Human Rotavirus Replicates in Salivary Glands and Primes Immune Responses in Facial and Intestinal Lymphoid Tissues of Gnotobiotic Pigs

Abstract

Human rotavirus (HRV) is a leading cause of viral gastroenteritis in children across the globe. The virus has long been established as a pathogen of the gastrointestinal tract, targeting small intestine epithelial cells and leading to diarrhea, nausea, and vomiting. Recently, this classical infection pathway was challenged by the findings that murine strains of rotavirus can infect the salivary glands of pups and dams and transmit via saliva from pups to dams during suckling. Here, we aimed to determine if HRV was also capable of infecting salivary glands and spreading in saliva using a gnotobiotic (Gn) pig model of HRV infection and disease. Gn pigs were orally inoculated with various strains of HRV, and virus shedding was monitored for several days post-inoculation. HRV was shed nasally and in feces in all inoculated pigs. Infectious HRV was detected in the saliva of four piglets. Structural and non-structural HRV proteins, as well as the HRV genome, were detected in the intestinal and facial tissues of inoculated pigs. The pigs developed high IgM antibody responses in serum and small intestinal contents at 10 days post-inoculation. Additionally, inoculated pigs had HRV-specific IgM antibody-secreting cells present in the ileum, tonsils, and facial lymphoid tissues. Taken together, these findings indicate that HRV can replicate in salivary tissues and prime immune responses in both intestinal and facial lymphoid tissues of Gn pigs.

Keywords: gnotobiotic pigs; rotavirus; salivary glands.

Figure 1

Rotavirus nasal and fecal shedding from PID1-7 in Gn pigs. Gn pigs inoculated with Rotarix, rRRV, or Wa AttHRV shed high levels of rotavirus in feces and nasally (A). The duration of virus shedding in both nasal and fecal samples ranged from one to five days (B). Genome copies detected in nasal and fecal swabs each day are shown in (C,D), respectively. Different letters indicate statistical significance, Kruskal–Wallis test followed by Dunn’s multiple comparisons test except Fisher exact test for percentage, p < 0.05. AUC: area under the curve.

Figure 2

Detection of rotavirus antigen in the nasal cavity of Rotarix (A), Wa AttHRV (B), rRRV (C), or mock (D) inoculated pigs. Representative images from each group are shown. Images were taken using 63× objective on Zeiss LSM 880 confocal. Blue: DAPI. Green: rotavirus VP6. Scale bar is 160 pixels.

Figure 3

Detection of rotavirus VP6 antigen in the parotid salivary gland of Rotarix (A), Wa AttHRV (B), rRRV (C), or mock (D) inoculated pigs. Representative images from each group are shown. Images were taken using 25× objective on Zeiss LSM 880 confocal. Blue: DAPI. Green: rotavirus VP6. Scale bar is 160 pixels.

Figure 4

Detection of rotavirus VP6 antigen in the ileum of Rotarix (A), Wa AttHRV (B), rRRV (C), or mock (D) inoculated pigs. Representative images from each group are shown. Images were taken using 25× objective on Zeiss LSM 880 confocal. Blue: DAPI. Green: rotavirus VP6. Scale bar is 160 pixels.

Figure 5

Rotavirus-specific IgM antibody titers in serum (A), small intestinal contents (SIC) (B), and large intestinal contents (LIC) (C). Error bars indicate standard error of the mean. Kruskall–Wallis test for multiple comparisons. * p < 0.05.

Figure 6

Rotavirus-specific IgM antibody-secreting cells detected in the ileum (A), tonsils (B), and facial lymph nodes (B) of Gn pigs. Error bars indicate standard error of the mean. Kruskall–Wallis test for multiple comparisons. ** p < 0.01.