS-CoV. b , Core residues of the CoV RdRp are highly conserved among CoV. The variation encompassed in panel a was modeled onto the RdRp structure of the SARS-CoV RdRp. c , Amino acid sequence of CoV in panel a at known resistance alleles to antiviral drug remdesivir (RDV). d , RDV resistant viruses are more susceptible to NHC antiviral activity. Virus titer reduction assay across a dose response of NHC with recombinant MHV bearing resistance mutations to RDV. Asterisks indicate statistically significant differences by Mann-Whitney test.

    Article Snippet: Previously published TaqMan primers were synthesized by Integrated DNA Technologies (IDT) to quantify MERS genomic RNA (targeting orf1a.

    Techniques: Sequencing, Activity Assay, Recombinant, MANN-WHITNEY

    NHC potently Inhibits MERS-CoV, SARS-CoV and newly emerging SARS-CoV-2 Replication. a , NHC antiviral activity and cytotoxicity in Calu3 cells infected with MERS-CoV. Calu3 cells were infected in triplicate with MERS-CoV nanoluciferase (nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a dose response of drug for 48 hours, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via Cell-Titer-Glo assay. Data is combined from 3 independent experiments. b , NHC antiviral activity and cytotoxicity in Vero cells infected with SARS-CoV-2. Vero cells were infected in duplicate with SARS-CoV-2 clinical isolate virus at an MOI of 0.05 in the presence of a dose response of drug for 48 hours, after which replication was measured through quantitation of cell viability by Cell-Titer-Glo assay. Cytotoxicity was measured as in a . Data is combined from 2 independent experiments. c , NHC inhibits MERS-CoV virus production and RNA synthesis in primary human lung epithelial cell cultures (HAE). HAE cells were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of NHC for 48 hours, after which apical washes were collected for virus titration. qRT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from cultures in c for qRT-PCR analysis. Representative data from three separate experiments with three different cell donors are displayed. PFU, plaque-forming units. d , NHC inhibits SARS-CoV virus production and RNA synthesis in primary human lung epithelial cell cultures (HAE). Studies performed as in c but with SARS-CoV green fluorescent protein (GFP). Representative data from two separate experiments with two different cell donors are displayed.

    Journal: bioRxiv

    Article Title: An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and multiple endemic, epidemic and bat coronavirus

    doi: 10.1101/2020.03.19.997890

    Figure Lengend Snippet: NHC potently Inhibits MERS-CoV, SARS-CoV and newly emerging SARS-CoV-2 Replication. a , NHC antiviral activity and cytotoxicity in Calu3 cells infected with MERS-CoV. Calu3 cells were infected in triplicate with MERS-CoV nanoluciferase (nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a dose response of drug for 48 hours, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via Cell-Titer-Glo assay. Data is combined from 3 independent experiments. b , NHC antiviral activity and cytotoxicity in Vero cells infected with SARS-CoV-2. Vero cells were infected in duplicate with SARS-CoV-2 clinical isolate virus at an MOI of 0.05 in the presence of a dose response of drug for 48 hours, after which replication was measured through quantitation of cell viability by Cell-Titer-Glo assay. Cytotoxicity was measured as in a . Data is combined from 2 independent experiments. c , NHC inhibits MERS-CoV virus production and RNA synthesis in primary human lung epithelial cell cultures (HAE). HAE cells were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of NHC for 48 hours, after which apical washes were collected for virus titration. qRT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from cultures in c for qRT-PCR analysis. Representative data from three separate experiments with three different cell donors are displayed. PFU, plaque-forming units. d , NHC inhibits SARS-CoV virus production and RNA synthesis in primary human lung epithelial cell cultures (HAE). Studies performed as in c but with SARS-CoV green fluorescent protein (GFP). Representative data from two separate experiments with two different cell donors are displayed.

    Article Snippet: Previously published TaqMan primers were synthesized by Integrated DNA Technologies (IDT) to quantify MERS genomic RNA (targeting orf1a.

