Journal: npj Viruses

Article Title: Respiratory syncytial virus glycoprotein G impedes CX 3 CR1-activation by CX 3 CL1 and monocyte function

doi: 10.1038/s44298-024-00075-9

Figure Lengend Snippet: 1 µg purified protein samples were resolved on a 10% SDS-PAGE gel and stained with PageBlue™ coomassie solution, showing total protein content (left). Western blot analyses using rabbit anti-BSA (1:1000; A11133; Thermo Fisher) verified BSA expression, rabbit anti-RSV G glycoprotein (1:1000; 40041-T62; Sino Biological) confirmed RSV G glycoprotein expression, and mouse anti-6xHis (1:1000; His.H8; Thermo Fisher) confirmed the presence of His-tagged proteins. Secondary antibodies were applied 1:20000 (goat anti-mouse IgG, Thermo Fisher; goat anti-rabbit IgG, Thermo Fisher). All membranes were treated with SuperSignal™ Western Blot Enhancer and detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher). Protein bands were visualized using a Chemidoc chemiluminescence imaging system (Bio-Rad). Black borders represent a division between separate blots.

Article Snippet: Primary antibodies were applied at a 1:1000 dilution: mouse anti-6xHis (Cat# MA1-21315; Thermo Fisher), rabbit anti-RSV G glycoprotein (Cat#40041-T62; Sino Biological), and rabbit anti-BSA (Cat#A11133; Thermo Fisher).

Techniques: Purification, SDS Page, Staining, Western Blot, Expressing, Imaging

Journal: Scientific Reports

Article Title: Ginkgolic acid inhibits orthopneumo- and metapneumo- virus infectivity

doi: 10.1038/s41598-024-58032-8

Figure Lengend Snippet: Respiratory syncytial virus spreading in culture in inhibited by Ginkgolic acid. ( A ) Cell cytotoxicity of Ginkgolic acid in Vero E6 cells. Cells were treated with Ginkgolic acid in a two-fold dilution series starting 50µM, for 5 days. CC 50 value was assessed based on cell viability percentage compared to DMSO treated cells. ( B ) Flow cytometry 24 h post Ginkgolic acid treatment of Vero E6 cells infected with hRSV-mKate2. Cells were infected for 2 h with hRSV-mKate2 at a MOI of 0.05. Post infection the cells were washed 3 times with PBS and media containing DMSO of Ginkgolic acid at the indicated concentrations were added to the cultures for 24 h. The percentage of cells scoring red-positive relative to DMSO control (set as 100) are plotted. The data are mean ± SEM from three independent experiments (n = 3). ( C ) hRSV-mKate2 spread in Vero E6 cultures post Ginkgolic acid treatment. Cells were infected as in (B) and were daily monitored for the spread of hRSV-mKate2 using fluorescence microscopy to image the presence of the mKate2 fluorescent reporter. Shown are representative fields of view from a single experiment out of three independent experiments (n = 3). B.F, brightfield. Scale bar, 100 μm.

Article Snippet: Human respiratory syncytial virus (hRSV) strain A2 nucleoprotein antibody was purchased from Sino Biologicals (Cat# 40821-T46).

Techniques: Virus, Flow Cytometry, Infection, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Ginkgolic acid inhibits orthopneumo- and metapneumo- virus infectivity

doi: 10.1038/s41598-024-58032-8

Figure Lengend Snippet: Infectivity of respiratory syncytial virus is inhibited post Ginkgolic acid treatment. ( A ) Cell cytotoxicity of Ginkgolic acid in A549 cells. Cells were treated with Ginkgolic acid in a two-fold dilution series starting 50 µM, for 96 h. CC 50 value was assessed based on cell viability percentage compared to DMSO treated cells. The data are mean ± SEM from three independent experiments (n = 3). Fluorescence microscopy monitoring the expression of mKate2 fluorescent protein in A549 cells ( B ) or Vero E6 ( C ) infected for 24 h with hRSV-mKate2 viruses that were pre-treated for 1 h with DMSO or the indicated Ginkgolic acid concentration. Shown are representative fields of view from a single experiment out of three independent experiments (n = 3). B.F, brightfield. Scale bar, 100 μm. ( D ) Flow cytometry of cells from ( B ) and ( C ) was used to determine the number of mKate2 expressing cells in each cultures and calculate the IC 50 values of Ginkgolic acid. The data are mean ± SEM from three independent experiments (n = 3).

Article Snippet: Human respiratory syncytial virus (hRSV) strain A2 nucleoprotein antibody was purchased from Sino Biologicals (Cat# 40821-T46).

Techniques: Infection, Virus, Fluorescence, Microscopy, Expressing, Concentration Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: Ginkgolic acid inhibits orthopneumo- and metapneumo- virus infectivity

doi: 10.1038/s41598-024-58032-8

Figure Lengend Snippet: Respiratory syncytial virus assembly and release are not affected by Ginkgolic acid treatment. A549 cells were infected with hRSV-mKate2 for 2 h, after which the cells were washed with PBS and media containing DMSO or GA at various concentration were added to the cultures for 24 h. ( A ) Fluorescence microscopy to detect the expression of mKate2 fluorescent protein in infected and treated cells. Shown are representative fields of view from a single experiment out of three independent experiments (n = 3). B.F, brightfield. Scale bar, 100 μm. ( B ) Flow cytometry of cells from (A) to quantify the number of mKate2 expressing cells. The percent cells scoring red-positive relative to DMSO control (set as 100) are plotted. The data are mean ± SEM from three independent experiments (n = 3). ( C ) hRSV Nucleocapsid expression levels from infected and treated cell lysates were assessed using western blot with specific anti-hRSV Nucleocapsid antibody. Shown are representative western blots from three independent experiments (n = 3). ( D ) viral genomic and anti-genomic RNA levels form infected and treated cell lysates were assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Two different primer sets were used for each RNA species—primers for N and L ORFs were used to measure genomic RNA levels, primers for the regions spanning between N and P ORFs or F and G ORFs were used to measure the levels of the anti-genomic RNA. The fold change of viral genomic or anti-genomic RNA levels normalized to microtubules mRNA levels in GA treated compared to DMSO treated are plotted. The data are mean ± SEM from three independent experiments (n = 3). ( E ) Virions from culture supernatants of (C) were isolated by pelleting through 25% sucrose cushions and hRSV Nucleocapsid expression levels were assessed as in (C). Shown is a representative western blot from three independent experiments (n = 3). ( F ) Virions from culture supernatants of (D) were isolated by pelleting through 25% sucrose cushions and qRT-PCR using two primer sets (amplifying in N and L ORFs) was used to measure the levels of genomic RNA in each sample. The fold change of genomic RNA levels in GA treated compared to DMSO treated cultures are plotted. The data are mean ± SEM from three independent experiments (n = 3).

Article Snippet: Human respiratory syncytial virus (hRSV) strain A2 nucleoprotein antibody was purchased from Sino Biologicals (Cat# 40821-T46).

Techniques: Virus, Infection, Concentration Assay, Fluorescence, Microscopy, Expressing, Flow Cytometry, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Isolation