Journal: Emerging Microbes & Infections

Article Title: A CRISPR activation screen identifies CH25H as a restriction factor against influenza viruses by targeting accessible cholesterol

doi: 10.1080/22221751.2026.2651464

Figure Lengend Snippet: CRISPR activation screen identified host restriction factors for involved in influenza virus infection in A549 cells. (A) Schematic of CRISPRa library construction in A549 cells and screening strategy for influenza virus infection. (B) Gene enrichment analysis in A549 cells transduced with the CRISPRa library after H7N9 infection. (C) Gene Ontology functional analysis of highly enriched genes ( P < 0.05). (D–E) H7N9 infection in HEK 293 T cells overexpressing (D) or with knockdown of (E) candidate genes. (F) H7N9 infection led to upregulation of CH25H in human cells, mouse cells and human lung organoids. (G) Spatial multi-omics profiling of H7N9-infected mouse lungs by in situ pairwise sequencing. Blue, Ch25 h . Yellow, Ifit2 . (H) A549 cells were infected with different subtypes of influenza viruses at MOI of 2. Cells were subsequently harvested at 12 h p.i. to measure viral M1 mRNA expression levels by qRT-PCR. Mean ± SD of n = 3. **** P < 0.0001, by Student’s t -test.

Article Snippet: In the K-604 dihydrochloride rescue experiment, A549 cells subjected to 25HC treatment with DMSO or 20 μM K-604 dihydrochloride (TargetMol, # T11733 ) for 12 h were washed and maintained with DMSO or K-604 dihydrochloride for an additional 4 h before exposure to H7N9 pseudovirus.

Techniques: CRISPR, Activation Assay, Virus, Infection, Transduction, Functional Assay, Knockdown, Biomarker Discovery, In Situ, Sequencing, Expressing, Quantitative RT-PCR

Journal: Emerging Microbes & Infections

Article Title: A CRISPR activation screen identifies CH25H as a restriction factor against influenza viruses by targeting accessible cholesterol

doi: 10.1080/22221751.2026.2651464

Figure Lengend Snippet: Identification of CH25H as a host antiviral factor against influenza viruses. (A) Multicycle replication of H7N9 influenza virus in A549 cells or cell lines stably expressing CH25H (MOI = 0 .1). Viral titers were measured by TCID 50 assay at 48 h p.i. (B–C) A549 cells were transfected with CH25H-Myc plasmids or empty vector control for 24 h and infected with H7N9 influenza virus at MOI = 1 . The relative level of NP RNA was detected by qPCR (B). NP proteins were detected by western blot (C). (D) Viral titers were measured by TCID 50 assay in WT and CH25H-deficient A549 cells at 48 h p.i. (E–F) A549 cells were transfected with CH25H-targeting siRNA or negative control (NC) siRNA for 48 h. Then cells were infected with H7N9 influenza virus at MOI = 1 . NP and M1 were measured by qPCR (E) and western blot (F). (G) Catalytic reaction scheme of CH25H and 25HC. (H–I) Cells were exposed to DMSO or 25HC (10 μM) for 12 h before being infected with H7N9 influenza virus at MOI = 1 . NP RNA (H) and NP protein (I) was analyzed. (J–K) A549 cells were treated with DMSO or 25HC, and then challenged with H7N9 influenza virus at MOI = 1 , NP RNA (J) and protein (K) were quantified after 12 h. (L) A549 cells were treated with 20 μM 25HC for 0, 3, 6, 12, and 18 h prior to H7N9 infection (MOI = 0 .3). Cells were subsequently harvested at 12 h p.i. to measure viral NP mRNA expression levels by qRT-PCR. (M) A549 cells transfected with CH25H or control siRNA were treated with 20 μM 25HC for 12 h prior to infection with H7N9 influenza virus. (N) A549 cells were challenged with H1N1, H5N6, H7N9 and H9N2 influenza viruses at MOI = 0 .3 for 6 h after treatment with 25HC. M1 proteins (green) were probed by immunofluorescence. Scale bars, 200 μm. Fluorescence dots inhibition was analyzed by high-content cell imaging system. Inhibition efficiency = 100 − ( total fluorescent dots in 25 HC total fluorescent dots in DMSO ) ∗ 100 . Mean ± SD of n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns P > 0.05, by Student’s t -test.

