ev a71  (ATCC)

Journal: Genes & Diseases

Article Title: Salt-inducible kinase 1 is a key gene in suppressing EVD68-induced asthma by modulating antiviral immunity

doi: 10.1016/j.gendis.2025.101845

Figure Lengend Snippet: SIK1 shows antiviral effects in various viral infections. (A, B) A549 cells were infected with EV-D68 (MOI = 0.1 or 1) for 24 h. (A) Quantitative reverse transcription PCR (RT-qPCR) analysis of relative SIK1 mRNA expression. The results were normalized to GAPDH expression. (B) Western blotting analysis of SIK1 and EV-D68 VP1 protein expression. β-actin was used as the loading control. Values were from three independent experiments and expressed as mean ± standard deviation. (C, D) RD cells were infected with EV-A71 (MOI = 0.1 or 0.5) for 24 h. (C) RT-qPCR analysis of relative SIK1 mRNA expression. The results were normalized to GAPDH expression. (D) Western blotting analysis of SIK1 and EV-A71 VP1 protein expression. β-actin was used as the loading control. Values were from three independent experiments and expressed as mean ± standard deviation. (E, F) A549 cells were transfected with si-SIK1 or si-NC and then infected with EV-D68 (MOI = 0.5) for 24 h. (E) The relative viral RNA copy numbers were determined by RT-qPCR and normalized to GAPDH. (F) The protein expression levels of EV-D68 VP1 and SIK1 were detected by western blotting. β-actin was used as the loading control. Values were from three independent experiments and expressed as mean ± standard deviation. (G, H) A549 cells were transfected with plasmid pCDH-SIK1 or empty pCDH vector and then infected with EV-D68 (MOI = 0.5) for 24 h. The viral replication and protein expression level of EV-D68 VP1 and SIK1 were detected as described above. Values were from three independent experiments and expressed as mean ± standard deviation. (I, J) A549 cells were infected with VSV-GFP for 6 h (MOI = 0.1, 0.5) or HSV-1 for 24 h (MOI = 0.1, 0.2), and then the relative mRNA expression of SIK1 was analyzed by RT-qPCR. Values were from three independent experiments and expressed as mean ± standard deviation. (K, L) Box plots represent the normalized expression levels of SIK1 using Z-score normalization in GSE157103 (for CV-A6) and GSE243200 (for SARS-COV-2) datasets. SIK1 expression correlation was analyzed using Spearman's method. (M, N) A549 cells were transfected with si-SIK1 or si-NC and then infected with VSV-GFP (MOI = 0.5) for 12 h. The replication of VSV-GFP was visualized by immunofluorescence microscopy (scale bar: 50 μm), and VSV-GFP RNA synthesis was determined by RT-qPCR analysis. Values were from three independent experiments and expressed as mean ± standard deviation. (O, P) A549 cells were transfected with plasmid pCDH-SIK1 or empty pCDH vector and then infected with VSV-GFP (MOI = 0.5) for 12 h. To detect viral replication by quantitative PCR, viral titers were determined by plaque assay as described in the methods section, and viral RNA synthesis was determined by RT-qPCR analysis. Values were from three independent experiments and expressed as mean ± standard deviation. (Q, R) SIK1 was interfered with by si-SIK1 or overexpressed via transfection of plasmid pCDH-SIK1 in A549 cells, and then the cells were infected with HSV-1. Viral replication was determined by RT-qPCR. Values were from three independent experiments and expressed as mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; ns, non-significant.

Article Snippet: The EV-D68 (ATCC VR-1826), EV-A71, HSV-1, and VSV-GFP were kept in our laboratory.

