Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Lung-Targeted Lipid Nanoparticle-Delivered siUSP33 Attenuates SARS-CoV-2 Replication and Virulence by Promoting Envelope Degradation.

doi: 10.1002/advs.202406211

Figure Lengend Snippet: Figure 6. LNP-siUSP33 reduces viral load and lung pathology in vivo. A) C57BL/6 mice were nasally infected with AAV-hACE2 and injected with indicated reagents via retro-orbital. On the second day of injection, the mice were infected nasally with 1 × 105 FFU WT SARS-CoV-2. All the lung tissues were collected on the 8th day after infection. B) The body weights of the mice were recorded daily for 8 days after infection. Quantification of body weights compared to pre-infection was shown as mean ± SD (n = 5 independent experiments). C) The lung tissues of mice were collected on the 8th day after infection and then lyzed to detect the levels of indicated proteins via immunoblotting. D) Total RNA was extracted from the lung tissues, and the relative levels of SARS-CoV-2 N genes were detected. E) The lungs were ground into homogenates and then infected Vero cells, and viral titers were calculated using the FFA. F) Immunohistochemistry analyses with anti-SARS-CoV-2 N antibody was performed to assess the relative amount of SARS- CoV-2 in the lungs. Scale bar, 100 μm. G) H&E staining was performed to observe the intensity of the inflammatory infiltrate. Scale bars, 1000 μm (up) and 50 μm (down). H) Total RNA was extracted from the lungs and relative levels of indicated inflammatory genes were detected using qRT-PCR. I) Schematic diagram of the K18-hACE2 mice of SARS-CoV-2 infection. J) Total RNA was extracted from the lung tissues collected on day 4 after infection, and the relative levels of SARS-CoV-2 N genes were detected. K–M) The viral loads of the lungs (K), livers (L), and kidneys (M) collected on day 4 were calculated using FFA. Student’s t test (unpaired, two-tailed) was used to compare two independent groups, and a two-way ANOVA test was performed for comparisons of multiple groups. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Article Snippet: All siRNAs were synthesized by RIBOBIO and the sense strand sequences are shown below. hUSP33#1: CCCAGUAAUACAACAUUAATT hUSP33#2: GGAGAAUAGAUGUUCAUAUTT hUSP33#3: GCUGCAUUCAUCAAGUCAUTT mUSP33#1: GCAGGAGACAAAGCAUUAUTT mUSP33#2: GCCGGCUAAUCUGUUCCAATT mUSP33#3: GCAGAGCCUCAGAAUCUAUTT mUSP33#4: GCUGAACCUGGCCCUAUUUTT Antibodies and Other Reagents: The following antibodies and reagents were used for immunoblotting and immunoprecipitation in this study: Rabbit anti-Flag monoclonal antibody (CST, 14 793); Rabbit anti-HA monoclonal antibody (CST, 3724); Mouse anti-Strep-Tag II monoclonal antibody (Abbkine, 8C12); Rabbit anti-GST monoclonal antibody (CST, 2625); Mouse anti-His monoclonal antibody (CST, 2366); Rabbit anti-Myc monoclonal antibody (CST, 13 987); Mouse anti-β-actin monoclonal antibody (TransGen Biotech, HC201-01); Rabbit anti-α-Tubulin polyclonal antibody (Proteintech, 11224-AP); Rabbit anti-Histone-H3 polyclonal antibody (Proteintech, 17168-1AP); Mouse anti-Ubiquitin monoclonal antibody (SantaCruz, sc-8017); Rabbit anti-USP33 polyclonal antibody (Proteintech, 20445-1-AP); Rabbit anti-RNF5 monoclonal antibody (ab308066); Rabbit anti-SARS-CoV-2 E monoclonal antibody (abcam, ab308371); Rabbit anti-SARS-CoV-2-M monoclonal antibody (abcam, ab308415); Rabbit anti-SARS-CoV-2-ORF7b monoclonal antibody (abcam, ab313933); Mouse anti-SARS-CoV-2-N monoclonal antibody (SinoBiological, 40143- MM05); Rabbit anti-SARS-CoV-2-S polyclonal antibody (SinoBiological, 40590-T46); Rabbit anti-SARS-CoV-2 NSP3 monoclonal antibody (CST, 88 086); Rabbit anti-hACE2 polyclonal antibody (CST, 4355); Goat antiRabbit and anti-Mouse HRP Conjugated secondary antibodies (TransGen Biotech); Goat anti-Rabbit Alexa Fluo 488 Conjugated secondary antibodies (Invitrogen, A-11008); Goat anti-Mouse Alexa Fluo 555 Conjugated secondary antibodies (Invitrogen, A-21422); DAPI (Beyotime, C1002); AntiFlag magnetic Beads (Sigma, M8823); Anti-HA magnetic beads (Sigma, SAE0197); Anti-Myc magnetic beads (Sigma, SAE0201); Protein A/G agarose (GE healthcare); Magnetic Beads-conjugated Rabbit anti GST-Tag mAb (ABclonal, AE122); Reduced Glutathione (Solarbio, G8180) and IPTG (Solarbio, I1020).

