Journal: bioRxiv

Article Title: Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay

doi: 10.64898/2026.03.17.712358

Figure Lengend Snippet: (A) The workflow is used to (i) verify dataset structure/QC; (ii) localize and rank conserved, feature-anchored windows; and (iii) guide MSA-based manual primer-probe design and specificity screening. Box colors indicate workflow phases; grey boxes denote intermediate artifacts/outputs; yellow boxes mark workflow components introduced in this study. (B) Family-level representation of dataset composition. Bars show the top 10 viral families by number of genomes, with Flaviviridae shown additionally for reference (highlighted), even though it falls outside the top 10. The cleaned dataset comprises 11,846 viral genomes, including 10,472 non-segmented and 1,374 segmented genomes. Most genomes fall into other taxa (6,292) and Unclassified (3,220), followed by Geminiviridae (591), Spinareoviridae (251), Sedoreoviridae (250), Phenuiviridae (219), Peribunyaviridae (205), Steitzviridae (204), Rhabdoviridae (180), Polydnaviriformidae (172), Fiersviridae (157), and Flaviviridae (105). (C) Genus-level composition of the family Flaviviridae in the RefSeq complete/near-complete genome dataset. Orthoflavivirus comprises the largest fraction (48.6%, n = 51), followed by Hepacivirus (8.1%, n = 19), unclassified Flaviviridae (16.2%, n = 17), Pestivirus (12.4%, n = 13), and Pegivirus (4.8%, n = 5). Genera are ranked by the number of complete/near-complete RefSeq genomes. (D) An alignment-free distance matrix was computed from preprocessed Orthoflavivirus genomes using a Mash-style transformed Jaccard metric (k = 11) and used to infer a neighbor-joining tree (rooted with a Pestivirus , orange branch in the bottom). The topology recapitulates expected intra-genus structure and coherent species-level clades, which we used to verify taxonomic assignments and flag outliers prior to conserved-signature analysis. The focused species within this study were labeled with their names on the tree. DENV1–4 = dengue virus serotype 1-4, JEV = Japanese encephalitis virus, WNV1,2 = West Nile virus linage 1,2, ZIKV_MR766 = Zika virus, ZIKV_ Natal_RGN = Zika virus

Article Snippet: Reference viruses were obtained from the American Type Culture Collection (ATCC), including DENV1 strain Hawaii (ATCC VR-1856), DENV2 strain TH-36 (ATCC VR-1810), DENV3 strain H87 (ATCC VR-3380), DENV4 H241 (ATCC VR-1490), and ZIKV strain PRVABC59 (ATCC VR-1843).

Techniques: Transformation Assay, Labeling, Virus

Journal: bioRxiv

Article Title: Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay

doi: 10.64898/2026.03.17.712358

Figure Lengend Snippet: (A) Average common feature shortest-unique k-mer (SUK) curves across k=9–51. Vertical guides highlight the onset of rapid feature loss from K17 and a sharper decline around K19, marking the transition from broadly shared to increasingly specific k-mer features. (B) SUK plot for the Orthoflavivirus considered genomes—dengue virus (DENV1–4), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV)—showing the decay in shared k-mer features as k increases. We selected k = 19 for downstream conserved-signature discovery because at k ≥ 21 the number of shared k-mers dropped below the genus-level panel size (n = 51), limiting detection of features conserved across the full panel. (C) Genome-presence rank plot of 19-mers within Orthoflavivirus . Bars show the number of genomes containing each 19-mer sequence, ranked from highest to lowest; the top 2 highest-presence 19-mers are labeled. (D) Unitig summary after compacted de Bruijn graph (cDBG) consolidation of 19-mers filtered to those present in ≥3 genomes. Unitig length is plotted against the number of genomes supporting each unitig. Point size indicates k-mer support (number of distinct 19-mers per unitig). Highly conserved unitigs (supported by more genomes) are generally shorter, whereas longer unitigs are typically supported by fewer genomes. (E) Genome-wide conservation hotspot profile along a pseudo-genomic coordinate. Bars indicate the number of genomes contributing conserved hits per window, with color encoding conservation score. Prominent peaks coincide with the NS5 region; terminal repeat/UTR regions are indicated in gray (0–0.3 kbp and 10.5–11.1 kbp). The gray boxes indicate the terminal repeat/UTR regions.

