Fig 1.
Porcine RVA (G5P[7], G9P[13]) levels in the small intestine (A) and large intestine (B); RVA shedding (rectal swabs) (C).
Six-day-old germ-free pigs were orally inoculated with 1 × 106 FFU of each RVA. Swabs were collected at designated time points (day post-infection, dpi, 1-4). For the small and large intestine contents pigs were euthanized at the post-inoculation times indicated and contents were collected. RVA quantification was performed using cell culture immunofluorescence (CCIF). Significant differences (*p < 0.05, ** p < 0.01, *** p < 0.001) are indicated as calculated by using two-way ANOVA with repeated measures and the Tukey-Kramer test for multiple comparisons.
Fig 2.
Tissue-specific analysis of RVA RNA levels (G5P[7], G9P[13]) in extraintestinal tissues.
Six-day-old germ-free pigs were orally inoculated with 1 × 10⁶ FFU of RVA. At the indicated post-inoculation time points, animals were euthanized, and tissues (lungs, salivary glands, liver, blood, and spleen) were collected. (A) RVA RNA levels in extraintestinal tissues across dpi 1–4. (B) RVA RNA levels in extraintestinal tissues at time points when significant differences were observed. Significant differences (*p < 0.05, ***p < 0.001, ****p < 0.0001) were calculated using two-way ANOVA with repeated measures and the Tukey–Kramer test for multiple comparisons. (C) RT-qPCR RVA RNA detection frequency in extraintestinal tissues across dpi 1–4. (D) RT-PCR Detection frequency at time points with significant differences. The frequency of RVA detection across tissues from dpi 1–4 was assessed using Fisher’s exact test. Significant differences (*p < 0.05) are indicated. (E) In situ hybridization (ISH)-based detection frequency of RVA RNA in extraintestinal tissues across dpi 1–4.(F) ISH Detection frequency at time points with significant differences. Statistical analysis was performed using Fisher’s exact test. Significant differences (*p < 0.05) are indicated.
Fig 3.
Strain-specific analysis of RVA RNA levels in extraintestinal tissues.
Six-day-old germ-free pigs were orally inoculated with 1 × 10⁶ FFU of each RVA. At the indicated post-inoculation time-points, animals were euthanized, and tissues (A: lungs; B: salivary glands; C: liver; D: spleen and E: blood) were collected. Significant differences (*p < 0.05, **p < 0.01) were determined using two-way ANOVA with repeated measures and the Tukey–Kramer test for multiple comparisons.
Fig 4.
Strain-specific analysis of RVA RNA levels in extraintestinal tissues.
Six-day-old germ-free pigs were orally inoculated with 1 × 10⁶ FFU of each RVA. At the indicated post-inoculation time points, animals were euthanized, and tissues (lungs, salivary glands, liver, blood, and spleen) were collected. (A) RT-qPCR RVA RNA detection frequency across dpi 1–4; (B) Detection frequency at time points where significant differences were observed. The frequency of RVA detection across tissues from dpi 1–4 was assessed using Fisher’s exact test. Significant differences (*p < 0.05) are indicated. (C) ISH-based RVA RNA detection frequency across dpi 1–4; (D) Detection frequency at time points with significant differences. Statistical analysis was performed using Fisher’s exact test. Significant differences (*p < 0.05) are indicated.
Fig 5.
Distribution of sialylated and non-sialylated glycans across the intestinal and extraintestinal tissues.
Representative fluorescence microscopy images (A) show DAPI-stained nuclei (blue), highlighting tissue architecture. Sialylated glycans were detected using FITC-labeled Sambucus nigra agglutinin (SNA) and Maackia amurensis I lectin (MAL I), which are specific to α2,6- and α2,3-linked sialic acids, respectively. Non-sialylated glycans were detected using FITC-labeled peanut agglutinin (PNA) and Griffonia simplicifolia lectin I (GSL I), which are specific to galactose and α-galactose, respectively (green).Histo-blood group antigens A (HBGA-A) and H (HBGA-H) were detected using specific antibodies, followed by FITC-labeled goat anti-mouse IgG/A/M (H + L) secondary antibody (1:750; Bio-Rad).The expression levels of glycans were analyzed (B) by calculating the corrected total cell fluorescence (CTCF) [47].
Fig 6.
Gross evaluation of extraintestinal organs detected changes in lungs from germ-free pigs challenged with RVA.
Lungs from negative control group pigs were fully collapsed and did not present any gross changes (left), while lungs from Rotavirus A–challenged pigs did not collapse and showed areas of red discoloration (black asterisks) and atelectasis (blue arrows).
Fig 7.
Histological changes and positive in situ hybridization signals were observed in germ-free pigs experimentally challenged with RVA.
(A) Ileum from a negative control piglet without histological changes. (B) Ileum from a Rotavirus A (G5P[7]) challenged piglet with villus atrophy and blunting (asterisks and degeneration of epithelial cells in the tip of the villi (insert). (C) Ileum villus epitheilal cells with multifocal positive signals (brown) for Rotavirus A (G5P[7]). (D) Lung from a negative control group piglet without histologic changes. (E) Lung of a Rotavirus A (G5P[7]) challenged piglet with severe hyperemia. (F) Lung of a Rotavirus A (G5P[7]) challenged piglet showing in situ hybridization positive signals (brown) in epithelial cells of bronchioles. (G) Salivary gland from a negative control piglet without histologic changes. (H) Salivary gland from a Rotavirus A (G5P[7]) challenged piglet without histological changes. (I) Salivary glands with focal positive in situ hybridization signal (brown) for Rotavirus A (G5P[7]).
Fig 8.
The scheme illustrates experimental design.
Six- to eight-day-old germ-free pigs were inoculated with an individual RVA strain at a dose of 1 × 10⁶ FFU/piglet. At 1–4 days post-inoculation (dpi), pigs (n = 3–6) were euthanized, and rectal swabs, blood, intestinal contents, and tissue samples (lungs, salivary glands, ileum, jejunum, spleen, liver, pancreas) were collected. Rectal swabs were used to assess RV shedding by cell culture immunofluorescence. Tissue fragments were either fixed in 10% neutral buffered formalin, embedded in paraffin, and stored at room temperature for future RNA in situ hybridization and hematoxylin and eosin staining or frozen at −20°C for future RNA extraction and RT-qPCR. Created in BioRender. Raev, S. (2025). https://BioRender.com/ctpuk1b.
Table 1.
Lectins and antibodies used in this study.