Article Snippet: In the K-604 dihydrochloride rescue experiment, A549 cells subjected to 25HC treatment with DMSO or 20 μM K-604 dihydrochloride (TargetMol, # T11733 ) for 12 h were washed and maintained with DMSO or K-604 dihydrochloride for an additional 4 h before exposure to H7N9 pseudovirus.

Techniques: Virus, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Control, Infection, Western Blot, Negative Control, Quantitative RT-PCR, Immunofluorescence, Fluorescence, Inhibition, Imaging

Journal: Emerging Microbes & Infections

Article Title: A CRISPR activation screen identifies CH25H as a restriction factor against influenza viruses by targeting accessible cholesterol

doi: 10.1080/22221751.2026.2651464

Figure Lengend Snippet: 25HC suppressed H7N9 infection in a murine model. (A) Experimental schematic of 25HC administration and influenza virus infection in mice. (B) Survival rates of H7N9 infection with 25HC treatment in mice (n = 6, with a log-rank test [Mantel-Cox], representative of 6 mice). (C) Body weight changes of H7N9 infection with 25HC treatment in mice (n = 6, representative of 6 mice). (D) Lung indices in mouse lungs at 4 d p.i. were calculated as lung wet weight/body weight*100 (n = 4, representative of 4 mice). (E) Virus titers in lungs of mice at 4 d p.i. were determined by TCID 50 assay (n = 4, representative of 4 mice). (F) H&E staining of lung tissues from mice at 4 d p.i. Scale bars, 1000 and 200 μm. (G) IHC staining (anti-NP) of lung tissues from mice at 4 d p.i. Scale bars, 1000 and 200 μm. (H) Total cholesterol levels in mouse serum at day-1 and day 2. Mean ± SD of n = 7.

Article Snippet: In the K-604 dihydrochloride rescue experiment, A549 cells subjected to 25HC treatment with DMSO or 20 μM K-604 dihydrochloride (TargetMol, # T11733 ) for 12 h were washed and maintained with DMSO or K-604 dihydrochloride for an additional 4 h before exposure to H7N9 pseudovirus.

Techniques: Infection, Virus, Staining, Immunohistochemistry

Journal: Emerging Microbes & Infections

Article Title: A CRISPR activation screen identifies CH25H as a restriction factor against influenza viruses by targeting accessible cholesterol

doi: 10.1080/22221751.2026.2651464

Figure Lengend Snippet: 25HC broadly inhibited HA-mediated membrane fusion of influenza viruses. (A) A549 cells were treated with DMSO or 25HC, then challenged with H7N9 influenza virus pseudovirus. (B) 293 T cells received CH25H-Myc or vector control together with RNP, NP-Luc, and RL-TK plasmids. Luciferase activity was measured 24 h post-transfection. (C) A flowchart of the binding and internalization assay. (D) For binding assay, A549 cells were transfected with CH25H-Myc plasmids and infected with H7N9 influenza virus (MOI = 10 ) at 4°C. For internalization assay, cells were maintained at 37°C for 1 h after viral binding. The relative level of vRNA was quantified by qPCR. (E) A flowchart of the acid-bypass assay. (F) After viral binding to 25HC treated A549 cells at 4°C, cells were exposed to either pH = 5.0 or pH = 7.0 at 37°C, then replaced in stop medium. The relative level of vRNA was quantified by qPCR. Mean ± SD of n = 3. (G) 24 h after co-transfection of H7N9-HA and EGFP plasmids in A549 cells, cells were treated with DMSO or 25HC for 12 h, followed by pH = 5.0 or pH = 7.0 treatment for 5 min. The formation of syncytium was observed using fluorescence microscopy. Mean ± SD of n = 20. (H) A549 cells were co-transfected with the indicated HA subtypes and EGFP plasmids. At 24 h post-transfection, the cells were subjected to DMSO or 25HC treatment for 12 h, and were incubated with TPCK-trypsin, followed by exposure to pH = 5.0. Mean ± SD of n = 20. Scale bars, 200 μm. *** P < 0.001, **** P < 0.0001, ns P > 0.05, by Student’s t -test.

