Journal: bioRxiv

Article Title: Generation of broad-spectrum vaccine platforms against tick-borne flaviviruses using ancestral sequence reconstruction

doi: 10.1101/2025.10.31.685878

Figure Lengend Snippet: (A) Plasmid constructs were designed to function as vaccine platforms for high level expression of ancestral TBF envelope antigen using an alphavirus infectious clone platform (SINV WT). A gBlock consisting of the TBF +E sequence was inserted into a Sindbis virus infectious clone following a 3’ subgenomic promoter (3’ SGP) via restriction enzyme digestion and Gibson Assembly (SINV +E). (B) Viral titers of passage 1 (p1) from plasmid-transfected BHK-21 cells. BHK cells were infected at MOI=0.01. Supernatant was collected at indicated times and viral titers were determined by standard plaque assay. (C) Cycle threshold (Ct) values of p1 SINV WT (red) and SINV +E (blue) stocks probing for the ancestral TBF E (ASR TBF E).

Article Snippet: Briefly, SINV WT plasmid was digested with XbaI restriction enzyme (RE) (New England Biolabs, R0145S) to generate a linear product and gel extracted.

Techniques: Plasmid Preparation, Construct, Expressing, Sequencing, Virus, Transfection, Infection, Plaque Assay

Journal: bioRxiv

Article Title: Generation of broad-spectrum vaccine platforms against tick-borne flaviviruses using ancestral sequence reconstruction

doi: 10.1101/2025.10.31.685878

Figure Lengend Snippet: Overview of animal studies in C57BL/6J mice. Female mice (n=8; 9 weeks old) were vaccinated with 10 5 PFU of SINV +E (blue) subcutaneously (SC) in 50 μL solution at day 0 and day 21. Controls include mice vaccinated with 10 5 PFU SINV WT (mock-vaccinated; red) and PBS (mock challenged; green). Whole blood was harvested and sera isolated by cardiac puncture at day 35 to assess neutralizing antibody titers. At day 35 mice are challenged with a lethal dose of 10 5 PFU of Powassan virus SC diluted in 50 μL solution PBS. Weight, clinical score, and survival were monitored over 21 days post-challenge until end of study. Figure created in https://BioRender.com .

Article Snippet: Briefly, SINV WT plasmid was digested with XbaI restriction enzyme (RE) (New England Biolabs, R0145S) to generate a linear product and gel extracted.

Techniques: Isolation, Virus

Journal: Virus Research

Article Title: Mosquito-borne alphaviruses in Zambia: Isolation and characterization of Eilat and Sindbis viruses

doi: 10.1016/j.virusres.2025.199604

Figure Lengend Snippet: Growth kinetics of Zambian SINV isolate M115 Vero (A) and BHK-21 (B) cells were infected with SINV isolate M115 and AR339 at an MOI of 0.0001. C6/36 cells (C) were infected with SINV at an MOI of 0.01. Titers at the indicated each time point were determined by plaque assay. The values shown are mean ± standard deviation (SD) of triplicate. * p < 0.05, *** p < 0.001 by two-way ANOVA test.

Article Snippet: The SINV AR339 strain (VR-1248) was obtained from the ATCC.

Techniques: Infection, Plaque Assay, Standard Deviation

Journal: Journal of Virology

Article Title: A BSL-2 chimeric system designed to screen SARS-CoV-2 E protein ion channel inhibitors

doi: 10.1128/jvi.02252-24

Figure Lengend Snippet: Generation and growth kinetics of SINV 6K ion-channel chimeras. ( A ) The amino acid sequences of 6K and 6K mutant chimeras—WT SINV, ∆6K SINV, SINV-ETM, SINV-ETM (N15A), SINV-ETM (V25F), and SINV-ETM (N15A, V25F). The ETM sequence is shown in red with channel-inactivating mutations in blue. ( B ) Plaque morphologies of viruses mentioned in ( A ) grown in BHK-21 cells for 2 days after electroporation. ( C ) Differences in mean plaque diameters of 10 randomly selected plaques for every mutant virus were compared to WT SINV using GraphPad Prism software. Dunnett’s multiple comparisons test as part of one-way ANOVA was used to determine statistical significance with a 95% confidence interval. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and ns-not significant. ( D ) One-step growth curves of wild-type and channel mutant viruses over 12 hours of infection in BHK-21 cells. Cells were infected at an MOI of 1, and virus-containing media were harvested at given time points to indicate the rate of virus release per hour. Data are representative of two independent experiments. ( E ) Comparison of viral titers and the total number of viral RNA genomes produced by wild-type and mutant viruses 12 hours after electroporation with in vitro transcribed RNA. The experiment was performed in triplicate, and error bars represent the standard deviation (SD). ( F ) One-step growth curves of wild-type and mCherry-tagged SINV and SINV-ETM chimera at an MOI of 2. Data are representative of two independent experiments.