Techniques: Infection, Reverse Transcription, Quantitative RT-PCR, Expressing, Western Blot, Control, Standard Deviation, Transfection, Plasmid Preparation, Immunofluorescence, Microscopy, Real-time Polymerase Chain Reaction, Plaque Assay

Journal: iScience

Article Title: Broad-spectrum antiviral activity of antisense oligonucleotides targeting GBF1 against SARS-CoV-2 and influenza viruses

doi: 10.1016/j.isci.2026.114851

Figure Lengend Snippet: Identification of host factors involved in SARS-CoV-2 and HCoV-229E replication (A) Schematic overview of the siRNA screening. HEK293 A/T cells were seeded in 24-well plates and transfected twice with siRNAs targeting 91 host genes previously identified as being involved in influenza virus replication. Cells were then infected with SARS-CoV-2 (100 plaque-forming unit [PFU]/100 μL) one day after the second transfection. Supernatants were collected at 2 days post-infection (dpi) and titrated by plaque assay. (B) Results of the SARS-CoV-2 siRNA screen. The 91 host factors were divided into four batches, each including a non-targeting siRNA as a negative control (N) and siRNA targeting SARS-CoV-2 nsp12 as a positive control (P). Viral titers were calculated based on the difference between each siRNA and its corresponding negative control. Each dot represents the mean of duplicate wells from a single independent experiment. Data are presented as the mean ± standard deviation (SD) of at least three independent experiments. (C) The seven host factors identified in the SARS-CoV-2 siRNA screen were further examined in HCoV-229E, along with the negative control siRNA (N) and positive control siRNA (P) targeting the HCoV-229E N gene. Following the same method described in (A), MRC-5 cells were infected with HCoV-229E (50 tissue culture infectious dose (TCID 50 )/100 μL). Supernatants were collected at 3 dpi and titrated by TCID 50 assay. Viral titers were calculated based on the difference between each siRNA and the negative control. Each dot represents the mean of duplicate wells from a single independent experiment. Data are presented as the mean ± SD of three independent experiments. (D) Cell viability was measured in duplicate wells across two independent experiments using CellTiter-Glo following siRNA transfection. See also and .

Article Snippet: HCoV-229E , ATCC , VR-740.

Techniques: Transfection, Virus, Infection, Plaque Assay, Negative Control, Positive Control, Standard Deviation

Journal: Genes & Diseases

Article Title: Salt-inducible kinase 1 is a key gene in suppressing EVD68-induced asthma by modulating antiviral immunity

doi: 10.1016/j.gendis.2025.101845

Figure Lengend Snippet: The common differentially expressed genes between EV-D68 infection and asthma. (A, B) Volcano plot of differentially expressed genes (DEGs) in the EV-D68-related GSE184488 dataset and asthma-related GSE143303 dataset. (C) Common DEGs in the GSE184488 dataset and the GSE143303 dataset were represented by Venn diagrams. (D) KEGG enrichment analysis of the 74 common DEGs identified in EVD68 infection and asthma. (E) GO enrichment analysis of the 74 common DEGs identified in EVD68 infection and asthma. BP, biological process; CC, cellular component; MF, molecular function.

Article Snippet: The EV-D68 (ATCC VR-1826), EV-A71, HSV-1, and VSV-GFP were kept in our laboratory.

Techniques: Infection

Journal: Genes & Diseases

Article Title: Salt-inducible kinase 1 is a key gene in suppressing EVD68-induced asthma by modulating antiviral immunity

doi: 10.1016/j.gendis.2025.101845

Figure Lengend Snippet: SIK1 expression is induced by EV-D68 infection in vivo . Eight-to-ten-week-old type I interferon receptor-deficient mice ( Ifna −/− ) were treated with EV-D68 (50 μL of 1 × 10 7 PFU/mL viral stock for each mouse) or an equal volume of phosphate-buffered saline through the intranasal route after anesthesia. Lungs were harvested 48 h after infection and then analyzed by hematoxylin-eosin staining, quantitative PCR, and western blotting. (A) Images showing lung inflammation 48 h after treatment with EV-D68 or mock phosphate-buffered saline, as visualized by hematoxylin-eosin staining (scale bar: 50 mm). (B–I) The indicated genes were detected by quantitative PCR and normalized to GAPDH expression. Values are from three independent experiments and expressed as mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (J) The protein expression level of SIK1 in the lungs of mice from the mock and EV-D68 groups was detected by western blotting.

Article Snippet: The EV-D68 (ATCC VR-1826), EV-A71, HSV-1, and VSV-GFP were kept in our laboratory.

