Experimental Porcine Astrovirus 4‐Associated Tracheitis and Bronchitis in Pigs

Figures

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  Figure 1. Experimental study timeline with inoculation and sampling timepoints highlighted. Figure was created with BioRender.com. Swab icon represents both fecal and nasal swabs. Blood tubes are representative of serum collection. Lungs indicate necropsy days.

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  Figure 2. PoAstV4 detection in swabs and respiratory tissues via RT‐qPCR. Nasal shedding of PoAstV4, including the percentage of pigs that tested positive in both inoculated and negative groups over time (A, B). Rectal shedding of PoAstV4, including the percentage of pigs that tested positive in both inoculated and negative groups over time (C, D). Presence of PoAstV4 within respiratory tissues was evaluated with PoAstV4 PCR at 5, 8, and 21 DPI. Data for graphs (A, C, E, and F) are presented as means with standard error mean bars. In graphs (E, F), data for individual animals are presented as individual symbols. p < 0.05 were considered significant ( ∗ < 0.05, ∗∗ < 0.01).

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  Figure 3. Microscopic lesions of tracheitis and bronchitis. Hematoxylin and eosin (H&E)‐stained slides display tracheal tissue from negative control (A) and PoAstV4‐inoculated pigs (B) at 5 DPI. Inoculated pigs had increased numbers of lymphocytes within the lamina propria with attenuation of the epithelium and loss of cilia (B) compared to negative controls (A). Sections of trachea and all three mainstem bronchi from pigs euthanized at 5 and 8 DPI were evaluated for histologic lesions affecting the epithelium and/or the infiltration of lymphocytes into the mucosa (C–F). Quantified data are presented as means with standard error mean bars, with data from individual animals represented by individual symbols. p‐Values were considered significant ( ∗ < 0.05).

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  Figure 4. Localization of virus within lesions via RNAscope ISH at 5 DPI. Nasal turbinates: PoAstV4 ISH staining (red) in negative control and PoAstV4‐inoculated pigs (A, B). The inoculated pig has red staining (detection of viral RNA) within the mucosa. Trachea: PoAstV4 ISH staining (red) in negative control and PoAstV4‐inoculated pigs (C, D). PoAstV4 ISH staining of the trachea and bronchi at 5 DPI, presented as positive tissue area divided by the evaluated tissue area in pigs euthanized on 5 DPI (E, F). Quantified data are presented as means with standard error mean bars with data from individual animals represented by individual symbols. Scale bars = 200 μm.

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  Figure 5. Analysis of lymphocytic infiltration into tracheal mucosa. CD3 IHC (A, C) with the detection of T cells with QuPath software and identification of individual cells in red (B, D). The evaluated mucosa is delineated by the yellow lines. CD20 IHC (E, G) with the detection of B cells with QuPath software and identification of individual cells in red (F, H). The evaluated mucosa is delineated by the yellow lines.

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  Figure 6. Quantification of lymphocytic infiltration of respiratory mucosa. T cell infiltration of the tracheal mucosa at 5 and 8 DPI was evaluated with CD3 IHCs quantified with QuPath as number of cells/area mm2 (A, B). B cell infiltration of the tracheal mucosa was evaluated with CD20 IHCs quantified with QuPath as number of cells/area evaluated (C, D). Bronchi were similarly assessed for T and B cell infiltration at 5 and 8 DPI (E–H). Data are presented as means and standard deviation error bars, with data from individual animals represented by individual symbols. p‐Values were considered significant ( ∗ < 0.05, ∗∗ < 0.01, ∗∗∗ < 0.001).

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  Figure 7. PoAstV4 spike IgM and IgG seroconversion. All results are presented as optical density. The 5 and 8 DPI results are from pigs that were euthanized on those days. Results from 10 to 21 DPI are from the same animals (PoAstV4‐inoculated n = 5, negative n = 3). Data are presented as means with standard mean error bars.