    Techniques: Activity Assay, Infection, Quantitation Assay, Glo Assay, Titration, Quantitative RT-PCR, Isolation

    NHC antiviral activity is associated with increased viral mutation rates. a , Both remdesivir (RDV) and NHC reduce MERS-CoV infectious virus production in primary human HAE. Cultures were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of vehicle, RDV or NHC for 48 hours, after which apical washes were collected for virus titration. Data is combined from two independent studies. b, A deep sequencing approach called Primer ID to gain accurate sequence data for single RNA genomes of MERS-CoV. c , The total error rate for MERS-CoV RNA isolated from cultures in panel a as determined by Primer ID. Error rate values are # mutations per 10,000 bases. Asterisks indicate significant differences as compared to untreated by 2-way ANOVA with a Dunnett’s multiple comparison test. d , description of potential NHC mutational spectra on both positive and negative sense viral RNA. e , Nucleotide transitions adenine (A) to guanine (G) and uridine (U) to cytosine (C) transitions are enriched in MERS-CoV genomic RNA in an NHC dose dependent manner.

    Journal: bioRxiv

    Article Title: An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and multiple endemic, epidemic and bat coronavirus

    doi: 10.1101/2020.03.19.997890

    Figure Lengend Snippet: NHC antiviral activity is associated with increased viral mutation rates. a , Both remdesivir (RDV) and NHC reduce MERS-CoV infectious virus production in primary human HAE. Cultures were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of vehicle, RDV or NHC for 48 hours, after which apical washes were collected for virus titration. Data is combined from two independent studies. b, A deep sequencing approach called Primer ID to gain accurate sequence data for single RNA genomes of MERS-CoV. c , The total error rate for MERS-CoV RNA isolated from cultures in panel a as determined by Primer ID. Error rate values are # mutations per 10,000 bases. Asterisks indicate significant differences as compared to untreated by 2-way ANOVA with a Dunnett’s multiple comparison test. d , description of potential NHC mutational spectra on both positive and negative sense viral RNA. e , Nucleotide transitions adenine (A) to guanine (G) and uridine (U) to cytosine (C) transitions are enriched in MERS-CoV genomic RNA in an NHC dose dependent manner.

    Article Snippet: Previously published TaqMan primers were synthesized by Integrated DNA Technologies (IDT) to quantify MERS genomic RNA (targeting orf1a.

    Techniques: Activity Assay, Mutagenesis, Infection, Titration, Sequencing, Isolation

    Prophylactic and therapeutic EIDD-2801 reduces MERS-CoV replication and pathogenesis coincident with increased viral mutation rates. Equivalent numbers of 10-14 week old male and female C57BL/6 hDPP4 mice were administered vehicle (10% PEG, 2.5% Cremophor RH40 in water) or NHC prodrug EIDD-2801 beginning at −2hr, +12, +24 or +48hr post infection and every 12hr thereafter by oral gavage (n = 10/group). Mice were intranasally infected with 5E+04 PFU mouse-adapted MERS-CoV M35C4 strain. a, Percent starting weight. Asterisks indicate differences by two-way ANOVA with Tukey’s multiple comparison test. b, Lung hemorrhage in mice from panel a scored on a scale of 0-4 where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. c , Virus lung titer in mice from panel a as determined by plaque assay. Asterisks in both panel b and c indicate differences by Kruskal-Wallis with Dunn’s multiple comparison test. d , MERS-CoV genomic RNA in lung tissue by qRT-PCR. Asterisks indicate differences by one-way ANOVA with a Dunnett’s multiple comparison test. e , Pulmonary function by whole body plethysmography was performed daily on four animals per group. Asterisks indicate differences by two-way ANOVA with Tukey’s multiple comparison test. f , Workflow to measure mutation rate in MERS-CoV RNA and host transcript ISG15 by Primer ID in mouse lung tissue. g , Number of template consensus sequences for MERS-CoV nsp10 and ISG15. h , Total error rate in MERS-CoV nsp10 and ISG15. i , The cytosine to uridine transition rate in MERS-CoV nsp10 and ISG15. In panels g-i, asterisks indicate differences by two-way ANOVA with Tukey’s multiple comparison test. j , Codon change frequency in MERS-CoV nsp10. Asterisks indicate differences on Kruskal-Wallis with Dunn’s multiple comparison test.