Techniques: Expressing, Infection, In Vivo, Saline, Staining, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: Genes & Diseases

Article Title: Salt-inducible kinase 1 is a key gene in suppressing EVD68-induced asthma by modulating antiviral immunity

doi: 10.1016/j.gendis.2025.101845

Figure Lengend Snippet: SIK1 shows antiviral effects in various viral infections. (A, B) A549 cells were infected with EV-D68 (MOI = 0.1 or 1) for 24 h. (A) Quantitative reverse transcription PCR (RT-qPCR) analysis of relative SIK1 mRNA expression. The results were normalized to GAPDH expression. (B) Western blotting analysis of SIK1 and EV-D68 VP1 protein expression. β-actin was used as the loading control. Values were from three independent experiments and expressed as mean ± standard deviation. (C, D) RD cells were infected with EV-A71 (MOI = 0.1 or 0.5) for 24 h. (C) RT-qPCR analysis of relative SIK1 mRNA expression. The results were normalized to GAPDH expression. (D) Western blotting analysis of SIK1 and EV-A71 VP1 protein expression. β-actin was used as the loading control. Values were from three independent experiments and expressed as mean ± standard deviation. (E, F) A549 cells were transfected with si-SIK1 or si-NC and then infected with EV-D68 (MOI = 0.5) for 24 h. (E) The relative viral RNA copy numbers were determined by RT-qPCR and normalized to GAPDH. (F) The protein expression levels of EV-D68 VP1 and SIK1 were detected by western blotting. β-actin was used as the loading control. Values were from three independent experiments and expressed as mean ± standard deviation. (G, H) A549 cells were transfected with plasmid pCDH-SIK1 or empty pCDH vector and then infected with EV-D68 (MOI = 0.5) for 24 h. The viral replication and protein expression level of EV-D68 VP1 and SIK1 were detected as described above. Values were from three independent experiments and expressed as mean ± standard deviation. (I, J) A549 cells were infected with VSV-GFP for 6 h (MOI = 0.1, 0.5) or HSV-1 for 24 h (MOI = 0.1, 0.2), and then the relative mRNA expression of SIK1 was analyzed by RT-qPCR. Values were from three independent experiments and expressed as mean ± standard deviation. (K, L) Box plots represent the normalized expression levels of SIK1 using Z-score normalization in GSE157103 (for CV-A6) and GSE243200 (for SARS-COV-2) datasets. SIK1 expression correlation was analyzed using Spearman's method. (M, N) A549 cells were transfected with si-SIK1 or si-NC and then infected with VSV-GFP (MOI = 0.5) for 12 h. The replication of VSV-GFP was visualized by immunofluorescence microscopy (scale bar: 50 μm), and VSV-GFP RNA synthesis was determined by RT-qPCR analysis. Values were from three independent experiments and expressed as mean ± standard deviation. (O, P) A549 cells were transfected with plasmid pCDH-SIK1 or empty pCDH vector and then infected with VSV-GFP (MOI = 0.5) for 12 h. To detect viral replication by quantitative PCR, viral titers were determined by plaque assay as described in the methods section, and viral RNA synthesis was determined by RT-qPCR analysis. Values were from three independent experiments and expressed as mean ± standard deviation. (Q, R) SIK1 was interfered with by si-SIK1 or overexpressed via transfection of plasmid pCDH-SIK1 in A549 cells, and then the cells were infected with HSV-1. Viral replication was determined by RT-qPCR. Values were from three independent experiments and expressed as mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; ns, non-significant.

Article Snippet: The EV-D68 (ATCC VR-1826), EV-A71, HSV-1, and VSV-GFP were kept in our laboratory.