Article Snippet: Reference viruses were obtained from the American Type Culture Collection (ATCC), including DENV1 strain Hawaii (ATCC VR-1856), DENV2 strain TH-36 (ATCC VR-1810), DENV3 strain H87 (ATCC VR-3380), DENV4 H241 (ATCC VR-1490), and ZIKV strain PRVABC59 (ATCC VR-1843).

Techniques: Virus, Sequencing, Labeling, Genome Wide

Journal: bioRxiv

Article Title: Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay

doi: 10.64898/2026.03.17.712358

Figure Lengend Snippet: (A) MAFFT multiple sequence alignment of 600 bps conserved windows from 51 Orthoflavivirus genomes, the red lines indicate the start and the end of the amplicon window used to place the primers and probe. The amplicon region shows high per-base identity, indicated by multiple, contiguous, or near-contiguous conserved bases at 100% identity, colored in forest green. (B) The top panel shows MSA of the considered genomes, including dengue virus (DENV1–4), Japanese encephalitis virus (JEV), West Nile virus (WNV1,2), yellow fever virus (YFV), and Zika virus (ZIKV) strains MR766 Natal RGN annotated with primer pair and probe position with mismatches in magenta underneath the genomes. The lower panel shows chemical properties of each oligonucleotide, including probe and primer concentration, Na+ concentration, length, degeneracy, GC content, salt-corrected T m , ΔG of the reaction, and product size.

Article Snippet: Reference viruses were obtained from the American Type Culture Collection (ATCC), including DENV1 strain Hawaii (ATCC VR-1856), DENV2 strain TH-36 (ATCC VR-1810), DENV3 strain H87 (ATCC VR-3380), DENV4 H241 (ATCC VR-1490), and ZIKV strain PRVABC59 (ATCC VR-1843).

Techniques: Sequencing, Amplification, Virus, Concentration Assay

Journal: bioRxiv

Article Title: Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay

doi: 10.64898/2026.03.17.712358

Figure Lengend Snippet: (A) Faceted bar charts show Ct values measured across input copy numbers (copies per µL) for each virus—dengue virus (DENV1–4), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV). Bars summarize Ct at each concentration, with points indicating individual reactions (technical replicates); red diamonds denote the mean (error bars, where shown, indicate variability across replicates). Across DENV1–4, ZIKV, and JEV (triplicates), Ct values generally decrease as input concentration increases. ZIKV shows greater replicate-to-replicate variation at some concentrations, reflected by wider error bars compared to other targets. WNV and YFV were detected at 1,000 copies per µL; only a single concentration was tested for these viruses due to limited sample material. Together, these results indicate broad target coverage and reliable amplification across a wide input range for the main panel members. (B) Limit of detection (LOD) assessment of the designed primer-probe set. Prior to testing clinical specimens, LOD was evaluated using nucleic acids from 6 arboviruses (DENV1–4, ZIKV, and JEV) at 1, 10, 1000 copies per µL, with 12 technical replicates per dilution. The assay detected DENV3, DENV4, and JEV down to 1 copy per µL, whereas DENV1, DENV2, and ZIKV were consistently detected down to 10 copies per µL. Points represent individual reaction; a diamond indicates the mean Ct, and error bars indicate ±SD. ND indicates not detected (did not meet the ≥95% positivity criterion and therefore did not qualify as the LOD). (C) Paired comparison of Ct values between assays in clinical samples. Ct values obtained with the commercial kit (Com; circles) and the designed primer–probe set (Des; triangles) are shown for each target, with paired measurements from the same sample connected by dashed lines, for 100 positive samples and 50 positive samples (20 positive with Chikungunya virus [CHIKV] and 30 others). Box plots summarize the distributions (center line, median; box, interquartile range; whiskers, range). Relative to the commercial kit, the designed primer–probe set detected DENV1–4 significantly earlier (lower Ct; p = 0.0004 [n = 25], 0.0013 [n=25], <0.0001 [n=11] and <0.0001 [n=25] for DENV1–4, respectively) but detected ZIKV later (higher Ct; p < 0.0001 [n =14]). P values were calculated using a two-sided paired t-test on matched Com–Des Ct values for each target. Overall diagnostic performance is reported in Tables 1,2 .

