Journal: Frontiers in Microbiology

Article Title: Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism

doi: 10.3389/fmicb.2023.1251065

Figure Lengend Snippet: Workflow for lipidomic and transcriptomic profiling of Vero E6 cells after SARS-CoV-2 infection with and without niclosamide treatment. We used a time-of-addition assay experimental design to (i) capture the lipidomic profile of SARS-CoV-2 infected Vero E6 cells, and (ii) explore the effect of niclosamide on the lipidomic profile of Vero E6 cells when added in the absence of infection, with SARS-CoV-2 virus, or at 24 h post-infection. Each replicate experimental condition ( n = 3) was processed for LC-HRMS/MS or RNASeq analyses. Samples were seeded 48 h prior to the start of the experiment ( t = 0 h). For all samples, media was changed at the start of the experiment ( t = 0 h) and infected with virus (for infected sample groups). Sample collection is denoted by an up arrow and tube above the timeline, media changes are denoted by red bottles, addition of virus and DMSO/drug are denoted as well. Separate samples for each condition were collected for LC-HRMS/MS and RNAseq analysis, respectively. Created with BioRender.com .

Article Snippet: Vero clone E6 cells (ATCC: CRL-1586) were obtained from Dr. Pei-Yong Shi (University of Texas Medical Branch).

Techniques: Infection, Virus

Journal: Frontiers in Microbiology

Article Title: Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism

doi: 10.3389/fmicb.2023.1251065

Figure Lengend Snippet: Niclosamide modulates lipid metabolism in Vero E6 cells in the absence of SARS-CoV-2 infection. (A,B) Bar graphs combining the log P-value (colored in blue) and the Log2 fold change (colored in orange) for total lipid expression at 16h vs 16h NIC (A) and 48h vs 48h NIC (B), data is sorted by low to high fold change. (C) Heatmap clustering of the top 50 lipids across all classes measured in Vero E6 cells showing the differential lipid regulation from 16h to 48h. NIC treatment increased several phosphatidylcholines (PC) and decreased several triglycerides (TG) comparing 16hr to 48hr. TG and PC Plasmalogens were increased at 16hr and 48hr without NIC but were decreased at 16hr and 48hr following NIC treatment.

Article Snippet: Vero clone E6 cells (ATCC: CRL-1586) were obtained from Dr. Pei-Yong Shi (University of Texas Medical Branch).

Techniques: Infection, Expressing

Journal: Frontiers in Microbiology

Article Title: Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism

doi: 10.3389/fmicb.2023.1251065

Figure Lengend Snippet: Clustering analysis of virus free Vero E6 cell cultures comparing early growth (16 h) and late growth (48 h) with and without NIC. Pearson correlation and PCA including all lipids identified (A,B) . Pearson correlation and PCA including only TG lipids detected (C,D) . Clustering was evident in all cases, but correlation with all lipid expressions was primarily based on time points while correlation using only TG expression was from NIC treatment.

Article Snippet: Vero clone E6 cells (ATCC: CRL-1586) were obtained from Dr. Pei-Yong Shi (University of Texas Medical Branch).

Techniques: Virus, Expressing

Journal: Frontiers in Microbiology

Article Title: Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism

doi: 10.3389/fmicb.2023.1251065

Figure Lengend Snippet: Global lipidomics analysis in SARS-CoV-2 infected Vero E6 cells at 16 h and 48 h. (A) Hierarchical cluster heatmap analysis depicting the major affected lipid clustering between 16 h and 48 h infection. (B) Volcano plot showing the differential lipid abundance with SARS-CoV-2 infection between 16 h and 48 h. The primary significantly upregulated lipids include plasmalogens, diglycerides and triglycerides. (C) Bar graph showing the total abundance of ether lipids between the time points. Lipid molecule abbreviations (shown in panel B ): PC-76 [1_PC (16,0_20:4) + Na], PC-114 [1_PC (18,1_20:4) + Na | 1_PC (16,0_22:5) + Na], PC-381 [2_PC (16,0_17:1) + Na | 2_PC (15,0_18:1) + Na], TG-584 [2_TG (18,1_22:4_22:4) + NH4], TG-590 [2_TG (20,2_22:3_22:4) + NH4], TG-598 [2_TG (22,3_22:3_22:3) + NH4], SM-524 [2_SM (d17:1/20:3) + H], Cer-NS-317 [2_Cer-NS (d17:1/14:0) + H], DG-15 [1_DG (16,0_18:1) + NH4 | 1_DG (16,1_18:0) + NH4], DG-19 [1_DG (18,1_18,1) + NH4 | 1_DG (16,0_20,2) + NH4 | 1_DG (16,1_20,1) + NH4 | 1_DG (18,0_18,2) + NH4], DG-341 [2_DG (16:1_18:1) + NH4 | 1_DG (16,1_18,1) + NH4 | 1_DG (16,0_18,2) + NH4 | 1_DG (14,0_20,2) + NH4], Ether-LPC-183 [1_Plasmanyl-LPC (O-18:0) + H], Ether-TG-493 [2_plasmanyl-TG (O-16:1_20:0_20:0) + NH4 | 2_plasmenyl-TG (P-16:0_20:0_20:0) + NH4], Plasmanyl-PC-167 [1_Plasmanyl-PC (O-18:0/16:0) + H], Plasmanyl-PC-170 [1_Plasmanyl-PC (O-16:0/22:2) + H], Plasmanyl-PC-175 [1_Plasmanyl-PC (O-16:0/22:1) + H], and Plasmanyl-PC-178 [1_Plasmanyl-PC (O-18:1/22:2) + H | 1_Plasmenyl-PC (P-18:0/22:2) + H].

