wild type t3d reovirus strain r124  (ATCC)

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

Journal: bioRxiv

Article Title: A type II Thoeris anti-phage defense enacts growth arrest independent of NAD depletion

doi: 10.64898/2026.01.22.701046

Figure Lengend Snippet: A : Overview of the different types of discovered Thoeris systems. Previously characterized Thoeris systems are grouped into four distinct types, with type I and type II further distinguished into 11 subfamilies. All types have in common one or more TIR-domain containing genes (called thsB in type I and II, thcB in type III and PsTIR in type IV). The categorization is based on the effector domain (highlighted with diagonal bars), which is unique for every type. B : Genome context of the ATCC 25922 type II Thoeris system: An overview of the 5, 132, 219 bp genome of E. coli ATCC 25922, with the locations of known phage-defense systems highlighted. The Thoeris system, highlighted in blue, is located in a part of the genome that is not homologous to other E. coli strains, but is not in proximity to other known phage defense systems. C : Domain annotation: The Thoeris locus, consisting of thsA , thsB2 and thsB1 , with their predicted functional domains annotated. The exact amino acid boundaries are shown on top. ThsB2 is predicted to contain a TIR-like domain (pf08937), similar to other Thoeris systems, while thsB1 is predicted to contain a TIR domain (pf13676). D : Alignment of the predicted structures of the ATCC 25922 ThsB2 protein (red) with the Streptococcus suis ThsB (blue; CYX44050. 1). CYX44050. 1 was chosen as representative of the subgroup 9 of type II Thoeris systems. TM-align of the two structures gave a TM-score of 0. 84431, where 1 would indicate a perfect match and < 0. 2 would correspond to randomly chosen unrelated proteins.

Article Snippet: To determine whether plasmid-based expression of thsAB2 also confers protection in the native host, we introduced the same pthsAB2 expression plasmid into both the wild-type (WT) ATCC 25922 strain and Δ thsAB2B1 mutant.

Techniques: Functional Assay

Journal: bioRxiv

Article Title: A type II Thoeris anti-phage defense enacts growth arrest independent of NAD depletion

doi: 10.64898/2026.01.22.701046

Figure Lengend Snippet: progressiveMAUVE alignment of ATCC 25922 and MG1655 genome sequences. The bottom shows a zoomed in view of the ATCC 25922 genomic region (1, 850, 000 - 2, 150, 000) containing the Thoeris locus, which is highlighted in blue. The height of the bar describes the similarity profile that corresponds to the average conservation between regions, while an empty space indicates a lack of detectable homology. The color of the bars show large regions that are presumably homologous and internally free from genomic rearrangement.

Article Snippet: To determine whether plasmid-based expression of thsAB2 also confers protection in the native host, we introduced the same pthsAB2 expression plasmid into both the wild-type (WT) ATCC 25922 strain and Δ thsAB2B1 mutant.

Techniques:

Journal: bioRxiv

Article Title: A type II Thoeris anti-phage defense enacts growth arrest independent of NAD depletion

doi: 10.64898/2026.01.22.701046

Figure Lengend Snippet: A: E. coli ATCC 25922 is resistant to most of the Basel phage collection, even in the absence of Thoeris. Number of phage plaques forming on the native ATCC 25922 host (WT), with the thsAB2B1 locus knocked out (KO) or on a susceptible host carrying an empty vector plasmid (pET28) or the same vector carrying the thsAB2 locus (pthsAB2). An asterisk highlights the phages showing >100-fold difference between pthsAB2 and pET28. A crossed out square means no plaques detected. Each square represents the average of two biological replicates. EOP: efficiency of plating. B: Susceptibility of E. coli ATCC 25922 to phage T4 infection. The E. coli strain contains the native Thoeris thsAB2B1 locus or expresses the thsAB2 locus from a plasmid, compared to the thsAB2B1 knockout strain and an empty vector control. C: Growth curves of ATCC 25922 WT and thsAB2B1- deletion strains, carrying thsAB2 plasmid, infected by phage T4 at varying MOIs. ATCC 25922 WT carrying an empty vector control is shown as negative control, being susceptible to phage T4. D: Heterologous expression of the thsB1B2A locus, with thsB1 under the control of an arabinose inducible promoter. Growth curves of E. coli MG1655 and ATCC 25922 strains heterologously expressing thsAB2B1 under inducing (+ L-arabinose) or repressing (+ D-glucose) conditions. Lines and shaded regions represent the mean and standard deviation of three independent cultures.

Article Snippet: To determine whether plasmid-based expression of thsAB2 also confers protection in the native host, we introduced the same pthsAB2 expression plasmid into both the wild-type (WT) ATCC 25922 strain and Δ thsAB2B1 mutant.

Techniques: Plasmid Preparation, Infection, Knock-Out, Control, Negative Control, Expressing, Standard Deviation