Article Snippet: Reference viruses were obtained from the American Type Culture Collection (ATCC), including DENV1 strain Hawaii (ATCC VR-1856), DENV2 strain TH-36 (ATCC VR-1810), DENV3 strain H87 (ATCC VR-3380), DENV4 H241 (ATCC VR-1490), and ZIKV strain PRVABC59 (ATCC VR-1843).

Techniques: Virus, Concentration Assay, Amplification, Comparison, Diagnostic Assay

Journal: bioRxiv

Article Title: Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay

doi: 10.64898/2026.03.17.712358

Figure Lengend Snippet: (A) Faceted bar charts show Ct values measured across input copy numbers (copies per µL) for each virus—dengue virus (DENV1–4), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV). Bars summarize Ct at each concentration, with points indicating individual reactions (technical replicates); red diamonds denote the mean (error bars, where shown, indicate variability across replicates). Across DENV1–4, ZIKV, and JEV (triplicates), Ct values generally decrease as input concentration increases. ZIKV shows greater replicate-to-replicate variation at some concentrations, reflected by wider error bars compared to other targets. WNV and YFV were detected at 1,000 copies per µL; only a single concentration was tested for these viruses due to limited sample material. Together, these results indicate broad target coverage and reliable amplification across a wide input range for the main panel members. (B) Limit of detection (LOD) assessment of the designed primer-probe set. Prior to testing clinical specimens, LOD was evaluated using nucleic acids from 6 arboviruses (DENV1–4, ZIKV, and JEV) at 1, 10, 1000 copies per µL, with 12 technical replicates per dilution. The assay detected DENV3, DENV4, and JEV down to 1 copy per µL, whereas DENV1, DENV2, and ZIKV were consistently detected down to 10 copies per µL. Points represent individual reaction; a diamond indicates the mean Ct, and error bars indicate ±SD. ND indicates not detected (did not meet the ≥95% positivity criterion and therefore did not qualify as the LOD). (C) Paired comparison of Ct values between assays in clinical samples. Ct values obtained with the commercial kit (Com; circles) and the designed primer–probe set (Des; triangles) are shown for each target, with paired measurements from the same sample connected by dashed lines, for 100 positive samples and 50 positive samples (20 positive with Chikungunya virus [CHIKV] and 30 others). Box plots summarize the distributions (center line, median; box, interquartile range; whiskers, range). Relative to the commercial kit, the designed primer–probe set detected DENV1–4 significantly earlier (lower Ct; p = 0.0004 [n = 25], 0.0013 [n=25], <0.0001 [n=11] and <0.0001 [n=25] for DENV1–4, respectively) but detected ZIKV later (higher Ct; p < 0.0001 [n =14]). P values were calculated using a two-sided paired t-test on matched Com–Des Ct values for each target. Overall diagnostic performance is reported in Tables 1,2 .

Article Snippet: Reference viruses were obtained from the American Type Culture Collection (ATCC), including DENV1 strain Hawaii (ATCC VR-1856), DENV2 strain TH-36 (ATCC VR-1810), DENV3 strain H87 (ATCC VR-3380), DENV4 H241 (ATCC VR-1490), and ZIKV strain PRVABC59 (ATCC VR-1843).

