Journal: Scientific Reports

Article Title: Sofosbuvir terminated RNA is more resistant to SARS-CoV-2 proofreader than RNA terminated by Remdesivir

doi: 10.1038/s41598-020-73641-9

Figure Lengend Snippet: Polymerase reactions with SOF-TP, UTP or UTP + RDV-TP to produce RNA extension products. The sequence of the RNA template-loop-primer used for these polymerase extension reactions is shown at the top of the figure. ( a ) MALDI-TOF mass spectrum of the unextended RNA. Polymerase extension reactions were performed by incubating ( b ) SOF-TP, ( c ) UTP and ( d ) UTP + RDV-TP with pre-assembled SARS-CoV-2 polymerase (nsp12, nsp7 and nsp8) and the indicated RNA template-loop-primer, followed by detection of reaction products by MALDI-TOF MS. The accuracy for m/z determination is approximately ± 10 Da.

Article Snippet: Other investigators have since demonstrated the ability of Sofosbuvir to inhibit SARS-CoV-2 replication in lung and brain cells; additionally, COVID-19 clinical trials with EPCLUSA and with Sofosbuvir plus Daclatasvir have been initiated in several countries.

Techniques: Sequencing

Journal: Scientific Reports

Article Title: Sofosbuvir terminated RNA is more resistant to SARS-CoV-2 proofreader than RNA terminated by Remdesivir

doi: 10.1038/s41598-020-73641-9

Figure Lengend Snippet: Treatment of the RNA products with exonuclease and analysis by MALDI-TOF MS to determine relative excision of Sofosbuvir, UMP and Remdesivir. Untreated products (0 min) are shown in ( a ) for SOF extended RNA, ( d ) for UMP extended RNA and ( g ) for UMP plus RDV extended RNA. Exonuclease reactions were performed by incubating the purified RNA products, generated using the same procedure as in Fig. , with pre-assembled SARS-CoV-2 exonuclease complex (nsp14 and nsp10) for 5 min ( b , e , h ) or 30 min ( c , f , i ), followed by detection of reaction products by MALDI-TOF MS. The signal intensities were normalized to the highest peak within each time series. The accuracy for m/z determination is approximately ± 10 Da.

Article Snippet: Other investigators have since demonstrated the ability of Sofosbuvir to inhibit SARS-CoV-2 replication in lung and brain cells; additionally, COVID-19 clinical trials with EPCLUSA and with Sofosbuvir plus Daclatasvir have been initiated in several countries.

Techniques: Purification, Generated

Journal: Scientific Reports

Article Title: Sofosbuvir terminated RNA is more resistant to SARS-CoV-2 proofreader than RNA terminated by Remdesivir

doi: 10.1038/s41598-020-73641-9

Figure Lengend Snippet: Treatment of the RNA products with exonuclease and then combined for analysis by MALDI-TOF MS to determine relative excision of Sofosbuvir, UMP and Remdesivir. Untreated products (0 min) are shown in ( a ) for SOF and UMP extended RNA and ( c ) for SOF and UMP + RDV extended RNA. Exonuclease reactions were performed by incubating the purified RNA products with pre-assembled SARS-CoV-2 exonuclease complex (nsp14 and nsp10). The SOF and UMP extended RNAs were treated with the exonuclease complex for 15 min and then combined for purification followed by MALDI-TOF–MS ( b ). The insets in ( a , b ) are an enlargement of the 8200–8300 Da portion of the spectrum. The SOF and UMP + RDV extended RNAs were treated with the exonuclease complex for 5 min or 30 min and combined for purification and MALDI-TOF–MS ( d , e , respectively). The signal intensities were normalized to the highest peak within each time series. The accuracy for m/z determination is approximately ± 10 Da.

Article Snippet: Other investigators have since demonstrated the ability of Sofosbuvir to inhibit SARS-CoV-2 replication in lung and brain cells; additionally, COVID-19 clinical trials with EPCLUSA and with Sofosbuvir plus Daclatasvir have been initiated in several countries.

