Group A rotavirus infection and age-dependent diarrheal disease in rats: a new animal model to study the pathophysiology of rotavirus infection

Abstract

Group A rotaviruses are major pathogens causing acute gastroenteritis in children and animals. To determine if group A rotavirus replicates and induces disease in rats, antibody-negative Lewis neonatal or adult rats were inoculated orally with tissue culture-adapted human (Wa, WI61, and HAL1166), simian (rhesus rotavirus [RRV] and SA11), bovine (WC3), lapine (ALA), or porcine (OSU) rotavirus strains, wild-type murine (EC(wt)) rotavirus strain, or phosphate-buffered saline (PBS). Rotavirus infection in rats was evaluated by (i) clinical findings, (ii) virus antigen shedding or infectious virus titers in the feces or intestinal contents measured by enzyme-linked immunosorbent assay or fluorescent-focus assay, (iii) histopathological changes in the small intestine, (iv) distribution of rotavirus antigen in small-intestine sections by immunofluorescence, and (v) growth rate. Rotavirus infection of 5-day-old but not > or =21-day-old rats resulted in diarrhea that lasted from 1 to 10 days postinoculation. The severity of disease and spread of infection to naIve littermates differed depending on the virus strain used for inoculation. The duration of virus antigen shedding following infection was considerably prolonged (up to 10 days) in neonatal rats compared to that in 21-day-old rats (1 or 2 days). Based on lack of virus antigen shedding and disease induction, the murine EC(wt) rotavirus was the only strain tested that did not infect rats. Histopathological changes in the small-intestine mucosa of 5-day-old RRV-inoculated rats but not of PBS-inoculated rats was limited to extensive enterocyte vacuolation in the ileum. In RRV-inoculated neonatal rats, rotavirus antigen was detected in the epithelial cells on the upper half of the intestinal villi of the jejunum and ileum. In addition, infection of neonatal rats with RRV but not with PBS resulted in reduced weight gain. Rats infected with group A rotaviruses provide a new animal model with unique features amenable to investigate rotavirus pathogenesis and the molecular mechanisms of intestinal development, including physiological factors that may regulate age-dependent rotavirus-induced diarrhea.

FIG. 1.

Viral antigen-shedding curves of individual fecal samples of 5-day-old rats inoculated with 0.5 ml of PBS (n = 10) (A), 2.2 × 10⁶ FFU of human rotavirus Wa (B), 3 × 10⁵ FFU of human rotavirus HAL1166 (C), 3 × 10⁶ FFU of human rotavirus WI61 (D), 10⁷ FFU of simian rotavirus SA11 (E), 2 × 10⁷ FFU of simian rotavirus RRV (F), 10⁶ FFU of lapine rotavirus ALA (G), or 4 × 10⁶ FFU of bovine rotavirus WC3 (H). Viral antigen shedding was assessed by ELISA from 0 to 10 DPI and was expressed as net readings for OD at 450 nm. Readings of ≥0.1 are considered positive. Fecal samples from individual 5-day-old rats could not be obtained every day; therefore, missing data points reflect a lack of sample and not a lack of detection of virus antigen shedding. Each virus-inoculated group consisted of eight virus-inoculated (▪, •, ▴, ⧫, □, •, ▵, and ◊) and two PBS-inoculated (white X in black box and black circle in white box) rat pups to monitor spread among mock-inoculated littermates, bringing the number of animals per group to 10. All PBS inoculations were performed prior to any virus inoculation.

FIG. 2.

Infectious virus-shedding curves of individual fecal samples of 5-day-old rats inoculated with 0.5 ml of PBS (n = 10) (A), 2.2 × 10⁶ FFU of human rotavirus Wa (B), 3 × 10⁵ FFU of human rotavirus HAL1166 (C), 3 × 10⁶ FFU of human rotavirus strain WI61 (D), 10⁷ FFU of simian rotavirus SA11 (E), 2 × 10⁷ FFU of simian rotavirus RRV (F), 10⁶ FFU of lapine rotavirus strain ALA (G), or 4 × 10⁶ FFU of bovine rotavirus strain WC3 (H). Infectious rotavirus shedding was assessed by FFA from 0 to 10 DPI and was expressed in FFU. Fecal samples from individual 5-day-old rats could not be obtained every day; therefore, missing data points reflect a lack of sample and not a lack of detection of virus antigen shedding. Each virus-inoculated group consisted of eight virus-inoculated (▪, •, ▴, ⧫, □, •, ▵, and ◊) and two PBS-inoculated (white X in black box and black circle in white box) rat pups to monitor spread among mock-inoculated littermates, bringing the number of animals per group to 10. All PBS inoculations were performed prior to any virus inoculation.

FIG. 3.

Percent diarrhea (▪) and mean diarrhea severity (•) in 5-day-old neonatal rat litters inoculated with PBS (A), human rotavirus strain Wa (B), human rotavirus strain HAL1166 (C), human rotavirus strain WI61 (D), simian rotavirus strain SA11 (E), simian rotavirus strain RRV (F), lapine rotavirus strain ALA (G), or bovine rotavirus strain WC3 (H) from 0 to 12 DPI. No diarrhea was observed in any rat beyond 12 DPI (data not shown). Percent diarrhea for each group per day was calculated by dividing the number of diarrheic samples by the number of total samples collected each day, since fecal samples from each 5-day-old neonatal rat could not be collected every day. A score of ≥2 was considered diarrhea, whereas a score of <2 was considered normal. The mean disease severity was determined by dividing the sum of all diarrhea or not-diarrhea scores (1 to 4) by the number of total samples scored each day.

