denv 2 rna  (Altona Diagnostics)

Journal: Microbiology Spectrum

Article Title: Defective Interfering Particles with Broad-Acting Antiviral Activity for Dengue, Zika, Yellow Fever, Respiratory Syncytial and SARS-CoV-2 Virus Infection

doi: 10.1128/spectrum.03949-22

Figure Lengend Snippet: Size and morphology of purified DENV DIP particles. (A) Samples containing unpurified DENV DIPs, purified DIPs, or infectious DENV-2 underwent ultracentrifugation. The pelleted material was solubilized and used for Western blot analysis using anti-DENV antibodies directed to the viral envelope (E) or capsid (CA) proteins. (B) Dynamic light scattering (DLS) analysis of purified DENV DIP particle size distribution or (C) DLS of the same DENV DIP preparation after an additional 7 days of storage at 4°C. (D) Transmission electron microscopy (TEM) images (scale bar = 100 nM) of purified DENV DIPs.

Article Snippet: Two μL of resuspended samples was used to quantify DI-290 RNA and DENV-2 NS5 RNA regions using a Luna Universal One-Step RT-qPCR kit (New England Biolabs) in a 10-μL reaction volume.

Techniques: Purification, Western Blot, Transmission Assay, Electron Microscopy

Journal: Microbiology Spectrum

Article Title: Defective Interfering Particles with Broad-Acting Antiviral Activity for Dengue, Zika, Yellow Fever, Respiratory Syncytial and SARS-CoV-2 Virus Infection

doi: 10.1128/spectrum.03949-22

Figure Lengend Snippet: DENV DIPs stimulate host innate immune responses. (A) Huh7 cells were incubated with DENV-2 (MOI [multiplicity of infection] = 0.01 CCID 50 [50% cell culture infective dose]/cell) alone, or DENV-2 mixed with DENV DIP (equivalent to 1,000 DI-290 RNA copies/cell) for 15 h, and then the culture medium was replaced. Culture supernatants were collected at 72 h postinfection (hpi). Viral titers in culture supernatants were measured by a CCID 50 assay. (B to F) To examine host innate immune response, Huh7 cells were treated with DENV-2 (MOI = 0.01 CCID 50 /cell, red line), DENV DIP (equivalent to 1,000 DI-290 RNA copies/cell, black line), or DENV-2 mixed with DENV DIP (green line) for 2, 24, and 72 h. Total RNA was extracted from the cells and the levels of interferon (IFN)-α, IFN-β, ISG15, protein kinase R (PKR), and 2′–5′ oligoadenylate synthetase 1 (OAS1) mRNA were quantified by RT-qPCR. The fold change relative to the untreated control cells was calculated (Ctrl, blue line). Data are shown as means ± SD from three replicate experiments. The P values were calculated using a two-tailed Student’s t test.

Article Snippet: Two μL of resuspended samples was used to quantify DI-290 RNA and DENV-2 NS5 RNA regions using a Luna Universal One-Step RT-qPCR kit (New England Biolabs) in a 10-μL reaction volume.

Techniques: Incubation, Infection, Cell Culture, Quantitative RT-PCR, Two Tailed Test