Techniques: Virus, Concentration Assay, Amplification, Comparison, Diagnostic Assay

Journal: bioRxiv

Article Title: Alignment-Free Guided Design of a Pan-Orthoflavivirus RT-qPCR Assay

doi: 10.64898/2026.03.17.712358

Figure Lengend Snippet: (A) Faceted bar charts show Ct values measured across input copy numbers (copies per µL) for each virus—dengue virus (DENV1–4), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV). Bars summarize Ct at each concentration, with points indicating individual reactions (technical replicates); red diamonds denote the mean (error bars, where shown, indicate variability across replicates). Across DENV1–4, ZIKV, and JEV (triplicates), Ct values generally decrease as input concentration increases. ZIKV shows greater replicate-to-replicate variation at some concentrations, reflected by wider error bars compared to other targets. WNV and YFV were detected at 1,000 copies per µL; only a single concentration was tested for these viruses due to limited sample material. Together, these results indicate broad target coverage and reliable amplification across a wide input range for the main panel members. (B) Limit of detection (LOD) assessment of the designed primer-probe set. Prior to testing clinical specimens, LOD was evaluated using nucleic acids from 6 arboviruses (DENV1–4, ZIKV, and JEV) at 1, 10, 1000 copies per µL, with 12 technical replicates per dilution. The assay detected DENV3, DENV4, and JEV down to 1 copy per µL, whereas DENV1, DENV2, and ZIKV were consistently detected down to 10 copies per µL. Points represent individual reaction; a diamond indicates the mean Ct, and error bars indicate ±SD. ND indicates not detected (did not meet the ≥95% positivity criterion and therefore did not qualify as the LOD). (C) Paired comparison of Ct values between assays in clinical samples. Ct values obtained with the commercial kit (Com; circles) and the designed primer–probe set (Des; triangles) are shown for each target, with paired measurements from the same sample connected by dashed lines, for 100 positive samples and 50 positive samples (20 positive with Chikungunya virus [CHIKV] and 30 others). Box plots summarize the distributions (center line, median; box, interquartile range; whiskers, range). Relative to the commercial kit, the designed primer–probe set detected DENV1–4 significantly earlier (lower Ct; p = 0.0004 [n = 25], 0.0013 [n=25], <0.0001 [n=11] and <0.0001 [n=25] for DENV1–4, respectively) but detected ZIKV later (higher Ct; p < 0.0001 [n =14]). P values were calculated using a two-sided paired t-test on matched Com–Des Ct values for each target. Overall diagnostic performance is reported in Tables 1,2 .

Article Snippet: Reference viruses were obtained from the American Type Culture Collection (ATCC), including DENV1 strain Hawaii (ATCC VR-1856), DENV2 strain TH-36 (ATCC VR-1810), DENV3 strain H87 (ATCC VR-3380), DENV4 H241 (ATCC VR-1490), and ZIKV strain PRVABC59 (ATCC VR-1843).

Techniques: Virus, Concentration Assay, Amplification, Comparison, Diagnostic Assay

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Comparison of the oncolytic effects of mutant jin-3 versus R124 reovirus in bladder cancer cell lines in vitro (A) Mean viral S4Q copy number (log fold change S4Q mRNA expression vs. mock treated cells) in bladder cancer cell lines exposed to a range of MOI (0.01–0.1–1–10) of either R124 or jin-3 reoviruses after 24 h. In red, the highest infection with virus is shown. Two-way ANOVA followed by Tukey’s post hoc test. n = 3 (2 replicates). UM-UC-3 cells R124 reovirus MOI1 vs. mock p = 0.0184; ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection, and dollar signs indicate R124 versus jin-3. (B) Viral load (% of viable, single cells expressing Sigma-3 protein) in bladder cancer cell lines exposed to MOI10 of either R124 or jin-3 after 72 h mean (SD) ( N = 3). Two-way ANOVA followed by Tukey’s post hoc test. ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection, and dollar signs indicate R124 versus jin-3. (C) Confocal images of viral protein (Sigma-3, green) and DAPI (blue)-stained bladder cancer cells treated with MOI10 of R124 or jin-3 for 72h. Scale bars, 25 μm. (D) Mean percentage of viable cells after exposure to a range of MOI of either R124 or jin-3 for 6 days. n = 3 (6 replicates). p values are depicted (vs. mock; when 2 depicted upper p value is R124 vs. jin-3 ); ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection, and dollar signs indicate R124 versus jin-3. (E) Percentage of single, viable, JAM-A protein expressing cells. Mean (SD), n = 3. MOI = multiplicity of infection.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Comparison, Mutagenesis, In Vitro, Expressing, Infection, Virus, Staining