Article Snippet: Vero clone E6 cells (ATCC: CRL-1586) were obtained from Dr. Pei-Yong Shi (University of Texas Medical Branch).

Techniques: Infection

Journal: Frontiers in Microbiology

Article Title: Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism

doi: 10.3389/fmicb.2023.1251065

Figure Lengend Snippet: The effect of SARS-CoV-2 infection and NIC treatment on host cell lipid metabolism. SARS-CoV-2 infection in Vero E6 cells alters host cell lipid metabolism during early and late stages of infection. Increased transcription of lipid receptors LRP2 and VLDLR, and phosphorylation signaling regulators are observed throughout viral infection. Changes in TG composition from unsaturated to saturated acyl-chains occur as a function of viral replication, with an overall decrease in TG lipids at late infection timepoints. This change corresponds with an increase to DG and BMP lipids that is indicative of energy consumption and incorporation into membranes and vesicles, activation of autophagy pathways, as well as impacting viral replication. Treatment of cells with NIC alters lipid composition and gene regulation corresponding to apoptosis and autophagy related pathways. Decreases to ether lipids (TGs and DGs) and BMP are observed and reflect a decrease to exocytosis pathways for viral egress. Created with BioRender.com .

Article Snippet: Vero clone E6 cells (ATCC: CRL-1586) were obtained from Dr. Pei-Yong Shi (University of Texas Medical Branch).

Techniques: Infection, Phospho-proteomics, Activation Assay

Journal: Advanced Science

Article Title: Potent Anti‐SARS‐CoV‐2 Efficacy of COVID‐19 Hyperimmune Globulin from Vaccine‐Immunized Plasma

doi: 10.1002/advs.202104333

Figure Lengend Snippet: Neutralization of COVID‐HIG against SARS‐CoV‐2 WIV04, Beta, and Delta strains in vitro. a) PRNTs showed that COVID‐HIG‐001, ‐002, and ‐003 significantly inhibited infection by SARS‐CoV‐2 WIV04, Beta, and Delta strains in Vero E6 cells. Viruses were incubated with COVID‐HIG at 37 °C for 1 h. Next, Vero E6 cells were infected with WIV04, Beta, and Delta strains and stained with hematoxylin/eosin at 48 h (Beta and Delta variants) or 72 h (WIV04 strain) postinfection. The y ‐axis represents percent inhibition. The mean from two independent replicates is shown ( n = 2). b) Comparison of the PRNT 50 of the variants and WIV04; statistical significance was analyzed using one‐way ANOVA. * P < 0.05. c) Microneutralization assays showed that COVID‐HIG‐001, ‐002, and ‐003 significantly inhibited the SARS‐CoV‐2 WIV04, Beta, and Delta strains in Vero E6 cells. Viruses were incubated with COVID‐HIG at 37 °C for 1 h. Next, Vero E6 cells were infected with the WIV04, Beta, and Delta strains. After 24 h, the infected cell supernatant was analyzed using real‐time reverse transcription‐PCR (qRT‐PCR). The y ‐axis represents percent inhibition. The mean from two independent replicates is shown ( n = 2). d) Comparison of the IC 50 of the variants and WIV04; statistical significance was analyzed using one‐way ANOVA. * P < 0.05. PRNT, plaque reduction neutralization tests.

Article Snippet: Vero E6 (American Type Culture Collection, Manassas, VA, USA; no. 1586) and Huh‐7 (National Collection of Authenticated Cell Cultures, Shanghai, China; TCHu182) cells were maintained in minimum Eagle's medium (Gibco) and Dulbecco's modified Eagle medium (Gibco, Grand Island, NY, USA), respectively.

Techniques: Neutralization, In Vitro, Infection, Incubation, Staining, Inhibition, Comparison, Reverse Transcription, Quantitative RT-PCR

Journal: Vaccines

Article Title: Development of an Inactivated Vaccine against SARS CoV-2

doi: 10.3390/vaccines9111266

Figure Lengend Snippet: Preparation of ERUCoV-VAC. ( a ) The viral kinetics of the hCoV-19/Turkey/ERAGEM-001/2020 strain at different multiplicities of infection (MOI). ( b ) Flowchart of ERUCoV-VAC preparation. ( c ) Protein profiles of the hCoV-19/Turkey/ERAGEM-001/2020 virus strain before and after purification. Total proteins were separated by Nu-page 10% bis-tris SDS-PAGE gel. Lane 1, protein molecular weight marker (M). Lane A, uninfected Vero E-6 cells as a negative control. Lane B, Vero E-6 cells infected with the hCoV-19/Turkey/ERAGEM-001/2020 virus strain. Lane C, purified hCoV-19/Turkey/ERAGEM-001/2020 virus strain. Western blot analysis of purified hCoV-19/Turkey/ERAGEM-001/2020 virus strain probed with a human antibody to the SARS-CoV-2 nucleocapsid protein (1:2500) (GenScript; HC2003) (Lane E) or a rabbit polyclonal to SARS-CoV-2 spike glycoprotein (lane G). Uninfected cell lysates probed with the SARS-CoV-2 nucleocapsid protein (Lane D) and a rabbit polyclonal to SARS-CoV-2 spike glycoprotein (F) were used as a negative control. The arrows indicate that the bands at approximately 180 kDa and 48 kDa represent spike glycoprotein and nucleocapsid protein, respectively.

Article Snippet: Vero E6 cells (African green monkey kidney) obtained from ATCC (CRL 1586) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 mM L-glutamine (Sigma–Aldrich, Darmstadt, Germany).

Techniques: Infection, Virus, Purification, SDS Page, Molecular Weight, Marker, Negative Control, Western Blot