Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: (A) Schematic representation of the SARS-CoV-2 reverse genetics platform used to generate the wild-type (WT) and ΔNsp2 recombinant viruses. The figure was created using BioRender. Overlapping genomic fragments were assembled and recombined into the YAC vector through TAR cloning. YAC DNA was nucleofected into Vero cells to reconstitute the viruses. (B) Viral protein expression following infection of Vero cells with WT or ΔNsp2 recombinant viruses. Cells were infected and harvested as described in the Materials and Methods section. Protein expression was analyzed by Western blot using antibodies against SARS-CoV-2 N (AF488), S(AF647), Nsp3(AF647), Nsp2(AF488) and Nsp1(AF488). Stain-free technology was used as a loading control (see Supplementary Figure 2). The experiment was performed twice, and a representative result is shown. (C–E) Growth kinetics of recombinant viruses in Vero (C), A549- hACE2 (D), and A549-ALI (E) cells. Viral shedding was quantified in cell culture supernatants (C and D) or mucus washes (E). Viral titers are expressed as TCID50/mL (mean ± SD; n = 3–6 per group). The dashed line indicates the limit of detection (LOD). The x-axis represents hours post-infection (hpi). Global growth kinetics were compared using two-way ANOVA. # P < 0.033. Individual time points were analyzed using uncorrected Fisher’s LSD test. ***P < 0.0002; ****P < 0.000

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Recombinant, Plasmid Preparation, Cloning, Expressing, Infection, Western Blot, Staining, Control, Cell Culture

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: (A) Schematic overview of the mouse infection protocol. This figure was created using BioRender. (B) Survival curves of K18- hACE2 mice following infection with wild-type or ΔNsp2 recombinant SARS-CoV-2. Survival is expressed as the percentage of mice not reaching the ethical endpoint at each day post-infection (DPI) (n = 7 per group). Statistical analysis was performed using the Mantel–Cox (log-rank) test. *P < 0.033. (C) Clinical disease scores following infection. Disease scores represent the cumulative score of individual parameters including body weight, activity, coat condition, and respiratory symptoms (each scored from 0 to 3). A total score of 4 corresponded to the ethical endpoint, as euthanasia was required when two or more parameters reached a score ≥2 (mean ± SD; n = 7 per group). (D) Body weight changes following infection, expressed as the percentage of initial body weight at each DPI (mean ± SD; n = 7 per group). For panels C and D, differences over the entire infection course were analyzed using mixed-effects models. # P < 0.033. Individual time points were compared using uncorrected Fisher’s LSD tests. (E) Infectious viral loads in lung tissues. Recombinant SARS-CoV-2 titers were determined in lung homogenates using the 50% tissue culture infectious dose (TCID₅₀) assay and normalized to total protein content (mean ± SD; n = 5 per group). (F) SARS-CoV-2 E gene RNA levels in lung tissues, quantified by RT-ddPCR and normalized to mouse Gapdh mRNA copy number (mean ± SD; n = 5 per group). For panels E and F, data across the infection course were analyzed using two-way ANOVA. ## P < 0.0021. Groups at individual time points were compared using uncorrected Fisher’s LSD tests. ****P < 0.0001.

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Infection, Recombinant, Activity Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: (A) Cytokine, chemokine, and interferon profiles in lung tissues following mock or rSARS-CoV-2 infection. Mediator concentrations were normalized to total protein content, and values were scaled from 0 to 10 for each mediator to facilitate visualization in the heatmap (mean; n = 5 per group). Differences between groups at the same time point for the mediator profile were assessed using two-way ANOVA (**P < 0.0021), followed by Fisher’s LSD test for individual mediators. (B) Carstairs staining of lung sections from mock-, wild-type–, or ΔNsp2-infected K18- hACE2 mice. Whole lung sections were scanned, and representative images from each group are shown. (C) Histological assessment of lung inflammation in perivascular, peribronchial, and parenchymal regions. Whole lung sections were independently scored for inflammation (scale 0–5) in each anatomical compartment (mean; n = 4–5 per group). Global inflammation across time points was analyzed using Fisher’s LSD test. (C) *P < 0.033; ****P < 0.0001