Techniques: Purification

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Schematic overview of the anti-SARS-CoV-2 drug screening procedure. ( a ) ACE2 targeted inhibitors and compounds from the natural product library were applied prior to infection with SARS-CoV-2 (pre-infection treatment) while compounds from the FDA-approved library and the flavonoid library were applied post infection (post- infection treatment). Out of 2191 compounds tested, 121 displayed viral CPE reductions and 7 were selected for further validations using Vero E6 and HuH7 cell lines and hNEC. ( b ) Uninfected cells, infected cells treated with 0.1% DMSO, and infected cells treated with remdesivir were included as controls in the screen. Following treatment with citicoline, pravastatin sodium, tenofovir alafenamide, imatinib mesylate, calcitriol, dexlansoprazole, and prochlorperazine dimaleate, reduced CPE was observed when compared to the DMSO control.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Drug discovery, Infection, Control

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Summary of compounds identified from the primary screen that exhibit potential antiviral effects against SARS-CoV-2 infection.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Infection, Reverse Transcription, Reflux

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Validation of primary hits. For validating compounds with activity pre-infection, Vero E6 cells were first pre-treated with increasing concentrations of ( a ) citicoline, ( b ) pravastatin sodium and ( c ) tenofovir alafenamide prior to infection with SARS-CoV-2. Similarly, Huh7 cells were pre-treated with ( d ) citicoline, ( e ) pravastatin sodium and ( f ) tenofovir alafenamide and subsequently infected with SARS-CoV-2. For post-infection treatment validation, Vero E6 cells were infected with SARS-CoV-2 and treated with increasing concentrations of ( g ) imatinib mesylate, ( h ) calcitriol, ( i ) dexlansoprazole and ( j ) prochlorperazine dimaleate. Similarly, HuH7 cells were also infected then treated with a range of concentrations of ( k ) imatinib mesylate, ( l ) calcitriol, ( m ) dexlansoprazole and ( n ) prochlorperazine dimaleate. The primary and secondary exes correspond to the viral titre and relative cell viability, and the dashed line represents the CC 50 cut-off for cell viability. One-way ANOVA and Dunnett’s post-test were used to determine statistical differences, with * denoting p < 0.05, ** denoting p < 0.01 and *** denoting p < 0.001. Error bars represent the standard deviation observed from the means of triplicates performed for both cell viability and dose-dependent inhibition studies.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Biomarker Discovery, Activity Assay, Infection, Standard Deviation, Inhibition

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Validation of primary hits in hNECs. Selected primary hits were further validated in hNECs. Cells were either pre-treated with 10 μM citicoline prior to SARS-CoV-2 infection or treated following SARS-CoV-2 infection with 10 μM imatinib mesylate and 10 μM calcitriol. One-way ANOVA and Dunnett’s post-test were used to determine statistical differences, with ** denoting that p < 0.01. Error bars represent the standard deviation observed from the means of triplicates performed for dose-dependent inhibition studies.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Biomarker Discovery, Infection, Standard Deviation, Inhibition

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: SARS-CoV-2 and calcitriol modulate vitamin D Receptor (VDR), 24-hydroxylase (24(OH)ase), and cathelicidin (LL-37) in Vero E6 and Huh7 cells. In the first part of the experiment, Vero E6 and Huh7 cells were either mock-infected with media, or SARS-CoV-2 infected and harvested at indicated timepoints in the absence of calcitriol for baseline VDR, 24(OH)ase, and LL-37 mRNA expression. Vero E6 expression of ( a ) VDR, ( b ) 24(OH)ase and ( c ) LL-37 and Huh7 expression of ( d ) VDR, ( e ) 24(OH)ase and ( f ) LL-37 were as shown. In the second part of the experiment, Vero E6 and Huh7 cells were either mock-infected with media or SARS-CoV-2 for 1 h as above, then treated with 0.01, 0.1, 1, 10 or 20 µM calcitriol following infection. At 48 h post infection, the cells were harvested to check for VDR, 24(OH)ase, and LL-37 mRNA expression. Vero E6 mRNA expression of ( g ) VDR, ( i ) 24(OH)ase and ( k ) LL-37 and Huh7 expression of ( h ) VDR, ( j ) 24(OH)ase and ( l ) LL-37 were as shown. Data are represented as relative quantity to 0.1% DMSO control, or as relative quantity to mock-infected control. One-way ANOVA and Dunnett’s post-test and paired t -test were used to determine statistical differences, with * denoting p < 0.05, ** denoting p < 0.01 and *** denoting p < 0.001. Error bars represent the standard deviation observed from the means of triplicates performed for mRNA expression studies.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Infection, Expressing, Control, Standard Deviation

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: Western blot analysis of SARS-CoV-2 spike and VDR proteins in Vero E6 cells. ( a ) Vero E6 cells are either mock infected with media (−) or with SARS-CoV-2 (+) for 1 h. After 1 h post-infection, the cells are then either untreated (−) or treated (+) with 20 µM calcitriol for 6-, 24- and 48- hours before harvesting of total cell lysate. ( b ) Densitometry quantitation of protein expression levels is shown as fold changes to beta-actin. Full-length blots are shown in .