    Journal: bioRxiv

    Article Title: An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and multiple endemic, epidemic and bat coronavirus

    doi: 10.1101/2020.03.19.997890

    Figure Lengend Snippet: Prophylactic and therapeutic EIDD-2801 reduces MERS-CoV replication and pathogenesis coincident with increased viral mutation rates. Equivalent numbers of 10-14 week old male and female C57BL/6 hDPP4 mice were administered vehicle (10% PEG, 2.5% Cremophor RH40 in water) or NHC prodrug EIDD-2801 beginning at −2hr, +12, +24 or +48hr post infection and every 12hr thereafter by oral gavage (n = 10/group). Mice were intranasally infected with 5E+04 PFU mouse-adapted MERS-CoV M35C4 strain. a, Percent starting weight. Asterisks indicate differences by two-way ANOVA with Tukey’s multiple comparison test. b, Lung hemorrhage in mice from panel a scored on a scale of 0-4 where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. c , Virus lung titer in mice from panel a as determined by plaque assay. Asterisks in both panel b and c indicate differences by Kruskal-Wallis with Dunn’s multiple comparison test. d , MERS-CoV genomic RNA in lung tissue by qRT-PCR. Asterisks indicate differences by one-way ANOVA with a Dunnett’s multiple comparison test. e , Pulmonary function by whole body plethysmography was performed daily on four animals per group. Asterisks indicate differences by two-way ANOVA with Tukey’s multiple comparison test. f , Workflow to measure mutation rate in MERS-CoV RNA and host transcript ISG15 by Primer ID in mouse lung tissue. g , Number of template consensus sequences for MERS-CoV nsp10 and ISG15. h , Total error rate in MERS-CoV nsp10 and ISG15. i , The cytosine to uridine transition rate in MERS-CoV nsp10 and ISG15. In panels g-i, asterisks indicate differences by two-way ANOVA with Tukey’s multiple comparison test. j , Codon change frequency in MERS-CoV nsp10. Asterisks indicate differences on Kruskal-Wallis with Dunn’s multiple comparison test.

    Article Snippet: Previously published TaqMan primers were synthesized by Integrated DNA Technologies (IDT) to quantify MERS genomic RNA (targeting orf1a.

    Techniques: Mutagenesis, Mouse Assay, Infection, Plaque Assay, Quantitative RT-PCR

    NHC potently inhibits MERS-CoV and newly emerging SARS-CoV-2 replication. (A) Percent inhibition of MERS-CoV replication and NHC cytotoxicity in Calu-3 cells. Calu-3 cells were infected in triplicate with MERS-CoV nanoluciferase (nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via Cell-Titer-Glo assay. Data are combined from 3 independent experiments. (B) NHC antiviral activity and cytotoxicity in Vero E6 cells infected with SARS-CoV-2. Vero E6 cells were infected in duplicate with SARS-CoV-2 clinical isolate 2019-nCoV/USA-WA1/2020 virus at an MOI of 0.05 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of cell viability by Cell-Titer-Glo assay. Cytotoxicity was measured as in A . Data are combined from 2 independent experiments. ( C) SARS-CoV-2 titer reduction (left) and percent inhibition (right) in Calu-3 cells. Cells were infected with at an MOI of 0.1 for 30 min, washed and exposed to a dose response of NHC in triplicate per condition. 72 hours post infection, virus production was measured by plaque assay. (D) SARS-CoV-2 genomic RNA reduction (left) and percent inhibition (right) in Calu-3 cells. Viral RNA was isolated from clarified supernatants from the study in panel C . Genome copy numbers were quantitated by qRT-PCR with primer/probes targeting the N gene. For A-D , the symbol is at the mean and the error bars represent the standard deviation.