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Comparison of the oncolytic effects of reovirus mutant jin-3 versus wild-type reovirus R124 in 3D cultures in vitro TM00024 bladder PDXOs were exposed to the indicated plaque-forming units (PFUs) of reoviruses for 3 or 7 days. (A) Mean (SD) viral copy number of TM00024 PDXOs treated for 3 days with the indicated PFUs of the reoviruses. Two-way ANOVA followed by Tukey’s post hoc comparison ( n = 3) ∗∗∗ p < .001. (B–F) Viability (mean [SD] was measured using cell titer glo 3D after days 3 and 7 of reovirus exposure. N = 3 (3 replicates). Two-way ANOVA followed by Tukey’s post hoc comparison. ∗∗∗ p < .001. (C) Confocal images of PDXOs (day 7) stained for respectively H&E, panKRT (red); Sigma-3, c-CASP3, or PCNA (green); and DAPI (blue). Scale bars, 20 μm. Cells expressing the respective proteins Sigma-3 (D), c-CASP-3 (E), and nuclear PCNA (F) were counted with ImageJ and divided by the amount of panKRT+_DAPI+ cells. At least five fields were scored per technical replicate. Mean (SD) of N = 3 (3 replicates). ∗∗∗ p < .001, asterisks indicate reovirus infection versus mock same day. Two-way ANOVA followed by Tukey’s post hoc comparison.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Comparison, Mutagenesis, In Vitro, Staining, Expressing, Infection

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Comparison of jin-3 and R124 reovirus infection in ex vivo cultured tumor tissue Explanted tissue slices from either RT-112 CDX (A–E) or TM00024 PDX (F–J) were exposed to the indicated PFU of R124 or jin-3 reovirus for 3 days. Tissues were stained for H&E, panKRT (red); Sigma-3, c-CASP3 or PCNA (green), type I collagen (white), and DAPI (blue). Representative confocal images are shown. Scale bars, 20 μm. Cells expressing the respective proteins were counted with ImageJ and divided by the number of panKRT+_DAPI+ cells. At least four fields were scored per technical replicate. Mean (SD) of N = 2 (4 replicates). The ratio fragmented tumor cells was measured by the number of fragmented cells/total tumor cells (E and J). ∗∗∗ p < .001, reovirus infection versus mock. Mean (SD), n = 2 (4 replicates). Two-way ANOVA followed by Tukey’s post hoc comparison.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Comparison, Infection, Ex Vivo, Cell Culture, Staining, Expressing

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Comparison of jin-3 and R124 reovirus infection in ex vivo cultured tumor tissue slices from human bladder cancer patients Explanted tissue slices from patients ( n = 15) diagnosed with bladder cancer were exposed to either mock, R124, or jin-3 reovirus for 3 days and stained for viral protein, c-CASP-3, PCNA (proliferation), and integrity of the cell (nuclear DAPI and panKRT tumor marker). (A–E) Representative confocal images of the mock and 10 7 PFU condition of both viruses from five patients ranging from low-risk NMIBC to MIBC (viral protein [green, Sigma-3], tumor markers [red, panKRT], and nuclei [DAPI, blue]. Scale bars, 20 μm). Cells expressing the respective proteins [(B) Sigma-3, (C) c-CASP3, or (D) nuclear PCNA] were counted with ImageJ and divided by the number of panKRT+_DAPI+ cells. At least four fields were scored per technical replicate. Mean (SD) (3–4 replicates) with each dot representing one bladder cancer patient. The ratio of fragmented tumor cells was measured by the number of fragmented cells/total tumor cells (E). ∗∗∗ p < 0.001, reovirus infection versus mock, one-way ANOVA followed by Tukey’ post hoc comparison.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Comparison, Infection, Ex Vivo, Cell Culture, Staining, Marker, Expressing