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: Western Blot, Infection, Quantitation Assay, Expressing

Journal: Pharmaceutics

Article Title: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants

doi: 10.3390/pharmaceutics15030925

Figure Lengend Snippet: In vivo study of calcitriol treatment. 8-week old K18-hACE2 male and female mice were separated into treatment and non-treatment groups. Treatment study group has been given 3 days pre-treatment and 5 days post-treatment of calcitriol (5 μg/kg) via intraperitoneal injection. The non-treatment group has been given a PBS as a treatment control. Next, 10 3 PFU of SARS-CoV-2 (L, Alpha, and Beta variants) were inoculated through intranasal delivery to all groups after pre-treatment. Physiological parameters: body weight ( a – c ), survival rate ( d – f ), and physiological conditions were monitored after infections. For comparison between individual physiological conditions, 5 dpi infected mice were compared between treatment groups of all three variant infected mice ( g – i ). Scoring of physiological conditions was based on five criteria: appearance of the mouse coat, level of consciousness, activity level, eye condition, and respiratory quality. To compare the severity of damage in mouse tissues after infection, left lung lobes from 4 dpi mice were processed for histological analyses and scored based on six criteria to determine severity ( j – l ). The six criteria are inflammatory cell infiltration, haemorrhage, oedema, bronchial epithelial cell damage, degeneration of alveolar epithelial cells, and parenchymal wall expansion. For viral load determination, right lung, brain, liver, and spleen tissues from the same 4 dpi mice were harvested and homogenised for virus titration ( m – o ). Data is not shown for liver and spleen tissues. Statistical significance is determined with a two-tailed unpaired t -test, with * denoting p < 0.05. Survival group: variant L: n = 6 (treatment and no treatment); Alpha: n = 6 (treatment), n = 4 (no treatment); Beta: n = 6 (treatment), n = 5 (no treatment). 4 dpi group: variant L: n = 7 (treatment and no treatment); Alpha and Beta: n = 5 each (treatment and no treatment). Mock infection, n = 3.

Article Snippet: A primary screen to identify novel compounds with potential antiviral effects against SARS-CoV-2 infection was performed on four different libraries, namely a 462-compound ACE2-targeted compound library (CADD) (TargetMol, Boston, MA, USA), a 57-compound natural product library, a 500-compound flavonoids library (TimTec, Tampa, FL, USA), and a 1172-compound FDA-approved drug library (Selleckchem, Houston, TX, USA).

Techniques: In Vivo, Injection, Control, Comparison, Infection, Variant Assay, Activity Assay, Virus, Titration, Two Tailed Test

Journal: Virologica Sinica

Article Title: Comparison of viral propagation and drug response among SARS-CoV-2 VOCs using replicons capable of recapitulating virion assembly and release

doi: 10.1016/j.virs.2022.06.008

Figure Lengend Snippet: Construction of the SARS-CoV-2 replicons. A diagram of the replicon construction. Vero E6 cells were infected with the three SARS-CoV-2 strains or variants. At 48 h post-infection, total RNA was extracted and served as a template for RT-PCR. Five primer pairs (1F/6195R, 6099F/12296R, 12188F/18407R, 18281F/21622R, and 25266F/29870R) were used to amplify the cDNA clone of the viral RNA genome and generated five fragments (F-1–F-5; F-3′ indicates F-3 without the RdRp coding sequence and F-5′ indicates F-5 without the viral sequence prior to the N gene). Viral cDNA amplicons were assembled into a pGF vector between a CMV promoter and an HDVrbz through a one-step isothermal assembly. The relative locations of the important primers used in this study are labeled (arrowhead). B Restriction assay of the replicon plasmids. The replicon plasmids were extracted from E. coli EPI300. Approximately 3 μg of each replicon was digested by Nco I. The resulting restriction fragments were separated by gel electrophoresis (1% agarose) and compared to a computer-generated gel simulation. The expected sizes of pRep-WIV04 restriction fragments were 19,076-, 8257-, 3710-, 3697-, 2793-, 1439-, 626-, 435-, 373-bp in length; for pRep-Beta, the sizes were 19,076-, 8257-, 4083-, 3697-, 2793-, 1439-, 626-, 435-bp; for pRep-Delta, the sizes were 19,112-, 8257-, 3915-, 3697-, 1439-, 626-, 373-bp; for pRep-mini, the sizes were 19,067-, 8257-, 5211-, 1439-, 626-bp; for pRep-Ctrl, the sizes were 16,679-, 8257-, 3710-, 3697-, 2793-, 1439-, 626-, 435-, 373-bp.

Article Snippet: In contrast to multistep methods that were previously attempted for the preparation of SARS-CoV-2 replicons, we developed a one-step method to assemble SARS-CoV-2 cDNA amplicons into a full-length replicon using the NEBuilder HiFi DNA assembly kit.