    Journal: Science Translational Medicine

    Article Title: An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice

    doi: 10.1126/scitranslmed.abb5883

    Figure Lengend Snippet: NHC potently inhibits MERS-CoV and newly emerging SARS-CoV-2 replication. (A) Percent inhibition of MERS-CoV replication and NHC cytotoxicity in Calu-3 cells. Calu-3 cells were infected in triplicate with MERS-CoV nanoluciferase (nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via Cell-Titer-Glo assay. Data are combined from 3 independent experiments. (B) NHC antiviral activity and cytotoxicity in Vero E6 cells infected with SARS-CoV-2. Vero E6 cells were infected in duplicate with SARS-CoV-2 clinical isolate 2019-nCoV/USA-WA1/2020 virus at an MOI of 0.05 in the presence of a range of drug for 48 hours, after which replication was measured through quantitation of cell viability by Cell-Titer-Glo assay. Cytotoxicity was measured as in A . Data are combined from 2 independent experiments. ( C) SARS-CoV-2 titer reduction (left) and percent inhibition (right) in Calu-3 cells. Cells were infected with at an MOI of 0.1 for 30 min, washed and exposed to a dose response of NHC in triplicate per condition. 72 hours post infection, virus production was measured by plaque assay. (D) SARS-CoV-2 genomic RNA reduction (left) and percent inhibition (right) in Calu-3 cells. Viral RNA was isolated from clarified supernatants from the study in panel C . Genome copy numbers were quantitated by qRT-PCR with primer/probes targeting the N gene. For A-D , the symbol is at the mean and the error bars represent the standard deviation.

    Article Snippet: Previously published TaqMan primers were synthesized by Integrated DNA Technologies (IDT) to quantify MERS genomic RNA (targeting orf1a: Forward: 5′- GCACATCTGTGGTTCTCCTCTCT-3′, Probe (6-FAM/ZEN/IBFQ): 5′- TGCTCCAACAGTTACAC-3′, Reverse: 5′-AAGCCCAGGCCCTACTATTAGC)( ). qRT-PCR was performed using 100ng total RNA compared to an RNA standard curve using TaqMan Fast Virus 1-Step Master Mix (ThermoFisher) on a Quant Studio 3 (Applied Biosystems).

    Techniques: Inhibition, Infection, Quantitation Assay, Glo Assay, Activity Assay, Plaque Assay, Isolation, Quantitative RT-PCR, Standard Deviation

    NHC is highly active against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary human airway epithelial cell cultures. (A) HAE were infected at an MOI of 0.5 with clinical isolate SARS-CoV-2 for 2 hours in the presence of NHC in duplicate after which virus was removed and cultures were washed in incubated in NHC for 48 hours when apical washes were collected for virus titration by plaque assay. The line is at the mean. Each symbol represents the titer from a single well. (B) HAE cells were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in triplicate and treated similarly to A . qRT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from cultures in C for qRT-PCR analysis. Representative data from three separate experiments with three different cell donors are displayed. PFU, plaque-forming units. (C) Studies performed as in A but with SARS-CoV green fluorescent protein (GFP). Representative data from two separate experiments with two different cell donors are displayed. Each symbol represents the data from one HAE culture, the line is at the mean and the error bars represent the standard deviation.

    Journal: Science Translational Medicine

    Article Title: An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice

    doi: 10.1126/scitranslmed.abb5883

    Figure Lengend Snippet: NHC is highly active against SARS-CoV-2, MERS-CoV, and SARS-CoV in primary human airway epithelial cell cultures. (A) HAE were infected at an MOI of 0.5 with clinical isolate SARS-CoV-2 for 2 hours in the presence of NHC in duplicate after which virus was removed and cultures were washed in incubated in NHC for 48 hours when apical washes were collected for virus titration by plaque assay. The line is at the mean. Each symbol represents the titer from a single well. (B) HAE cells were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in triplicate and treated similarly to A . qRT-PCR for MERS-CoV ORF1 and ORFN mRNA. Total RNA was isolated from cultures in C for qRT-PCR analysis. Representative data from three separate experiments with three different cell donors are displayed. PFU, plaque-forming units. (C) Studies performed as in A but with SARS-CoV green fluorescent protein (GFP). Representative data from two separate experiments with two different cell donors are displayed. Each symbol represents the data from one HAE culture, the line is at the mean and the error bars represent the standard deviation.

    Article Snippet: Previously published TaqMan primers were synthesized by Integrated DNA Technologies (IDT) to quantify MERS genomic RNA (targeting orf1a: Forward: 5′- GCACATCTGTGGTTCTCCTCTCT-3′, Probe (6-FAM/ZEN/IBFQ): 5′- TGCTCCAACAGTTACAC-3′, Reverse: 5′-AAGCCCAGGCCCTACTATTAGC)( ). qRT-PCR was performed using 100ng total RNA compared to an RNA standard curve using TaqMan Fast Virus 1-Step Master Mix (ThermoFisher) on a Quant Studio 3 (Applied Biosystems).