Article Snippet: Bacterial clones of full-length SINV 6K, SARS-CoV-2 E, EGFP, and E mutant proteins (N15A, V25F, and N15A, V25F double mutant) in pET His6 MBP N10 TEV (Addgene #29706 ) were used for bacterial expression and purification.

Techniques: Mutagenesis, Sequencing, Electroporation, Virus, Software, Infection, Comparison, Produced, In Vitro, Standard Deviation

Journal: Journal of Virology

Article Title: A BSL-2 chimeric system designed to screen SARS-CoV-2 E protein ion channel inhibitors

doi: 10.1128/jvi.02252-24

Figure Lengend Snippet: SINV 6K and SARS-CoV-2 E proteins bind to amiloride derivatives. UV-Vis spectra of ( A ) HMA, EIPA, and DMA. UV-Vis spectra of compounds bound to ( B ) SINV 6K, ( C ) SARS-CoV-2 E, and ( D ) EGFP proteins. Blank refers to the absorbance of the dialysis buffer after unbound compounds have been removed from each sample. The data shown represent two independent experiments.

Article Snippet: Bacterial clones of full-length SINV 6K, SARS-CoV-2 E, EGFP, and E mutant proteins (N15A, V25F, and N15A, V25F double mutant) in pET His6 MBP N10 TEV (Addgene #29706 ) were used for bacterial expression and purification.

Techniques:

Journal: Journal of Virology

Article Title: A BSL-2 chimeric system designed to screen SARS-CoV-2 E protein ion channel inhibitors

doi: 10.1128/jvi.02252-24

Figure Lengend Snippet: Inhibition of SINV and SINV-ETM mutant virus infection by amilorides and amantadine in BHK cells at MOI of 1.0 and 12 h incubation. BHK cells were treated with indicated concentrations of ( A, E ) HMA, ( B, F ) EIPA, ( C, G ) DMA, and ( D, H ) amantadine and infected with ( A–D ) mCherry E2 WT SINV (green), mCherry E2 Δ6K SINV (pink) and ( E–H ) mCherry E2 SINV-ETM (orange), ETM (N15A) (blue), ETM (V25F) (red), ETM (N15A, V25F) (purple) viruses at an MOI of 1.0 for 12 hours. ( I ) BHK-21 cells were infected with untagged WT SINV, ∆6K SINV, and SINV-ETM chimera at an MOI of 1.0 treated with indicated concentrations of inhibitors and plaqued after 12 hours.

Article Snippet: Bacterial clones of full-length SINV 6K, SARS-CoV-2 E, EGFP, and E mutant proteins (N15A, V25F, and N15A, V25F double mutant) in pET His6 MBP N10 TEV (Addgene #29706 ) were used for bacterial expression and purification.

Techniques: Inhibition, Mutagenesis, Virus, Infection, Incubation

Journal: Journal of Virology

Article Title: A BSL-2 chimeric system designed to screen SARS-CoV-2 E protein ion channel inhibitors

doi: 10.1128/jvi.02252-24

Figure Lengend Snippet: Time of inhibitor addition assay at high MOI. ( A ) Schematic representation of the time of addition assay protocol. ( B ) Time of addition assay in BHK-21 cells with channel inhibitors. Cells were infected with either mCherry-E2-tagged SINV or SINV-ETM virus at t = 0 with a high MOI of 10. 25 µM HMA, 25 µM EIPA, 50 µM DMA, and 500 µM Amantadine were added to the cells at the indicated time points. Cells were fixed and imaged at 15 hpi at 4× magnification. The fluorescence intensity signal (TRITC channel) was measured for each sample relative to the DMSO-control signal and plotted for graphical representation. Error bars represent the standard error of the mean (SEM) of two independent experiments. Statistical significance was determined for each time point between inhibitor-treated and DMSO control values by two-tailed unpaired t-test with a 95% confidence interval. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and ns-not significant.

Article Snippet: Bacterial clones of full-length SINV 6K, SARS-CoV-2 E, EGFP, and E mutant proteins (N15A, V25F, and N15A, V25F double mutant) in pET His6 MBP N10 TEV (Addgene #29706 ) were used for bacterial expression and purification.

Techniques: Infection, Virus, Fluorescence, Control, Two Tailed Test