Techniques: In Vivo, Infection, Injection, Western Blot, Immunohistochemistry, Staining, Quantitative RT-PCR, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Generation of the SARS-CoV-2 N conditional knock-in mouse model. a Schematic diagrams illustrating the knock-in strategy, in which the CAG-loxP-Stop-loxP-SARS-CoV-2 N-WPRE-PolyA sequence was inserted into the Tigre locus on chromosome 9, using the K18-hACE2 KI mouse background (N-hACE2 mouse). The stop sequence was flanked by two loxP sites, and upon expression of Cre recombinase, the stop sequence located between these loxP sites was excised. N-hACE2 mice were crossed with Rosa26 SA-CreERT2 or Sftpc-IRES-iCre mice, resulting in the generation of SA-N-hACE2 (TAM-inducible systemic Cre expression) and Sftpc-N-hACE2 (lung-specific Cre expression) mice. b N-hACE2 mouse genotyping by PCR showed the presence of N gene and hACE2 gene fragments. M: GimiRun DM5000 DNA Marker. c PCR confirmed excision of the stop sequence between loxP sites in SA-N-hACE2 mice after 5 days of TAM injection. Western blot analysis of SARS-CoV-2 N protein expression in multiple tissues from SA-N-hACE2 mice after TAM induction ( d ) and from Sftpc-N-hACE2 mice ( e ). f Immunofluorescence staining of lung sections was conducted using 4,6-diamidino-2-phenylindole (DAPI, blue), an anti-SARS-CoV-2 N antibody (red), and an anti-hACE2 antibody (green) to evaluate the expression of SARS-CoV-2 N and hACE2 in lung cells

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Knock-In, Sequencing, Expressing, Marker, Injection, Western Blot, Immunofluorescence, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: SA-N-hACE2 mice are susceptible to SARS-CoV-2 ΔN/GFP-HiBiT infection. a Experimental design for intranasal infection. After TAM treatment, SA-N-hACE2 mice were infected either with 5 × 10 4 or 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. Tissue samples were collected at the indicated dpi. b Changes in the weights of the mice are shown ( n = 4 per group). c qRT‒PCR was used to quantify viral loads in tissues at 7 dpi ( n = 4 per group). The viral loads ( d ) and luminescence ( e ) were measured in the lungs collected at 2, 4, and 7 dpi ( n = 4 per group). f Immunofluorescence staining of lung sections at 2, 4, and 7 dpi was conducted using DAPI (blue) and an anti-GFP antibody (green) specific to GFP in SARS-CoV-2 ΔN/GFP-HiBiT. g – j Pathological changes observed using H&E staining in lung ( g ) and brain ( i ) tissues from SA-N-hACE2 mice challenged with 1 × 10 6 TCID 50 at 0, 2, 4, and 7 dpi. Pathology scores for the lungs ( h ) and brain ( j ) were calculated ( n = 4 per group). Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( h ). ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ND not detected

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Immunofluorescence, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Sftpc-N-hACE2 mice are susceptible to SARS-CoV-2 ΔN/GFP-HiBiT infection. a Illustration of the protocol for intranasal infection with tissue samples collected at the indicated dpi. Sftpc-N-hACE2 mice were infected with 5 × 10 4 or 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. b Changes in the body weights of the mice are shown ( n = 4 per group). c Viral loads in the tissues obtained at 7 dpi were quantified via qRT‒PCR ( n = 4 per group). The viral loads ( d ) and luminescence ( e ) were measured in the lungs collected at 2, 4, and 7 dpi ( n = 4 per group). f Immunofluorescence staining of lung sections at 2, 4, and 7 dpi was conducted using DAPI (blue) and an anti-GFP antibody (green) specific to GFP in SARS-CoV-2 ΔN/GFP-HiBiT. g – j Pathological changes in the lungs ( g ) and brains ( i ) of Sftpc-N-hACE2 mice challenged with 1 × 10 6 TCID 50 at 0, 2, 4, and 7 dpi were assessed using H&E staining. Pathology scores for the lungs ( h ) and brains ( j ) are shown ( n = 4 per group). Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( h ). ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ND not detected