Article Snippet: In the K-604 dihydrochloride rescue experiment, A549 cells subjected to 25HC treatment with DMSO or 20 μM K-604 dihydrochloride (TargetMol, # T11733 ) for 12 h were washed and maintained with DMSO or K-604 dihydrochloride for an additional 4 h before exposure to H7N9 pseudovirus.

Techniques: Membrane, Virus, Plasmid Preparation, Control, Luciferase, Activity Assay, Transfection, Binding Assay, Infection, Cotransfection, Fluorescence, Microscopy, Incubation

Journal: Emerging Microbes & Infections

Article Title: A CRISPR activation screen identifies CH25H as a restriction factor against influenza viruses by targeting accessible cholesterol

doi: 10.1080/22221751.2026.2651464

Figure Lengend Snippet: 25HC blocked viral fusion through accessible cholesterol by activating ACAT. (A) 25HC induces the depletion of accessible cholesterol on the PM. (B) A549 cells were treated with DMSO, 25HC or HP-β-CD and subsequently incubated with ALOD4. The quantities of bound ALOD4 were measured by western blot. (C–D) A549 cells were exposed to DMSO or 25HC, then treated with PBS or cholesterol. ALOD4 were measured by western blot (C). Detection of H7N9 influenza virus pseudovirus (D). Mean ± SD of n = 3. (E) A549 cells were co-transfected with H7N9-HA and EGFP plasmids, then were subjected to DMSO or 25HC treatment. After cholesterol incubation, then stimulated at pH = 5. Mean ± SD of n = 20. (F) Huh7 cells were pretreated with DMSO or 25HC, then incubated with PBS or cholesterol to infection with ZIKA. Mean ± SD of n = 3. (G) Inhibition of cholesterol transport by the ACAT inhibitor K-604 dihydrochloride. (H–I) A549 cells were treated with DMSO or 25HC in the presence of DMSO or K-604 dihydrochloride, then incubated with DMSO or K-604 dihydrochloride. Cells were then subjected to incubation with ALOD4 (H). Cells were infected with H7N9 influenza virus pseudovirus (I). Mean ± SD of n = 3. (J) A549 cells were treated with DMSO or 25HC in the presence of DMSO or K-604 dihydrochloride, followed by stimulated at pH = 5. Mean ± SD of n = 20. Scale bars, 200 μm. * P < 0.05, *** P < 0.001, **** P < 0.0001, by Student’s t -test.

Article Snippet: In the K-604 dihydrochloride rescue experiment, A549 cells subjected to 25HC treatment with DMSO or 20 μM K-604 dihydrochloride (TargetMol, # T11733 ) for 12 h were washed and maintained with DMSO or K-604 dihydrochloride for an additional 4 h before exposure to H7N9 pseudovirus.

Techniques: Incubation, Western Blot, Virus, Transfection, Infection, Inhibition

Journal: Emerging Microbes & Infections

Article Title: A CRISPR activation screen identifies CH25H as a restriction factor against influenza viruses by targeting accessible cholesterol

doi: 10.1080/22221751.2026.2651464

Figure Lengend Snippet: 25HC modulated accessible cholesterol by Aster-mediated non-vesicular transport. (A–B) A549 cells received DMSO or 25HC together with DMSO or U18666A before being exposed to ALOD4 (A). After incubation in pH = 5, syncytium formation was observed (B). Mean ± SD of n = 20. (C–D) CH25H-knockdown cells were treated with 25HC. Analysis of ALOD4 bound (C) and syncytia formation (D). Mean ± SD of n = 20. (E) Cells were transfected with siRNA, then after treatment with DMSO or 25HC infected with H7N9 influenza virus to quantify NP RNA. Mean ± SD of n = 3. Scale bars, 200 μm. * P < 0.05 *** P < 0.001, **** P < 0.0001, by Student’s t -test.