Techniques: Infection, Reverse Transcription, Quantitative RT-PCR, Expressing, Western Blot, Control, Standard Deviation, Transfection, Plasmid Preparation, Immunofluorescence, Microscopy, Real-time Polymerase Chain Reaction, Plaque Assay

Journal: Genes & Diseases

Article Title: Salt-inducible kinase 1 is a key gene in suppressing EVD68-induced asthma by modulating antiviral immunity

doi: 10.1016/j.gendis.2025.101845

Figure Lengend Snippet: Metformin-mediated activation of SIK1 protects against EV-D68-driven asthma exacerbation in house dust mite (HDM)-sensitized mice. (A) C57BL/6 mice (6–8 weeks) were administered metformin at doses of 100 mg/kg or 250 mg/kg once daily via intraperitoneal injection on day 1 and day 2. On day 3, lung tissues were collected, and the protein level of SIK1 was determined by western blotting analysis. (B) Experimental timeline. C57BL/6 mice (6–8 weeks) were intranasally sensitized with 250 μg kg −1 HDM extract on day 0 and challenged daily with the same dose on days 7–11. On days 12–13, animals received EV-D68 (1 × 10 6 PFU/kg) or DMEM (vehicle) intranasally. Metformin (100 mg/kg, intraperitoneal) was administered once daily on days 12–14. Airway hyper-responsiveness measurements and broncho-alveolar lavage fluid (BALF) collection were performed on day 15; lung tissue was used for quantitative PCR analyses. (C) Airway responsiveness to increasing doses of methacholine. (D) Differential cell counts of BALF by Wright-Giemsa staining. (E – H) The indicated genes were detected by quantitative PCR and normalized to GAPDH expression. Values were from three independent experiments and expressed as mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The EV-D68 (ATCC VR-1826), EV-A71, HSV-1, and VSV-GFP were kept in our laboratory.

Techniques: Activation Assay, Injection, Western Blot, Real-time Polymerase Chain Reaction, Staining, Expressing, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Functional decoupling of crRNA enables customizable CRISPR diagnostics

doi: 10.1093/nar/gkag189

Figure Lengend Snippet: Customizable one-pot diagnostic system. ( A ) Schematic illustration comparing two design approaches for one-pot reactions. Created in BioRender, Park, H. ( https://BioRender.com/jaoj8es ). (B–D) Fluorescence analysis of one-pot reactions across varying concentrations of SARS-CoV-2 RNA using Cas12a complexes formed with ( B ) crRNA1, ( C ) crRNA2, and ( D ) crRNA3. For each condition, two plots are shown: conventional (left), and decoupling based (right) one-pot strategy at 20 min. All experiments were performed in triplicate; error bars represent mean ± standard deviation ( n = 3). P- values were calculated by unpaired two-tailed Student’s t -test (**** P < .0001, *** P < .001, ** P < .01, * P < .05, ns = not significant).

Article Snippet: SARS-CoV-2 (cat. #VR-1986HK) and influenza A virus (cat. #VR-1938) were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Diagnostic Assay, Fluorescence, Standard Deviation, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Functional decoupling of crRNA enables customizable CRISPR diagnostics

doi: 10.1093/nar/gkag189

Figure Lengend Snippet: Clinical feasibility of the CLUE system. ( A ) Clinical application of the CLUE system using 120 nasopharyngeal swab samples: 30 SARS-CoV-2-positive, 30 influenza A-positive, 30 influenza B-positive, 30 negative control specimens. Error bars represent mean ± standard deviation ( n = 3). The dashed line represents the threshold, determined by the maximum Youden’s index. ( B ) Comparison of fluorescence signals between different patient swab samples, P- values were calculated by unpaired two-tailed Student’s t -test (**** P < .0001, *** P < .001, ** P < .01, * P < .05, ns = not significant). ( C ) ROC curve generated from clinical samples. The AUC was 1.0. ( D ) Confusion matrix of the clinical results. The sensitivity was 100% (95% CIs: 88.7%–100%) and the specificity was 100% (95% CIs: 95.9%–100%). Statistical significance between the test results and clinical status was determined by Fisher’s exact test (two-sided P < .0001; odds ratio = ∞, 95% CIs 514.4 to ∞).

Article Snippet: SARS-CoV-2 (cat. #VR-1986HK) and influenza A virus (cat. #VR-1938) were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Negative Control, Standard Deviation, Comparison, Fluorescence, Two Tailed Test, Generated