Journal: bioRxiv

Article Title: The human DEAD-box protein DDX3X regulates host and viral mRNA translation during Sendai Virus infection

doi: 10.64898/2026.03.08.707086

Figure Lengend Snippet: (A) Overview of the different SeV RNA species. (B) Mean count of forward vs. reverse reads in 16hr SeV infected DDX3X PAR-CLIP samples aligned to SeV Cantell strain genome. (C) Distribution of RNAseq reads and DDX3X-crosslinked PAR-CLIP reads aligning to regions of the SeV Cantell strain genome at 16hpi (single RNAseq replicate of n = 6 and PAR-CLIP replicate of n = 2 shown). Transcription start sites and start codons of SeV genes, as well as a region presumed to correspond to SeV dcRNA, are shown. PAR-CLIP T-to-C conversions are indicated as T-to-C mis-matches. Colored sites possess a large proportion of reads with nucleotide mismatches, with the ratio of A,T,C,G shown by the ratio of green, red, blue and orange, respectively. (D) Effects of DDX3X knockdown on FLUC/RLUC signal from cells transfected with pGL3-Prom Firefly luciferase 5’UTR reporter constructs and pGL3-Renilla luciferase. The FLUC signal was normalised to RLUC signal for each sample, and median values of triplicates were then normalised to a pGL3-Prom control with no 5’UTR insert. Reporters containing SeV 5’UTRs were transfected into non-silencing control (NSC) or short-hairpin DDX3X knockdown (shDDX3X) HEK293T cells treated with doxycycline for 72h to induce DDX3X knockdown. P values are calculated via Mann-Whitney U test paired by biological replicate. * = p < 0.05, NS = p > 0.05.

Article Snippet: For SeV infections, cells were inoculated at a concentration of 80 Hemagglutinating-units/mL of Cantell-strain SeV purchased from Charles River Laboratories.

Techniques: Infection, RNA sequencing, Knockdown, Transfection, Luciferase, Construct, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: A Stable Reporter Cell Line to Study Respiratory Syncytial Virus NS2-Mediated Inhibition of IFNB Promoter Activation

doi: 10.64898/2026.02.24.706909

Figure Lengend Snippet: In A) A549 cells were infected with RSV A2 (MOI=3; 8 hrs) before analysis of WCE by immunoblotting using an anti-RSV NS1/NS2 antibody (left panel). The specificity of the anti-RSV NS1/NS2 antibody was confirmed by transfection of siRNA-targeting NS1 or NS2 before infection with RSV A2 (right panel). Anti-actin was used as a loading control. B) A549 cells were transiently transfected with an empty vector or the hNS2-pcDNA5 plasmid containing a humanized version of the RSV Long NS2 coding sequence and analyzed by immunoblotting using anti-RSV NS1/NS2 antibodies. C) A549 cells were co-transfected with an IFNBprom-pGL3 firefly luciferase plasmid, a pRL-null renilla luciferase control plasmid (pRL-Null, Promega) and increasing amounts of hNS2-pcDNA5 plasmid before infection with Sendai virus (SeV) at 80HAU/10 6 cells. Luciferase activities were expressed as fold activation over the corresponding NS condition after normalization with renilla luciferase activities. Data (mean ±SEM; n=3) were analyzed using a two-tailed paired Student’s t-test.

Article Snippet: Alternatively, subconfluent monolayers of cells were infected with Sendai virus (SeV) Cantell strain (Charles River Laboratories) at 40 hemagglutinin units (HAU)/10 6 cells in the minimum volume of serum free medium.

Techniques: Infection, Western Blot, Transfection, Control, Plasmid Preparation, Sequencing, Luciferase, Virus, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: A Stable Reporter Cell Line to Study Respiratory Syncytial Virus NS2-Mediated Inhibition of IFNB Promoter Activation

doi: 10.64898/2026.02.24.706909

Figure Lengend Snippet: A) A549 cells transfected with either 3xFlag-pCMV-3Tag1a control or hNS2-3xFlag-pCMV-3Tag1a plasmids, followed by antibiotic selection to establish stable population and isolate control monoclonal cell lines (A549-ctrl) and NS2-expressing monoclonal cell lines (A549-Flag-NS2). The expression of Flag-tagged NS2 in the resulting monoclonal cells was confirmed by immunoblotting using anti-Flag antibody. Anti-actin was used as a loading control. Transiently transfected cells were used as positive control of Flag-NS2 expression. B) Pools of four A549-Ctrl or of A549-Flag-NS2 monoclonal cells were infected with Sendai virus (SeV) for the indicated times. IFNβ, TNF and SeV N transcript levels were quantified by RT-qPCR. C) A549-Ctrl and A549-Flag-NS2 cells were stimulated with recombinant human IFNβ for the indicated times. WCE were analyzed by immunoblotting using antibodies against total STAT2, phosphorylated STAT2 (Tyr689). Anti-actin was used as a loading control.

Article Snippet: Alternatively, subconfluent monolayers of cells were infected with Sendai virus (SeV) Cantell strain (Charles River Laboratories) at 40 hemagglutinin units (HAU)/10 6 cells in the minimum volume of serum free medium.

Techniques: Transfection, Control, Selection, Expressing, Western Blot, Positive Control, Infection, Virus, Quantitative RT-PCR, Recombinant

Journal: medRxiv

Article Title: Clinical validation of a novel metagenomic nanopore sequencing method for detecting viral respiratory pathogens

doi: 10.64898/2026.02.06.26345651

Figure Lengend Snippet: Red indicates passed and black failed. RNA from murine respirovirus, orthoreovirus and Zika virus were spiked as cDNA synthesis and SISPA controls, MS2 phage was spiked as an extraction, cDNA synthesis and SISPA control. Within the derivation set MS2 was not required to pass QC due to poor performance of the laboratory MS2 stock; in both validation and derivation only detection of 2/3 virus spikes was required to pass QC.

Article Snippet: Extracted RNA (8μl) was treated with DNAse, as recommended, then spiked with three reverse transcription/amplification controls (1μl each, containing approximately 10 4 genome copies each of Zika virus (ATCC-VR-1838DQ), murine respirovirus (ATCC-VR-907DQ) and orthoreovirus (ATCC-VR-824DQ) (ATCC Manassas, Virginia, USA). cDNA synthesis was primed randomly using the 3’ terminal N 9 segment of custom oligonucleotide primers (5’-GAT-GAT-AGT-AGG-GCT-TCG-TCA-CNN-NNN-NNN N-3’; Integrated DNA Technologies, Leuven, Belgium), final concentration 5μM.

Techniques: Virus, cDNA Synthesis, Extraction, Control, Biomarker Discovery