Article Snippet: In the K-604 dihydrochloride rescue experiment, A549 cells subjected to 25HC treatment with DMSO or 20 μM K-604 dihydrochloride (TargetMol, # T11733 ) for 12 h were washed and maintained with DMSO or K-604 dihydrochloride for an additional 4 h before exposure to H7N9 pseudovirus.

Techniques: Incubation, Knockdown, Transfection, Infection, Virus

Journal: Nature Communications

Article Title: Intranasal replicon vaccine establishes mucosal immunity and protects against H5N1 and H7N9 influenza

doi: 10.1038/s41467-025-64829-6

Figure Lengend Snippet: For all assays, n = 6 (3 male and 3 female) animals per group. A Animal survival after challenge with 1 × 10 6 pfu of intranasally-delivered A/Anhui/PA-1/2013. B Averaged clinical scores. C Percent change in body weight from date of challenge. Two statistical hypotheses were tested within each figure, with filled lines showing comparisons to the vector control and dotted lines representing tests between key experimental groups. D – F Viral load in nasal wash at Day D 1, E 3, and F 5 post-challenge. Statistical analysis performed on log-transformed data using one-way ANOVA with Dunnett’s multiple comparisons test. G H7-binding IgG antibody titers in pre-challenge ferret serum. H H7 pseudovirus neutralization capacity (IC 50 ) of pre-challenge ferret serum. G Statistical analysis performed on log-transformed data used one-way ANOVA with Dunnett’s multiple comparisons test. H Statistical analysis performed on log-transformed data using the Kruskal–Wallis test with Dunn’s multiple comparisons. Data are presented as D – G mean +/− SD or H geometric mean +/− geometric SD. IN Intranasal, IM Intramuscular, LLOD lower limit of detection, NIBSC NIBSC Influenza Antigen A/Anhui/1/2013 (H7N9), ns not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Associated body temperature data in Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: Plates were coated with either H5(H5N1)(A/Vietnam/1203/2004) obtained from Immune Technology (#IT-003-0051p) or H7N9 (A/Anhui/1/2013) HA from Sino Biological (#40103-V08H) followed by blocking for 1 h. Each serum or BAL sample was diluted 1:40 or 1:4, respectively, and then serially 1:2 to create a 14-point dilution curve for each sample.

Techniques: Plasmid Preparation, Control, Transformation Assay, Binding Assay, Neutralization

Journal: Nature Communications

Article Title: Intranasal replicon vaccine establishes mucosal immunity and protects against H5N1 and H7N9 influenza

doi: 10.1038/s41467-025-64829-6

Figure Lengend Snippet: For all assays, n = 6 (3 male and 3 female) animals per group. A Animal survival after challenge with 1 × 10 6 pfu of intranasally-delivered A/Anhui/PA-1/2013. B Averaged clinical scores. C Percent change in body weight from date of challenge. Two statistical hypotheses were tested within each figure, with filled lines showing comparisons to the vector control and dotted lines representing tests between key experimental groups. D – F Viral load in nasal wash at Day D 1, E 3, and F 5 post-challenge. Statistical analysis performed on log-transformed data using one-way ANOVA with Dunnett’s multiple comparisons test. G H7-binding IgG antibody titers in pre-challenge ferret serum. H H7 pseudovirus neutralization capacity (IC 50 ) of pre-challenge ferret serum. G Statistical analysis performed on log-transformed data used one-way ANOVA with Dunnett’s multiple comparisons test. H Statistical analysis performed on log-transformed data using the Kruskal–Wallis test with Dunn’s multiple comparisons. Data are presented as D – G mean +/− SD or H geometric mean +/− geometric SD. IN Intranasal, IM Intramuscular, LLOD lower limit of detection, NIBSC NIBSC Influenza Antigen A/Anhui/1/2013 (H7N9), ns not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Associated body temperature data in Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: The Anti-H7N9 Hemagglutinin/HA Antibody (Sino Biological #11082-MM04) was used as a positive control for H7-specific ELISAs.

Techniques: Plasmid Preparation, Control, Transformation Assay, Binding Assay, Neutralization