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Pathogenesis of a genogroup II human norovirus in gnotobiotic pigs - PubMed

Pathogenesis of a genogroup II human norovirus in gnotobiotic pigs

Sonia Cheetham et al. J Virol. 2006 Nov.

Abstract

We evaluated the gnotobiotic (Gn) pig as a model to study the pathogenesis of human norovirus (HuNoV) and to determine the target cells for viral replication. Sixty-five Gn pigs were inoculated with fecal filtrates of the NoV/GII/4/HS66/2001/US strain or with pig-passaged intestinal contents (IC) and euthanized acutely (n = 43) or after convalescence (n = 22). Age-matched Gn piglets (n = 14) served as mock-inoculated controls. Seventy-four percent (48/65) of the inoculated animals developed mild diarrhea compared to 0 of 14 controls. Pigs from postinoculation days (PID) 1 to 4 tested positive for HuNoV by reverse transcription-PCR of rectal swab fluids (29/65) and IC (9/43) and by antigen (Ag) enzyme-linked immunosorbent assay (ELISA) using antiserum to virus-like particles of HuNoV GII/4. No control pigs were positive. Histopathologic examination showed mild lesions in the proximal small intestine of only one pig (1/7). Seroconversion after PID 21 was detected by antibody ELISA in 13 of 22 virus-inoculated pigs (titers, 1:20 to 1:200) but not in controls. Immunofluorescent microscopy using a monoclonal antibody to HuNoV GII capsid revealed patchy infection of duodenal and jejunal enterocytes of 18 of 31 HuNoV-inoculated pigs with a few stained cells in the ileum and no immunofluorescence (IF) in mock-inoculated controls. Immunofluorescent detection of the viral nonstructural N-terminal protein antigen in enterocytes confirmed translation. Transmission electron microscopy of intestines from HuNoV-inoculated pigs showed disrupted enterocytes, with cytoplasmic membrane vesicles containing calicivirus-like particles of 25 to 40 nm in diameter. In summary, serial passage of HuNoV in pigs, with occurrence of mild diarrhea and shedding, and immunofluorescent detection of the HuNoV structural and nonstructural proteins in enterocytes confirm HuNoV replication in Gn pigs.

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Figures

FIG. 1.
FIG. 1.

Confocal microscopy showing indirect immunofluorescent localization of the HuNoV capsid protein in small intestinal tissues from virus-inoculated Gn pigs. (A to G) IF on whole-mount tissues. (H to I) IF on tissue paraffin sections. Primary antibody NS14 MAb was used to detect the capsid protein of HuNoV GII. Secondary antibodies: goat anti-mouse IgG Alexa488 (green) (nuclear counterstain [A to G] SYTOX orange [red] or propidium iodide [H to I] [red]) and actin stained with phallotoxin Alexa633 (blue). (A) Jejunum of a P2-inoculated Gn pig at PID 3 showing scattered IF-positive cells on the villi tips or sides. (B) Jejunum of a mock-inoculated pig with no IF-positive cells evident. (C) Jejunum of a P1-inoculated Gn pig at PID 3, showing individually infected enterocytes on the side of a villus. (D) Tip of a villus in the duodenum of a P1-inoculated Gn pig at PID 3 with several contiguous infected cells. (E) Tip of a villus on the jejunum of a P1-inoculated Gn pig at PID 3 with positive signal in the apical portion of the enterocyte cytoplasm. (F) Enterocytes from the duodenum of a P0-inoculated Gn pig at PID 3, showing nuclear displacement and positive signal throughout the cytoplasm of individual cells. (G) Tip of a villus in the duodenum of a P0-inoculated pig at PID 2 showing contiguous infected cells. (H) Paraffin section of the duodenum of a P0-inoculated Gn pig at PID 4 showing contiguous infected enterocytes on the sides of the villi and some individual enterocytes on the tips. (I) Higher magnification, most enterocytes showed perinuclear IF. Bars: A and B, 100 μm; C to H, 20 μm.

FIG. 2.
FIG. 2.

Confocal microscopy of immunofluorescent colocalization of viral capsid and N-terminal protein antigens in whole-mount small intestinal tissue. (A) Anti-capsid NS14 MAb with the secondary goat anti-mouse IgG antibody conjugated to Alexa488 (green). (B) Antiviral N-terminal protein guinea pig serum with the secondary goat anti-guinea pig IgG labeled with AlexaFluor576/603 (red). (C) Cell nuclei were counterstained with TO-PRO-3 iodide (642/661) dye (blue). (D) Merged image where the yellow color indicates colocalization of viral capsid and N-terminal protein antigens. Asterisks indicate cells where only the capsid protein was detected, and arrows indicate apical globular-like structures detected with the anti-N-terminal protein serum. Bars, 20 μm.

FIG. 3.
FIG. 3.

TUNEL reaction to detect apoptotic cells in small intestinal tissues of HuNoV-inoculated and mock-inoculated pigs. (A) More cells with dark-staining apoptotic nuclei (arrows) were observed in HuNoV P0-inoculated Gn pig duodenum. (B) TUNEL results showing fewer apoptotic cells and more goblet cells (not evident in the infected pig) in a mock-inoculated control Gn pig. Bars, 50 μm.

FIG. 4.
FIG. 4.

Transmission electron microscopy of (A) the jejunum of a HuNoV GII/4 P0-inoculated Gn pig at PID 3 with the nucleus displaced opically. The arrows indicate vesicles (Ve) not seen in the control Gn pig. (B) Cells showed changes in their intracellular organization, with cellular organelles decreased in number. (B) Jejunum of an age-matched, mock-inoculated control pig. (C to E) Duodenum of a P0-inoculated Gn pig at PID 3. (C) The overall intracellular morphology of the enterocyte is disrupted, with vesicles in the cytoplasm containing calicivirus-like particles (box) and nuclear (Nu) displacement; mitochondria (Mi) are indicated. A goblet cell (Gc) can be observed. (D) Detail of the membrane vesicle, from the box in panel C, containing calicivirus-like particles. (E) Aggregates of calicivirus-like particles in the cytoplasm of another cell. Bars: A to C, 1 μm; D, 200 nm; E, 225 nm.

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