Journal: Journal of Virology

Article Title: Stealth replication of SARS-CoV-2 Omicron in the nasal epithelium at physiological temperature

doi: 10.1128/jvi.02008-25

Figure Lengend Snippet: Temperature-dependent replication of SARS-CoV-2 variants in reconstructed human nasal epithelia. The viral load in apical supernatants was quantified by RT-qPCR for samples infected by the following SARS-CoV-2 lineages: Wuhan, D614G, Alpha, Delta, and Omicron BA.1. Infections were performed at an input equivalent to 10 8 viral RNA copies. ( A, B ) Kinetics of viral replication at 37°C ( A ) and at 33°C ( B ), with means and SD reported ( n = 4 to 14 independent samples per point). ( C,D ) Comparison of apical viral loads measured at 1 day post-infection (dpi) at 37°C ( C ) and 33°C ( D ). Medians are reported. ( E ) The ratio of the number of viral RNA copies measured at 33°C to that measured at 37°C is shown at different dpi, with means and SD reported ( n = 4 to 14 independent samples per point).( C, D, E ) Statistical comparisons were done between the D614G reference and all other variants or the mock condition, using the Kruskal-Wallis test with Dunn’s correction; ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. In ( E ), the only significant differences in ratios were seen between BA.1 and D614G at 2 dpi and 4 dpi.

Article Snippet: For infection experiments, we used reconstructed human nasal epithelia generated from pooled human donors (MucilAir, Epithelix) to minimize donor-dependent variability.

Techniques: Quantitative RT-PCR, Infection, Comparison

Journal: Journal of Virology

Article Title: Stealth replication of SARS-CoV-2 Omicron in the nasal epithelium at physiological temperature

doi: 10.1128/jvi.02008-25

Figure Lengend Snippet: Temperature-dependent cytopathic effects induced by different SARS-CoV-2 variants. ( A ) Reconstructed nasal epithelia were Mock-infected (top line) or infected with different SARS-CoV-2 variants (D614G, Delta, and Omicron BA.1) at 37°C (left) or 33°C (right) and were fixed at 4 dpi and immunolabeled for cleaved caspase-3 (red), Spike (green) and nuclei (DAPI, blue). The scale bar represents 40 µm. The * symbol labels large syncytia. ( B–C ) Quantification of the cleaved caspase 3-positive area (casp3+) at 37°C ( B ) and 33°C ( C ), with values normalized to the D614G or Wuhan values (D614G/Wu) at 37°C. ( A–C ) Infections were performed at an input equivalent to 10 8 viral RNA copies. Medians and 95%CI are reported. Statistical comparisons were done between the D614G/Wu reference and all other variants or the Mock condition, using the Kruskal-Wallis test with Dunn’s correction; ns: not significant; * P < 0.05.

Article Snippet: For infection experiments, we used reconstructed human nasal epithelia generated from pooled human donors (MucilAir, Epithelix) to minimize donor-dependent variability.

Techniques: Infection, Immunolabeling

Journal: Journal of Virology

Article Title: Stealth replication of SARS-CoV-2 Omicron in the nasal epithelium at physiological temperature

doi: 10.1128/jvi.02008-25

Figure Lengend Snippet: SARS-CoV-2 variants perturb the motile cilia layer in a temperature-dependent fashion. ( A ) Reconstructed nasal epithelia were mock-infected (top line) or infected with different SARS-CoV-2 variants (D614G, Delta, and Omicron BA.1) at an input equivalent to 10 8 viral RNA copies. Cultures propagated at 37°C (left) or 33°C (right) were fixed at 4 dpi and immunolabeled for acetylated α-tubulin (cyan), which preferentially detects motile cilia. The scale bar represents 40 µm. ( B–C ) Quantification of the area positive for acetylated α-tubulin at 37°C ( B ) and 33°C ( C ), with values normalized to the Mock-infected condition. Medians and 95%CI are reported. Statistical comparisons were done between the Mock-infected sample and all the SARS-CoV-2 variant-infected samples, using the Kruskal-Wallis test with Dunn’s correction; ns: not significant; * P < 0.05; ** P < 0.01.

Article Snippet: For infection experiments, we used reconstructed human nasal epithelia generated from pooled human donors (MucilAir, Epithelix) to minimize donor-dependent variability.