    Techniques: Infection, Incubation, Titration, Plaque Assay, Quantitative RT-PCR, Isolation, Standard Deviation

    Remdesivir (RDV) resistance mutations in the highly conserved RNA-dependent RNA polymerase increase susceptibility to NHC. (A) Neighbor-joining trees created with representatives from all four CoV genogroups showing the genetic similarity of CoV nsp12 (RdRp) and CoV spike glycoprotein, which mediates host tropism and entry into cells. Text color of the virus strain label corresponds to virus host species on the left. The heatmap adjacent to each neighbor-joining tree depicts percent amino acid identity (% A.A. similarity) against mouse hepatitis virus (MHV), SARS-CoV, or MERS-CoV. (B) The variation encompassed in panel A was modeled onto the RdRp structure of the SARS-CoV RdRp. (C) Amino acid sequence of CoV in panel A at known resistance alleles to antiviral drug RDV. (D) Virus titer reduction assay in DBT cells across a range of NHC with recombinant MHV bearing resistance mutations to RDV. Data shown are combined from three independent experiments performed with biological duplicates or triplicates per condition. Asterisks indicate statistically significant differences by Mann-Whitney test as indicated on the graph.

    Journal: Science Translational Medicine

    Article Title: An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice

    doi: 10.1126/scitranslmed.abb5883

    Figure Lengend Snippet: Remdesivir (RDV) resistance mutations in the highly conserved RNA-dependent RNA polymerase increase susceptibility to NHC. (A) Neighbor-joining trees created with representatives from all four CoV genogroups showing the genetic similarity of CoV nsp12 (RdRp) and CoV spike glycoprotein, which mediates host tropism and entry into cells. Text color of the virus strain label corresponds to virus host species on the left. The heatmap adjacent to each neighbor-joining tree depicts percent amino acid identity (% A.A. similarity) against mouse hepatitis virus (MHV), SARS-CoV, or MERS-CoV. (B) The variation encompassed in panel A was modeled onto the RdRp structure of the SARS-CoV RdRp. (C) Amino acid sequence of CoV in panel A at known resistance alleles to antiviral drug RDV. (D) Virus titer reduction assay in DBT cells across a range of NHC with recombinant MHV bearing resistance mutations to RDV. Data shown are combined from three independent experiments performed with biological duplicates or triplicates per condition. Asterisks indicate statistically significant differences by Mann-Whitney test as indicated on the graph.

    Article Snippet: Previously published TaqMan primers were synthesized by Integrated DNA Technologies (IDT) to quantify MERS genomic RNA (targeting orf1a: Forward: 5′- GCACATCTGTGGTTCTCCTCTCT-3′, Probe (6-FAM/ZEN/IBFQ): 5′- TGCTCCAACAGTTACAC-3′, Reverse: 5′-AAGCCCAGGCCCTACTATTAGC)( ). qRT-PCR was performed using 100ng total RNA compared to an RNA standard curve using TaqMan Fast Virus 1-Step Master Mix (ThermoFisher) on a Quant Studio 3 (Applied Biosystems).

    Techniques: Sequencing, Recombinant, MANN-WHITNEY

    NHC is effective against multiple genetically distinct Bat-CoV. Top: Antiviral efficacy of NHC in HAE cells against SARS-like (HKU3, SHC014, group 2b) and MERS-like (HKU5, group 2c) bat-CoV. HAE cells were infected at an MOI of 0.5 in the presence of NHC in duplicate. After 48 hours, virus produced was titrated via plaque assay. Each data point represents the titer per culture. Bottom: qRT-PCR for CoV ORF1 and ORFN mRNA in total RNA from cultures in the top panel. Mock, mock-treated. Representative data from two separate experiments with two different cell donors are displayed.

    Journal: Science Translational Medicine

    Article Title: An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice

    doi: 10.1126/scitranslmed.abb5883

    Figure Lengend Snippet: NHC is effective against multiple genetically distinct Bat-CoV. Top: Antiviral efficacy of NHC in HAE cells against SARS-like (HKU3, SHC014, group 2b) and MERS-like (HKU5, group 2c) bat-CoV. HAE cells were infected at an MOI of 0.5 in the presence of NHC in duplicate. After 48 hours, virus produced was titrated via plaque assay. Each data point represents the titer per culture. Bottom: qRT-PCR for CoV ORF1 and ORFN mRNA in total RNA from cultures in the top panel. Mock, mock-treated. Representative data from two separate experiments with two different cell donors are displayed.

