Fig 1.
Transcriptomic response of lungs and blood of SARS-CoV-2 infected hamsters.
(A) Volcano plots indicate the number of significantly upregulated (red arrows) or downregulated (blue arrows) genes in lungs or blood of hamsters infected with the indicated variant at each timepoint (compared to mock-infected controls). (B-C) Pathway analysis highlighting the enriched ontologies of differentially expressed genes in the lungs (B) and blood (C) of infected hamsters at 2—and 6-days post-infection (dpi). The number of genes involved are indicated beside each pathway. (D-E) Scatterplots representing the relative expression of interferon stimulated genes (ISG) in lungs (D) and blood (E) of hamsters experimentally infected with either the Delta or BA.1 variant at the indicated timepoints. ISG with relative higher expression in BA.1 compared to Delta (FDR<0.05) are shown in red. ISG with relative higher expression in Delta compared to BA.1 (FDR<0.05) are shown in blue. Data derived from n = 8 hamsters (4 females and 4 males per group), infected in two independent experiments.
Fig 2.
Distribution of Delta and BA.1 in organs of the respiratory tract of experimentally infected hamsters.
Golden Syrian hamsters were infected intranasally with either Delta or BA.1 (3.75x106 genome copies equivalent per animal) (or mock-infected). Control animals received media alone. Animals were culled 2- or 6-days post infection (dpi) and the nose, turbinates, larynx, trachea, and lungs were collected for digital pathology analyses (A). Tissues were assessed for the presence of spike RNA by in situ hybridisation (B) or for the expression of MX1 by immunohistochemistry (C). For signal quantification, slides were scanned with an Aperio VERSA 8 Brightfield, Fluorescence & FISH Digital Pathology Scanner (Leica Biosystems) at 200 x brightfield magnification. Tissues to be analysed were outlined using QuPath (Version 0.3.2 or newer). The algorithm to detect the percentage of positively stained pixels was tuned individually before analysis. Statistical analysis was performed using a Two-Way ANOVA, *<0.05, **<0.01, ***<0.001, ****<0.0001. Data were derived from two independent experiments (n = 8 in total; n = 4 per experiment). Black: uninfected; red: Delta-infected; blue: BA.1-infected (male animals, triangles; female animals, circles). Blue scale bar: 100 μm; green scale bar: 1 mm. Graphics made using biorender.com.
Fig 3.
Expression of interferon stimulated genes in lungs of experimentally infected hamsters.
(A) In situ hybridisation of serial lung sections obtained from hamsters experimentally infected with either Delta or BA.1 and culled at 2 days post-infection (2 dpi). Probes used included those for SARS-CoV-2 spike, RSAD2, IFIT1, MX1 and OAS1. (B) Signal was quantified as in Fig 2 and statistical analysis was performed using a One-Way ANOVA, *<0.05, **<0.01, ***<0.001, ****<0.0001. Data represents two independent experiments using a total of n = 6 hamsters (3 females and 3 males per group). Black: uninfected; red: SARS-CoV-2 (Delta) infected; blue: SARS-CoV-2 (BA.1) infected (male animals, triangles; female animals, circles). Scale bar: 3 mm.
Fig 4.
Lung histopathology of SARS-CoV-2 experimentally infected hamsters.
Low (left) and high (right) magnification of lung sections stained with haematoxylin and eosin of hamsters experimentally infected with either the Delta or BA.1 (3.75x106 genome equivalent copies per animal), or mock-infected controls. At 2 days post-infection (dpi), Delta causes a higher level of infiltration of macrophages in and around bronchi, while BA.1 causes only mild infiltrations (white arrow) compared to mock-treated hamsters. On the same day, Delta-infected animals showed a moderate vasculitis (inset, right panel) and a moderate sloughing of bronchial epithelial cells (asterisk). Vascular and bronchi pathology was minimal in BA.1-infected animals at 2 dpi. At 6 dpi the lungs of Delta-infected animals show a severe infiltration of macrophages, lymphocytes, plasma cells and neutrophils/heterophils as well as a severe alveolar epithelial hyperplasia covering large areas of the lung lobe (black arrows; see also S4 Fig). BA.1-infected animals show in some cases the same lesions, including type 2 pneumocytes hyperplasia (asterisk) but covering only limited amounts of the lung lobes (black arrows). (Empty scale bars: 2 mm; filled scaled bars: 100 μm).
