Pathogenicity of porcine G9P[23] and G9P[7] rotaviruses in piglets

Abstract

G9 group A rotaviruses (RVAs) are considered important pathogens in pigs and humans, and pigs are hypothesized to be a potential host reservoir for human. However, intestinal and extra-intestinal pathogenicity and viremia of porcine G9 RVAs has remained largely unreported. In this study, colostrum-deprived piglets were orally infected with a porcine G9P[23] or G9P[7] strain. Histopathologically, both strains induced characteristic small intestinal lesions. Degeneration and necrosis of parenchymal cells were observed in the extra-intestinal tissues, but most predominantly in the mesenteric lymph nodes (MLNs). RVA antigen was continuously detected in the small intestinal mucosa and MLNs, but only transiently in cells of the liver, lung, and choroid plexus. Viral RNA levels were much higher in the feces and the MLNs compared to other tissues. The onset of viremia occurred at day post infection (DPI) 1 with the amount of viral RNA reaching its peak at DPI 3 or 5, before decreasing significantly at DPI 7 and remaining detectable until DPI 14. Our data suggest that porcine G9 RVAs have a strong small intestinal tropism, are highly virulent for piglets, have the ability to escape the small intestine, spread systemically via viremia, and replicate in extra-intestinal tissues. In addition, MLNs might act as a secondary site for viral amplification and the portal of systemic entry. These results add to our understanding of the pathogenesis of human G9 RVAs, and the validity of the pig model for use with both human and pig G9 RVAs in further studies.

Keywords: Experimental model; G9 genotype; Group A rotaviruses; Pathogenicity; Piglets.

Fig. 1

Histopathological changes in the small intestine of piglets inoculated with PRG942 (G9P[23]) strain. Duodenum (A), jejunum (B), and ileum (C) from a mock-inoculated piglet had normal structure of mucosal membrane. Duodenum (D), jejunum (E), and ileum (F) sampled from a piglet inoculated with PRG942 (G9P[23]) strain at DPI 1 showed mild to moderate mucosal changes. Duodenum (G), jejunum (H), and ileum (I) sampled from a piglet inoculated with PRG942 (G9P[23]) strain at DPI 3 showed marked mucosal changes including the widespread villous atrophy (up-down arrow) and fusion (arrows), and increased crypt depth (up-wards arrow). Duodenum (J), jejunum (K), and ileum (L) sampled from a piglet inoculated with PRG942 (G9P[23]) strain at DPI 7 had marked mucosal changes. Samples were stained with hematoxylin and eosin stain. Bars A–L = 200 μm.

Fig. 2

Histopathological changes in the small intestine of piglets inoculated with PRG9121 (G9P[7]) strain. Duodenum (A), jejunum (B), and ileum (C) from a mock-infected piglet had normal structure of mucosal membrane. Duodenum (D), jejunum (E), and ileum (F) sampled from a piglet infected by PRG9121 (G9P[7]) strain at DPI 1 showed mild to moderate mucosal changes. Duodenum (G), jejunum (H), and ileum (I) sampled from a piglet inoculated with PRG9121 (G9P[7]) strain at DPI 3 showed marked mucosal changes including the widespread villous atrophy (up-down arrow) and fusion (arrows), and increased crypt depth (up-wards arrow). Duodenum (J), jejunum (K), and ileum (L) sampled from a piglet inoculated with PRG9121 (G9P[7]) strain at DPI 7 had marked mucosal changes. Samples were stained with hematoxylin and eosin stain. Bars A–L = 200 μm.

Fig. 3

Distribution of RVA-positive cells of PRG942 (G9P[23]) and PRG9121 (G9P[7]) strains in the duodenum. Piglets inoculated with either chloroform-inactivated PRG942 (G9P[23]) strain (A) or PRG9121 (G9P[7]) strain (B) contained no RVA-positive cells in the villi of each duodenum. RVA-positive cells (arrows) were scattered in the villi of duodenum sampled from piglets inoculated with PRG942 (G9P[23]) strain (C) or PRG9121 (G9P[7]) strain (D) at DPI 3. Indirect immunofluorescence assay was performed with monoclonal antibody against the VP6 protein of strain OSU. Bars A–D = 100 μm.