    Article Snippet: Previously published TaqMan primers were synthesized by Integrated DNA Technologies (IDT) to quantify MERS genomic RNA (targeting orf1a: Forward: 5′- GCACATCTGTGGTTCTCCTCTCT-3′, Probe (6-FAM/ZEN/IBFQ): 5′- TGCTCCAACAGTTACAC-3′, Reverse: 5′-AAGCCCAGGCCCTACTATTAGC)( ). qRT-PCR was performed using 100ng total RNA compared to an RNA standard curve using TaqMan Fast Virus 1-Step Master Mix (ThermoFisher) on a Quant Studio 3 (Applied Biosystems).

    Techniques: Infection, Produced, Plaque Assay, Quantitative RT-PCR

    Prophylactic and therapeutic EIDD-2801 reduces MERS-CoV replication and pathogenesis coincident with increased viral mutation rates . Equivalent numbers of 10-14 week old male and female C57BL/6 hDPP4 mice were administered vehicle (10% PEG, 2.5% Cremophor RH40 in water) or NHC prodrug EIDD-2801 beginning at -2 hours, +12, +24 or +48 hours post infection and every 12 hours thereafter by oral gavage (n = 10/group). Mice were intranasally infected with 5E+04 PFU mouse-adapted MERS-CoV M35C4 strain. ( A) Percent starting weight. Asterisks indicate differences between -2 hours and +12 hours group from vehicle by two-way ANOVA with Tukey’s multiple comparison test. ( B) Lung hemorrhage in mice from panel A scored on a scale of 0-4 where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. (C) Virus lung titer in mice from panel A as determined by plaque assay. Asterisks in both panel B and C indicate differences from vehicle by Kruskal-Wallis with Dunn’s multiple comparison test. (D) MERS-CoV genomic RNA in lung tissue by qRT-PCR. Asterisks indicate differences by one-way ANOVA with a Dunnett’s multiple comparison test. (E) Pulmonary function by whole body plethysmography was performed daily on four animals per group. Asterisks indicate differences from vehicle by two-way ANOVA with Tukey’s multiple comparison test. (F) Workflow to measure mutation rate in MERS-CoV RNA and host transcript ISG15 by Primer ID in mouse lung tissue. (G) Number of template consensus sequences (TCS) for MERS-CoV nsp10 and ISG15. (H) Total error rate in MERS-CoV nsp10 and ISG15. (I) The cytosine to uridine transition rate in MERS-CoV nsp10 and ISG15. In panels G-I, asterisks indicate differences from vehicle by two-way ANOVA with Tukey’s multiple comparison test. (J) Codon change frequency in MERS-CoV nsp10. Asterisks indicate differences from vehicle by Kruskal-Wallis with Dunn’s multiple comparison test. For all panels, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range.

    Journal: Science Translational Medicine

    Article Title: An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice

    doi: 10.1126/scitranslmed.abb5883

    Figure Lengend Snippet: Prophylactic and therapeutic EIDD-2801 reduces MERS-CoV replication and pathogenesis coincident with increased viral mutation rates . Equivalent numbers of 10-14 week old male and female C57BL/6 hDPP4 mice were administered vehicle (10% PEG, 2.5% Cremophor RH40 in water) or NHC prodrug EIDD-2801 beginning at -2 hours, +12, +24 or +48 hours post infection and every 12 hours thereafter by oral gavage (n = 10/group). Mice were intranasally infected with 5E+04 PFU mouse-adapted MERS-CoV M35C4 strain. ( A) Percent starting weight. Asterisks indicate differences between -2 hours and +12 hours group from vehicle by two-way ANOVA with Tukey’s multiple comparison test. ( B) Lung hemorrhage in mice from panel A scored on a scale of 0-4 where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. (C) Virus lung titer in mice from panel A as determined by plaque assay. Asterisks in both panel B and C indicate differences from vehicle by Kruskal-Wallis with Dunn’s multiple comparison test. (D) MERS-CoV genomic RNA in lung tissue by qRT-PCR. Asterisks indicate differences by one-way ANOVA with a Dunnett’s multiple comparison test. (E) Pulmonary function by whole body plethysmography was performed daily on four animals per group. Asterisks indicate differences from vehicle by two-way ANOVA with Tukey’s multiple comparison test. (F) Workflow to measure mutation rate in MERS-CoV RNA and host transcript ISG15 by Primer ID in mouse lung tissue. (G) Number of template consensus sequences (TCS) for MERS-CoV nsp10 and ISG15. (H) Total error rate in MERS-CoV nsp10 and ISG15. (I) The cytosine to uridine transition rate in MERS-CoV nsp10 and ISG15. In panels G-I, asterisks indicate differences from vehicle by two-way ANOVA with Tukey’s multiple comparison test. (J) Codon change frequency in MERS-CoV nsp10. Asterisks indicate differences from vehicle by Kruskal-Wallis with Dunn’s multiple comparison test. For all panels, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range.