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Generated, Sequencing, Plasmid Preparation, Labeling, Restriction Assay, Nucleic Acid Electrophoresis

Journal: Virologica Sinica

Article Title: Comparison of viral propagation and drug response among SARS-CoV-2 VOCs using replicons capable of recapitulating virion assembly and release

doi: 10.1016/j.virs.2022.06.008

Figure Lengend Snippet: The replicon recapitulates viral genome replication and sgRNA transcription. Vero E6 cells were infected with the three SARS-CoV-2 strains or variants, and HEK-293T cells were transfected with the indicated replicon plasmids. Total RNA was extracted from both the infected and transfected cells and served as the template for RT-PCR. To detect −fgRNA, reverse transcription was primed with 20674F, and the resulting cDNA was amplified with the primer pair 20674F/21555R ( A ). To detect the sgRNA encoding the N protein, reverse transcription was primed with oligo(dT), and RT-PCR amplified the resulting cDNA with the primer pair TRS-L-F/29533R ( B ). The abovementioned RT-PCR amplicons were subjected to Sanger sequencing, and a common TRS-L motif and a fusion site were identified in all the sequenced samples ( C ).

Article Snippet: In contrast to multistep methods that were previously attempted for the preparation of SARS-CoV-2 replicons, we developed a one-step method to assemble SARS-CoV-2 cDNA amplicons into a full-length replicon using the NEBuilder HiFi DNA assembly kit.

Techniques: Infection, Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing

Journal: Virologica Sinica

Article Title: Comparison of viral propagation and drug response among SARS-CoV-2 VOCs using replicons capable of recapitulating virion assembly and release

doi: 10.1016/j.virs.2022.06.008

Figure Lengend Snippet: Comparison of viral intracellular propagation among SARS-CoV-2 variants. A Quantification of replicon RNA in the supernatant. HEK-293T cells were transfected with the indicated replicons. At 6, 12, 24, 48, and 72 h post-transfection (hpt), ERR in the supernatants was quantified by qPCR. The relative increase in individual replicon RNA is presented as the ratio of fgRNA copy number at the indicated time points to that at 6 hpt. B Quantification of intracellular replicon RNA. HEK-293T cells were transfected with the indicated replicons. At 6, 12, 24, 48, and 72 hpt, total RNA was extracted from transfected cells and was reverse-transcribed using the gene-specific primer 20674F. The resulting −fgRNA-derived cDNA was then quantified by qPCR. The relative increase in individual −fgRNA is presented in a way similar to that of ( A ). All experiments were performed in triplicate. The data points indicate the averages of triplicate experiments. The error bars indicate the standard derivation from the mean. n.s., not significant; u.d., under detected.

Article Snippet: In contrast to multistep methods that were previously attempted for the preparation of SARS-CoV-2 replicons, we developed a one-step method to assemble SARS-CoV-2 cDNA amplicons into a full-length replicon using the NEBuilder HiFi DNA assembly kit.

Techniques: Transfection, Derivative Assay

Journal: Virologica Sinica

Article Title: Comparison of viral propagation and drug response among SARS-CoV-2 VOCs using replicons capable of recapitulating virion assembly and release

doi: 10.1016/j.virs.2022.06.008

Figure Lengend Snippet: Comparison of viral response to RDV treatment among SARS-CoV-2 variants. A Evaluation of the drug response using viral replicons. HEK-293T cells were transfected with indicated replicons. RDV was added to the culture medium at 6 h post-transfection. After 24 h of drug treatment, ERR was extracted from the supernatant and quantified by qPCR. The replicon response to RDV treatment is presented as the ratio of fgRNA copy number at the indicated RDV concentrations to that of the no drug treatment control. B Evaluation of drug response using infectious viruses. Vero E6 cells were infected with the three SARS-CoV-2 strains or variants at an MOI of 0.02. The infected cells were incubated with culture medium containing RDV at the indicated concentrations for 24 h. The viral yield in the cell supernatant was then quantified by qPCR. The Y-axis represents the mean % reduction in viral genome copy number. All experiments were performed in triplicate. The data points indicate the averages of triplicate experiments. The error bars indicate the standard derivation from the mean. The IC 50 values were calculated by GraphPad PRISM. u.d., under detected.

Article Snippet: In contrast to multistep methods that were previously attempted for the preparation of SARS-CoV-2 replicons, we developed a one-step method to assemble SARS-CoV-2 cDNA amplicons into a full-length replicon using the NEBuilder HiFi DNA assembly kit.

Techniques: Transfection, Infection, Incubation