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Comparison of immune modulation induced by jin-3 and R124 reovirus treatment of bladder cancer cell lines Heat maps of various inflammatory cytokines and interferon-stimulated genes (log fold change mRNA expression [2 −ΔΔCt ] vs. mock treated cells) in bladder cancer cell lines UM-UC-3 (A), T24 (B), HT-1197 (C), RT-112 (D), RT-4 (E), TCCSUP (F), J82 (G), and PDXOs (H) exposed to a range of either R124 or jin-3 reoviruses after respectively 24 h (cell lines) and 3 days (PDXOs). p values are depicted (vs. mock; when 2 depicted upper p value is R124 vs. jin-3 ) ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection, and dollar signs indicate R124 versus jin-3.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Comparison, Expressing, Infection

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Comparison of immunogenic cell death induced by jin-3 and R124 reovirus treatment of bladder cancer cell lines (A–C) Cell lines were treated with oncolytic viruses for 48 h at an MOI of 10, and the DAMPs ecto-calreticulin (A) and ecto-HSP90 (B) were measured using flow cytometry. Secreted HMGB1 protein was measured with an ELISA after 48 h of treatment with a dose range of oncolytic viruses (C). ∗∗∗ p < 0.001, $$$ p < 0.001, asterisks indicate reovirus infection versus mock same day, and dollar signs indicate R124 vs. jin-3. Mean (SD), N = 3 (2 replicates). Two-way ANOVA followed by Tukey’s posthoc comparison. (D) Correlation graph of the HMGB1 release ( x axis), percentage of viable cells (size of the dots), and fold change of ecto-CRT ( y axis)-positive cells at MOI 10 of the indicated virus.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Comparison, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Infection, Virus

Journal: Molecular Therapy Oncology

Article Title: Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

doi: 10.1016/j.omton.2026.201128

Figure Lengend Snippet: Activation of PBMCs in RT-112 co-cultures (A) Brightfield images of RT-112 bladder cancer cells that were cultured in 60% Matrigel and allowed to form 3D structures for approximately 3 days. Subsequently, PBMCs were added at an effector: target ratio of 20:1 in absence or presence of either R124 or jin-3 reovirus at the indicated PFU for an additional 3 days. (B) Fragmented KRT18 was determined as an outcome measure for tumor cell killing 3 days after OV exposure. (C–E) CXCL10 levels and (D) IFN-γ levels were measured 3 days after OV exposure. ( n = 3, 2 replicates). Mean (SD). Two-way ANOVA with Tukey’s post hoc comparison. (E) Heatmap of various inflammatory cytokines and interferon-stimulated genes (log fold change mRNA expression [2 −ΔΔCt ] vs. mock treated cells) in 3D cultured RT-112 cells or RT-112 cells co-cultured with PBMCs exposed to either R124 or jin-3 reoviruses. Mean (SD). p values are depicted (vs. mock; when two depicted upper p value is R124 vs. jin-3 ) ∗∗∗ p < .001, $$$ p < .001, asterisks indicate mock versus reovirus infection and dollar signs R124 versus jin-3. , n = 2 (2 replicates). Two-way ANOVA followed by Tukey’s post hoc comparison.

Article Snippet: Wild-type T3D reovirus strain R124 was plaque-purified from the wild-type reovirus T3D (ATCC) on HER911 cells., Reovirus mutant jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.

Techniques: Activation Assay, Cell Culture, Comparison, Expressing, Infection