Pathogenesis and immune responses in gnotobiotic calves after infection with the genogroup II.4-HS66 strain of human norovirus

Abstract

We previously characterized the pathogenesis of two host-specific bovine enteric caliciviruses (BEC), the GIII.2 norovirus (NoV) strain CV186-OH and the phylogenetically unassigned NB strain, in gnotobiotic (Gn) calves. In this study we evaluated the Gn calf as an alternative animal model to study the pathogenesis and host immune responses to the human norovirus (HuNoV) strain GII.4-HS66. The HuNoV HS66 strain caused diarrhea (five/five calves) and intestinal lesions (one/two calves tested) in the proximal small intestine (duodenum and jejunum) of Gn calves, with lesions similar to, but less severe than, those described for the Newbury agent 2 (NA-2) and NB BEC. Viral capsid antigen was also detected in the jejunum of the proximal small intestine of one of two calves tested by immunohistochemistry. All inoculated calves shed virus in feces (five/five calves), and one/five had viremia. Antibodies and cytokine (proinflammatory, tumor necrosis factor alpha [TNF-alpha]; Th1, interleukin-12 [IL-12] and gamma interferon [IFN-gamma]; Th2, IL-4; Th2/T-regulatory, IL-10) profiles were determined in serum, feces, and intestinal contents (IC) of the HuNoV-HS66-inoculated calves (n = 5) and controls (n = 4) by enzyme-linked immunosorbent assay in the acute (postinoculation day 3 [PID 3]) and convalescent (PID 28) stages of infection. The HuNoV-HS66-specific antibody and cytokine-secreting cells (CSCs) were quantitated by ELISPOT in mononuclear cells of local and systemic tissues at PID 28. Sixty-seven percent of the HuNoV-HS66-inoculated calves seroconverted, and 100% coproconverted with immunoglobulin A (IgA) and/or IgG antibodies to HuNoV-HS66, at low titers. The highest numbers of antibody-secreting cells (ASC), both IgA and IgG, were detected locally in intestine, but systemic IgA and IgG ASC responses also occurred in the HuNoV-HS66-inoculated calves. In serum, HuNoV-HS66 induced higher peaks of TNF-alpha and IFN-gamma at PIDs 2, 7, and 10; of IL-4 and IL-10 at PID 4; and of IL-12 at PIDs 7 and 10, compared to controls. In feces, cytokines increased earlier (PID 1) than in serum and TNF-alpha and IL-10 were elevated acutely in the IC of the HS66-inoculated calves. Compared to controls, at PID 28 higher numbers of IFN-gamma and TNF-alpha CSCs were detected in mesenteric lymph nodes (MLN) or spleen and Th2 (IL-4) CSCs were elevated in intestine; IL-10 CSCs were highest in spleen. Our study provides new data confirming HuNoV-HS66 replication and enteropathogenicity in Gn calves and reveals important and comprehensive aspects of the host's local (intestine and MLN) and systemic (spleen and blood) immune responses to HuNoV-HS66.

FIG. 1.

Histopathology. (A) Jejunum of a mock-inoculated calf at PID 3. No abnormalities were evident (VH/CD ratio = 7). Original magnification, ×150. (B) Jejunum of a HuNoV-HS66-inoculated calf showing moderate to severe atrophic enteritis (moderate = VH/CD ratio of 4 to 5; severe = VH/CD ratio of 2 to 4) characterized by loss (50 to 70%) of villi and mild to moderate proliferation of crypt cell layers (arrow) and increase of cell populations in the lamina propria. Original magnification, ×200. Hematoxylin and eosin stain was used.

FIG. 2.

IHC for detection of GII HuNoV capsid antigens. (A) Jejunum of mock-inoculated calf at PID 3. No positive cells were observed (magnification, ×150). (B) Jejunum of HuNoV-HS66-inoculated calf at PID 3. Viral capsid antigens (stained red) were observed in the cytoplasm of a few intestinal epithelial cells attached to the villi or exfoliated enterocytes (arrow) (original magnification, ×300). IHC, 3,3′-diaminobenzidine, and Mayer's hematoxylin counterstain were used.

FIG. 3.

Isotype-specific (IgM, IgA, and IgG) GMTs in the serum and mean numbers of ASC in intestine, MLN, spleen, and blood MNCs of Gn calves inoculated with HuNoV-HS66 (solid bars) or controls (open bars, baseline values).

FIG. 4.

Th1 (IFN-γ and IL-12), proinflammatory (TNF-α), Th2 (IL-4), and Th2/T-regulatory (IL-10) cytokine concentrations in serum of Gn calves inoculated with HuNoV-HS66 (solid squares) or mock controls (open triangles).

FIG. 5.

Th1 (IFN-γ and IL-12), proinflammatory (TNF-α), Th2 (IL-4) and Th2/T-regulatory (IL-10) cytokine concentrations in fecal samples of Gn calves inoculated with HuNoV-HS66 (solid squares) or mock controls (open triangles).

FIG. 6.

Th1 (IFN-γ and IL-12), proinflammatory (TNF-α), Th2 (IL-4), and Th2/T-regulatory (IL-10) mean CSC numbers in intestine, MLN, spleen, and blood of Gn calves inoculated with HuNoV-HS66 (solid bars) or mock controls (open bars).