Fig 5.
Quantification of tissue histopathology in Delta or BA.1-experimentally infected hamsters.
Images of whole lung sections of hamsters experimentally infected with either Delta or BA.1 (3.75x106 genome equivalent copies per animal) or mock-infected controls, and culled at either 2- or 6-days post-infection (dpi). Expression of CD3 (A), IBA1 (B) and TTF1 (C-D) was assessed by immunohistochemistry. Animals were culled 2- or 6-days post infection (dpi) and the lungs were collected for histological analysis. (A-C) Representative photomicrographs for each marker and associated quantification are shown. (D) Mock-infected animals show a positive signal for TTF1 only in bronchial epithelium and in single cells scattered throughout the lung parenchyma. (E) Lungs of Delta-infected hamsters shows severe proliferation of TTF1+ positive cells (arrows). The inset shows a higher magnification of type 2 pneumocytes hyperplasia surrounding centrally located bronchi (asterisk). (F) Same image as in E after analysis with the HALO software algorithm trained to detect alveolar epithelial hyperplasia. The software shows negative areas in green and positive ones in yellow; only proliferating alveolar epithelial cells are identified as positive areas, while TTF1+ type-2 pneumocytes and bronchial epithelial cells are excluded by the software. CD3- and IBA1-positive cells in experimentally infected animals were quantified using QuPath (Version 0.3.2 or newer), while HALO was used to quantify alveolar epithelial hyperplasia. Statistical analysis was performed using a Two-Way ANOVA, *<0.05, **<0.01, ***<0.001, ****<0.0001. Data were obtained from two independent experiments using 8 hamsters (4 females and 4 males infected in two independent experiments). Black: uninfected; red: Delta-infected; blue: BA.1-infected (males, triangles; females: circles). Blue scale bar: 1 mm; green scale bar: 400 μm.
Fig 6.
Quantification of Omicron sub-lineages virulence in experimentally infected hamsters.
(A) Photomicrographs of lung sections collected at 6 days post-infection (dpi) from hamsters experimentally infected with either Delta, BA.1, BA.5 (virus load equivalent to 3.75x106 genome copies per hamster) or mock-infected. Expression of TTF1+ hyperplastic epithelial cells and infiltrating macrophages (IBA1+) was assessed by in situ-hybridisation. Expression of MX1 was instead assessed by immunohistochemistry. Data were quantified as in Fig 5. (B) Differences in the weights between these animals were recorded daily. (C) Daily recorded weight changes in animals mock-infected or infected with either BA.1, XBB, BQ.1.18 or BA.2.75. (D) Lung whole scanned sections were analysed for the presence of IBA1+ cells or (E) hyperplastic alveolar epithelial cells as in A. (F) Daily weight changes of mock-infected hamsters or animals infected with either BA.1, BA.2 or BA.2.75. (G) Quantification of cells expressing IBA1 or (H) hyperplastic TTF1+ cells. Comparison of weight loss (I), macrophage infiltration (J), alveolar epithelial hyperplasia (K) and T cells infiltration (L) in the lungs of BA2.75 or EG.5.1-infected hamsters. Statistical analysis was performed using a One-Way ANOVA with Tukey’s multiple comparisons test. Weight comparisons were performed using a Two-Way ANOVA. Data are shown as mean +/- standard deviation. Significance is indicated with *<0.05, **<0.01, ***<0.001, ****<0.0001. * Denote comparisons between Delta and mock, or BQ.1.18 and mock, or BA.2.75 and mock; # denote comparisons between BA.1 and delta, or mock and BA.2.75; $ denotes comparisons between Delta and BA.5, or mock and XBB; + denotes comparisons between uninfected and BA.5; & denotes comparisons between BA.5 and BA.1. n = 6 (3 females and 3 males per group). **€ denotes the differences between mock and EG.5.1, and *€€ denotes differences between mock and BA2.75, as well as mock and EG.5.1. Black: uninfected; red: Delta-infected; blue: BA.1-infected; green inverted triangles: SARS-CoV-2 (BA.5) infected; orange inverted triangles: SARS-CoV-2 (XBB) infected; purple squares: SARS-CoV-2 (BQ.1.18) infected; pink squares: SARS-CoV-2 (BA.2) infected; green diamonds: SARS-CoV-2 (BA.2.75) infected. Males: triangles, females: circles. Scale bar: 500 μm. Graphics made using biorender.com.