Techniques: Infection, Immunolabeling, Variant Assay

Journal: Journal of Virology

Article Title: Stealth replication of SARS-CoV-2 Omicron in the nasal epithelium at physiological temperature

doi: 10.1128/jvi.02008-25

Figure Lengend Snippet: Infection by SARS-CoV-2 variants induces a loss of motile cilia. SEM images of reconstructed nasal epithelia at 4 dpi. The epithelia were infected at 37°C (left) or at 33°C (right) with the D614G, Delta, or Omicron, BA.1 variant (shown in rows 2, 3, and 4, respectively) or were Mock-infected (first row). The scale bar represents 10 µm. Red symbols: * membrane protrusion surrounding cilia; ** blebbing cell.

Article Snippet: For infection experiments, we used reconstructed human nasal epithelia generated from pooled human donors (MucilAir, Epithelix) to minimize donor-dependent variability.

Techniques: Infection, Variant Assay, Membrane

Journal: Journal of Virology

Article Title: Stealth replication of SARS-CoV-2 Omicron in the nasal epithelium at physiological temperature

doi: 10.1128/jvi.02008-25

Figure Lengend Snippet: Temperature-dependent alteration of the cellular transcriptomic profile upon SARS-CoV-2 infection: A bulk RNA-seq analysis was performed at 2 dpi on reconstructed nasal epithelia either Mock-infected or infected with SARS-CoV-2 variants, using cultures grown at 37°C or 33°C. ( A ) Distribution of SARS-CoV-2 reads at 37°C (top graph) and 33°C (bottom graph), showing the mean normalized counts per million for the different SARS-CoV-2 transcripts ( n = 2 replicates). ( B ) The percentage of viral reads among total mapped reads (cellular and viral) is reported for n = 2 replicate samples infected at 37°C (top) and 33°C (bottom). The median and 95%CI are reported. ( C ) Venn diagram showing the number of differentially expressed cellular genes (DEGs) shared between the different infected samples at 37°C (left graph) and 33°C (right graph). DEGs were determined with the DESeq2 package, with Mock-infected samples used as a reference. The total number of DEGs for each variant is reported below the variant name.

Article Snippet: For infection experiments, we used reconstructed human nasal epithelia generated from pooled human donors (MucilAir, Epithelix) to minimize donor-dependent variability.

Techniques: Infection, RNA Sequencing, Variant Assay

Journal: Journal of Virology

Article Title: Stealth replication of SARS-CoV-2 Omicron in the nasal epithelium at physiological temperature

doi: 10.1128/jvi.02008-25

Figure Lengend Snippet: Differential induction of type I/III IFNs by SARS-CoV-2 variants ( A ) The induction of IFN-β (left), IFN-λ1 (middle), and IFN-λ2/3 (right) release in apical supernatants of reconstructed nasal epithelia infected at 37°C (top panels) and 33°C (bottom panels) was measured by digital ELISA (Simoa) and Legendplex assays. The mean ± SD are reported for N = 3 to 6 independent biological replicates. The induction of IFN between 2 dpi (D2) and 4 dpi (D4) was evaluated with a paired t -test. ( B ) The ratio of IFN measured at 33°C to that measured at 37°C is reported for the four variants tested (colored symbols) and the three IFNs measured (IFN-β, left; IFN-λ1, middle; IFN-λ2/3, right) at the D4 time point. The mean ± SD are reported for N = 4 to 5 independent biological replicates. The dashed line represents a ratio value of 1. ( C ) The ratio of IFN released at 4 dpi to the viral RNA released at 2 dpi is reported at 37°C ( C ) and 33°C ( D ) for IFN-β (left plots), IFN-λ1 (middle plots), and IFN-λ2/3 (right plots). Medians are reported. Statistical comparisons were done between the D614G reference variants and all the other variants tested, using the Kruskal-Wallis test with Dunn’s correction; ns: not significant; * P < 0.05; ** P < 0.01.

Article Snippet: For infection experiments, we used reconstructed human nasal epithelia generated from pooled human donors (MucilAir, Epithelix) to minimize donor-dependent variability.

Techniques: Infection, Enzyme-linked Immunosorbent Assay