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Immunofluorescence, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: SARS-CoV-2 ΔN/GFP-HiBiT infection in Sftpc-N-hACE2 mice can lead to lethal disease. a The schematic outlines the procedure for infecting the mice with 5 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. At 0, 7, 10, 14, and 21 dpi, four mice were euthanized at each time point for sample collection. Mice that experienced a loss of more than 20% of their initial body weight were euthanized as a humane endpoint. Mice were monitored for body weight changes ( b ) and survival ( c ) ( n = 16). E gene copies in lung ( d ) and brain ( e ) tissues were quantified via qRT‒PCR. f – i Pathological changes were assessed using H&E staining in the lungs ( f ) and brains ( g ). Pathology scores for the lungs ( h ) and brains ( i ) were recorded ( n = 4 per group). j Immunofluorescence staining of brain sections was performed using DAPI (blue) and an anti-GFP antibody (green) specific to GFP in SARS-CoV-2 ΔN/GFP-HiBiT. k Immunofluorescence analysis was performed on brain sections from mock-infected mice, K18-hACE2 KI mice challenged with live SARS-CoV-2, and both SA-N-hACE2 and Sftpc-N-hACE2 mice exposed to SARS-CoV-2 ΔN/GFP-HiBiT. The sections were stained with anti-IBA1 (red) and anti-CD68 (green) antibodies. l Light-sheet imaging of cleared lung tissues from Sftpc-N-hACE2 mice at 7 dpi confirmed the distribution of SARS-CoV-2 N in lung epithelial cells and SARS-CoV-2 ΔN/GFP-HiBiT infection. Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( h ). ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ND not detected

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Staining, Immunofluorescence, Imaging

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Transcriptomic profiling of the lungs of SA-N-hACE2 and Sftpc-N-hACE2 mice revealed distinct molecular features. a Scheme illustrating the transcriptome sequencing. Gene expression heatmaps of IFN-I-related genes ( b ) and cytokines and chemokines ( c ) in the lungs of the mice. Mock: PBS-treated mice; the SA-N-hACE2 and Sftpc-N-hACE2 mice were infected with 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. d Bubble plot showing gene set enrichment analysis (GSEA) results for pathways enriched in the lungs of the infected mice. The color scale indicates normalized enrichment scores (NES), while the size of the bubbles corresponds to −log 10 ( p values)

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Sequencing, Gene Expression, Infection

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Assessment of anti-SARS-CoV-2 therapeutics using SA-N-hACE2 and Sftpc-N-hACE2 mouse models. a This schematic diagram illustrates the procedure for evaluating the neutralizing antibody 7B3 and NMV. SA-N-hACE2, Sftpc-N-hACE2, and K18-hACE2 KI mice received 10 mg/kg of 7B3 at 1 dpi or 300 mg/kg NMV via oral gavage from 0–3 dpi. SA-N-hACE2 and Sftpc-N-hACE2 mice were challenged with 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT, whereas K18-hACE2 KI mice were infected with 3.57 × 10 2 TCID 50 of SARS-CoV-2 WT at 0 dpi. At 4 dpi, all mice were sacrificed, and lung samples were collected. The expression of the viral E gene in lung tissues from mock-, NMV- and 7B3-treated SA-N-hACE2 ( b ), Sftpc-N-hACE2 ( c ) and K18-hACE2 KI mice ( d ) was quantified via qRT‒PCR ( n = 4 per group). e – j Lung tissues from mock-, NMV-, and 7B3-treated SA-N-hACE2 ( e ), Sftpc-N-hACE2 ( f ), and K18-hACE2 KI ( g ) mice were analyzed using H&E staining to assess pathological changes, with histological scores documented for each group ( n = 4 per group) ( h – j ). Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( b – d , h – j ). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Expressing, Staining