FIG. 4.

Group A simian RRV replication in neonatal rats. (A) Growth curves of simian RRV inoculated into 5-day-old neonatal rats. Titers of infectious virus (FFU) from the contents of the small intestine (⧫), large intestine (▪), and cecum (•) after inoculation with 2 × 10⁷ FFU of RRV were determined by FFA from 0 to 216 hpi. For infectious titers of <100, 50 was used to calculate the mean infectious titer. An infectious titer of 50 was considered negative. No infectious virus from the intestinal contents of the small intestine (□), large intestine (•), and cecum (▵) was detected after inoculation with PBS. (B) Total intestinal content yield of RRV-inoculated (•) rat pups measured in FFU. The total amount of infectious RRV recovered (9 × 10⁷ FFU) from the intestinal contents surpassed the amount of the virus inoculum (2 × 10⁷ FFU). No infectious virus was detected in the intestinal contents of PBS-inoculated (▪) rat pups. Each data point represents the average infectious rotavirus titers of a total of three independent experiments ± standard error of the mean (total PBS n = 6 and total RRV n = 12 per time point).

FIG. 5.

Photomicrograph of small intestine of duodenal (A), jejunal (B), and ileal (C) mucosa of 5-day-old PBS mock-infected control neonatal rat or duodenal (D), jejunal (E), and ileal (F) mucosa of 5-day-old group A simian RRV-infected neonatal rat at 72 hpi. In RRV-infected rat pups, histopathological lesions are limited to large vacuoles in the enterocytes lining most of the surface of the villi in the ileum. Hematoxylin and eosin staining; original magnification, ×240.

FIG. 6.

Electron micrographs of ultrastructural appearance of the ileum of PBS-inoculated (A) or RRV-inoculated (B) 5-day-old rats at 48 hpi. Extensive cytoplasmic vacuolization occurred after RRV infection (B), including relocation of the nucleus. Vacuoles are devoid of virus particles, and the brush border system appears intact. Original magnification, ×2,400.

FIG. 7.

Photomicrograph of the distribution of specific immunofluorescence staining against rotavirus VP6 antigen of duodenum (A), jejunum (B), or ileum (C) of 5-day-old PBS mock-infected control neonatal rat and duodenum (D), jejunum (E), or ileum (F) of 5-day-old RRV-infected neonatal rat at 24 hpi. Rotavirus VP6 antigen is distributed in the epithelial cells of the upper half of the intestinal villi of the jejunum and ileum. Original magnification, ×240.

FIG. 8.

Photomicrograph of the distribution of specific immunofluorescence staining against rotavirus NSP4 antigen of duodenum (A), jejunum (B), or ileum (C) of 5-day-old PBS mock-infected control neonatal rat and duodenum (D), jejunum (E), or ileum (F) of 5-day-old RRV-infected neonatal rat at 24 hpi. Rotavirus NSP4 antigen is distributed in the epithelial cells of the upper half of the intestinal villi of the jejunum and ileum. Original magnification, ×240.

FIG. 9.

Effect of group A RRV infection in body weight gain of 5-day-old neonatal rats. Following infection with 2 × 10⁷ FFU of RRV, individual neonatal rats were weighed daily to monitor weight gain compared to that of PBS-inoculated, 5-day-old neonatal rats. All RRV-inoculated (n = 10, four males and six females) but not PBS-inoculated (n = 10, six males and four females) neonatal rats shed virus antigen (as measured by ELISA) or infectious virus (as measured by FFA) and developed diarrhea from 1 to 9 DPI (data not shown). Each group of rats was from the same litter and was sexed at 16 DPI.

FIG. 10.

Fecal virus antigen-shedding curves of individual 21-day-old rats inoculated with PBS (A), simian RRV (4 × 10⁷ FFU) (B), lapine rotavirus ALA (2 × 10⁶ FFU) (C), bovine rotavirus WC3 (8 × 10⁶ FFU) (D), human rotavirus Wa (4.4 × 10⁶ FFU) (E), or human rotavirus HAL1166 (6 × 10⁵ FFU) (F). Fecal rotavirus antigen shedding was assessed by ELISA from 0 to 10 DPI and expressed as net readings of OD at 450 nm. Readings of ≥0.1 are considered positive.

FIG. 11.

Fecal infectious virus-shedding curves of individual 21-day-old rats inoculated with PBS (A), simian RRV (4 × 10⁷ FFU) (B), lapine rotavirus ALA (2 × 10⁶ FFU) (C), bovine rotavirus WC3 (8 × 10⁶ FFU) (D), human rotavirus Wa (4.4 × 10⁶ FFU) (E), or human rotavirus HAL1166 (6 × 10⁵ FFU) (F). Fecal infectious virus shedding was assessed by FFA from 0 to 10 DPI and was expressed in FFU.