Fig. 4

Histopathological findings and antigen distribution in the extra-intestinal organs of piglets inoculated with PRG942 (G9P[23]) strain. (A) Normal mesenteric lymph node (MLN) sampled from a mock-inoculated piglet showed intact cortex with densely concentrated lymphocytes (asterisk). (B) MLN sampled from a piglet inoculated with PRG942 (G9P[23]) strain at DPI 3 showed lymphoid cell depletion in the cortex (asterisk). (C) RVA-positive lymphoid cells (arrows) were scattered in the MLN from a piglet inoculated with PRG942 (G9P[23]) strain at DPI 3. (D) Normal lung tissue sampled from a mock-inoculated piglet had thin alveolar walls (arrows). (E) Lung tissue sampled from a piglet inoculated with PRG942 (G9P[23]) strain at DPI 3 showed interstitial pneumonia (arrows). (F) RVA antigens were detected in a pneumocyte (arrowhead) and lymphoid cell (arrow) in the lung from a piglet inoculated with PRG942 (G9P[23]) strain at DPI 3. (G) Normal liver sampled from a mock-inoculated piglet showed fat-stored hepatocytes (arrow). (H) Multiple necrotic hepatocytes scattered in the liver sampled from a piglet inoculated with PRG942 (G9P[23]) strain at DPI 3 showed pyknosis of nucleus and increased eosinophilia of cytoplasm (arrows). (I) RVA antigens were detected in a hepatocyte (arrow) in the liver from a piglet inoculated with PRG942 (G9P[23]) strain at DPI 3. (J) Intact epithelium (arrowheads) was observed in the choroid plexus sampled from a mock-inoculated piglet. (K) Choroid plexus sampled from a piglet inoculated with PRG942 (G9P[23]) strain at DPI 3 showed epithelial degeneration (arrowheads), necrosis (arrows), and lymphocyte infiltration (double arrows). (L) RVA antigens were detected in the epithelial cells (arrow) of the choroid plexus from a piglet inoculated with PRG942 (G9P[23]) strain at DPI 3. Bars A–L = 100 μm.

Fig. 5

Histopathological findings and antigen distribution in the extra-intestinal organs of piglets inoculated with PRG9121 (G9P[7]) strain. (A) Normal mesenteric lymph node (MLN) sampled from a mock-inoculated piglet showed intact cortex with densely concentrated lymphocytes (asterisk). (B) MLN sampled from a piglet inoculated with PRG9121 (G9P[7]) strain at DPI 3 showed marked lymphoid cell depletion in the cortex (asterisk). (C) RVA-positive lymphoid cells (arrows) were scattered in the MLN from a piglet inoculated with PRG9121 (G9P[7]) strain at DPI 3. (D) Normal lung tissues sampled from a mock-inoculated piglet had thin alveolar walls (arrows). (E) Lung tissues sampled from a piglet inoculated with PRG9121 (G9P[7]) strain at DPI 3 showed interstitial pneumonia (arrows). (F) RVA antigen was detected in a pneumocyte (arrow) in the lung from a piglet inoculated with PRG9121 (G9P[7]) strain at DPI 3. (G) Normal liver sampled from a mock-inoculated piglet showed fat-stored hepatocytes (arrows). (H) Multiple necrotic hepatocytes scattered in the liver sampled from a piglet inoculated with PRG942 (G9P[23]) strain at DPI 3 showed pyknosis of nucleus and increased eosinophilia of cytoplasm (arrows). (I) RVA antigens were detected in a few hepatocytes (arrows) in the liver from a piglet inoculated with PRG9121 (G9P[7]) strain at DPI 3. (J) Intact epithelium (arrowheads) was observed in the choroid plexus sampled from a mock-inoculated piglet. (K) Choroid plexus sampled from a piglet inoculated with PRG9121 (G9P[7]) strain at DPI 3 showed epithelial degeneration (arrowheads), necrosis (arrows), and lymphocyte infiltration (double arrows). (L) RVA antigen was detected in a lymphoid cell (arrow) in the choroid plexus from a piglet inoculated with PRG9121 (G9P[7]) strain. Bars A–L = 100 μm.

Fig. 6

Quantification of RVA RNA by SYBR Green real-time RT-PCR. Real-time RT-PCR was performed with the feces, serum, mesenteric lymph node (MLN), liver, lung, choroid plexus, and nasal swab sampled from piglets inoculated with porcine G9 RVA strain PRG942 (G9P[23]) (A) and PRG9121 (G9P[7]) (B). The geometric means of viral RNA copy number per mg of tissue are displayed.