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: (A) Systemic inflammatory response in plasma from mock-, wild-type–, or ΔNsp2-infected mice. Chemokines and interferons were quantified in Triton X-100–inactivated, platelet-depleted plasma and expressed as pg/mL (mean; n = 4–5 per group). Mean mediator levels were compared between groups at the same time point using uncorrected Fisher’s LSD test. (B) Quantification of circulating leukocytes. Citrate-treated whole blood was subjected to red blood cell lysis, and leukocytes were stained and analyzed by flow cytometry. Results are expressed as CD45⁺ cells per 100 µL of whole blood. (C–I) Abundance of circulating T helper (Th) cells (C), cytotoxic T lymphocytes (CTLs) (D), B cells (E), natural killer (NK) cells (F), monocytes (Mo) (G), neutrophils (Neutro) (H), and PD-L1–expressing neutrophils (I). Data are presented as percentages of CD45⁺ cells (mean ± SD; n = 4–5 per group). For all panels, comparisons across the infection course were performed using two-way ANOVA. # P < 0.033. Group comparisons at individual time points were assessed using uncorrected Fisher’s LSD test. *P < 0.033; **P < 0.0021; ***P < 0.0002; ****P < 0.0001.

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Clinical Proteomics, Infection, Red Blood Cell Lysis, Staining, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: (A) Dimension reduction of the transcriptome. Transcript counts were normalized using DESeq2 to stabilize variance, and Uniform Manifold Approximation and Projection (UMAP) was applied to visualize sample clustering and distances between conditions (B–D) Differential gene expression between ΔNsp2 and wild-type SARS-CoV-2-infected mice. Differential expression analysis was performed using DESeq2 from raw transcript counts. Results are presented as log2 fold change (log2FC) for ΔNsp2 relative to wild-type infection at 3 days post-infection (DPI) (B), 5 DPI (C), and 7 DPI (D) (mean; n = 4–5 per group). The y-axis represents the adjusted p-value (padj), corrected for multiple testing using a false discovery rate (FDR) threshold of 0.05. Transcripts with a padj below 0.05 and an absolute log 2 (FC) above 1.5 were colorized in red (up-regulated) or blue (down-regulated).

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Gene Expression, Infection, Quantitative Proteomics

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: Functional enrichment was performed using Gene Set Enrichment Analysis (GSEA) with the MSigDB M2 curated gene sets. DESeq2 Wald statistics from the ΔNsp2 versus wild-type comparison at each DPI were used as input for GSEA. The top 50 most differentially enriched pathways across all time points are shown in the heatmap. Pathways were hierarchically clustered based on enrichment patterns, and colors represent normalized enrichment scores (NES), with red indicating pathways enriched (upregulated) and blue indicating pathways depleted (downregulated) in ΔNsp2-infected mice relative to wild-type infection (n = 4–5 per group). The 4 major clusters were highlighted with dotted frame.

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Functional Assay, Comparison, Infection

Journal: bioRxiv

Article Title: SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

doi: 10.64898/2026.05.06.723222

Figure Lengend Snippet: (A) Schematic representation of Cross-linking and Immunoprecipitation Sequencing (CLIP-seq) procedure. The figure was created using BioRender. (B) Functional enrichment of transcript interacting with Nsp2. Genes with peak identified by both peak caller algorythms were analysed using Metascape. Term with a P adjusted value for false discovery rate (FDR) below 0.05 were retained. One representative term per cluster were presented and the top 15 are shown. Dot size represents the number of gene detected within the term (Gene Count), color scale represents the P adjusted value (padj) and x-axis represents the enrichment score.

Article Snippet: The fragment encompassing Nsp2 was subcloned into a standard pUC19 vector using NEBuilder® HiFi DNA Assembly.

Techniques: Immunoprecipitation, Sequencing, Functional Assay