    Article Snippet: Previously published TaqMan primers were synthesized by Integrated DNA Technologies (IDT) to quantify MERS genomic RNA (targeting orf1a: Forward: 5′- GCACATCTGTGGTTCTCCTCTCT-3′, Probe (6-FAM/ZEN/IBFQ): 5′- TGCTCCAACAGTTACAC-3′, Reverse: 5′-AAGCCCAGGCCCTACTATTAGC)( ). qRT-PCR was performed using 100ng total RNA compared to an RNA standard curve using TaqMan Fast Virus 1-Step Master Mix (ThermoFisher) on a Quant Studio 3 (Applied Biosystems).

    Techniques: Mutagenesis, Mouse Assay, Infection, Plaque Assay, Quantitative RT-PCR

    NHC antiviral activity is associated with increased viral mutation rates . ( A ) HAE cultures were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of vehicle, RDV, or NHC for 48 hours, after which apical washes were collected for virus titration. Data are combined from two independent studies. The boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. (B) Schematic of Primer ID deep sequencing for single RNA genomes of MERS-CoV. (C) The total error rate for MERS-CoV RNA isolated from cultures in panel A as determined by Primer ID. Error rate values are # mutations per 10,000 bases. Asterisks indicate significant differences as compared to untreated group by two-way ANOVA with a Dunnett’s multiple comparison test. (D) Description of potential NHC mutational spectra on both positive and negative sense viral RNA. (E) Nucleotide transitions in cDNA derived from MERS-CoV genomic RNA.

    Journal: Science Translational Medicine

    Article Title: An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice

    doi: 10.1126/scitranslmed.abb5883

    Figure Lengend Snippet: NHC antiviral activity is associated with increased viral mutation rates . ( A ) HAE cultures were infected with MERS-CoV red fluorescent protein (RFP) at an MOI of 0.5 in duplicate in the presence of vehicle, RDV, or NHC for 48 hours, after which apical washes were collected for virus titration. Data are combined from two independent studies. The boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. (B) Schematic of Primer ID deep sequencing for single RNA genomes of MERS-CoV. (C) The total error rate for MERS-CoV RNA isolated from cultures in panel A as determined by Primer ID. Error rate values are # mutations per 10,000 bases. Asterisks indicate significant differences as compared to untreated group by two-way ANOVA with a Dunnett’s multiple comparison test. (D) Description of potential NHC mutational spectra on both positive and negative sense viral RNA. (E) Nucleotide transitions in cDNA derived from MERS-CoV genomic RNA.

    Article Snippet: Previously published TaqMan primers were synthesized by Integrated DNA Technologies (IDT) to quantify MERS genomic RNA (targeting orf1a: Forward: 5′- GCACATCTGTGGTTCTCCTCTCT-3′, Probe (6-FAM/ZEN/IBFQ): 5′- TGCTCCAACAGTTACAC-3′, Reverse: 5′-AAGCCCAGGCCCTACTATTAGC)( ). qRT-PCR was performed using 100ng total RNA compared to an RNA standard curve using TaqMan Fast Virus 1-Step Master Mix (ThermoFisher) on a Quant Studio 3 (Applied Biosystems).

    Techniques: Activity Assay, Mutagenesis, Infection, Titration, Sequencing, Isolation, Derivative Assay