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.

Detection of genogroup II (GII) norovirus (NoV) RNA from adult pigs in Japan and Europe and GII NoV antibodies in US swine raises public health concerns about zoonotic transmission of porcine NoVs to humans, although no NoVs have been detected in US swine. To detect porcine NoVs and to investigate their genetic diversity and relatedness to human NoVs, 275 fecal samples from normal US adult swine were screened by reverse transcription–polymerase chain reaction with calicivirus universal primers. Six samples were positive for NoV. Based on sequence analysis of 3 kb on the 3´ end of 5 porcine NoVs, 3 genotypes in GII and a potential recombinant were identified. One genotype of porcine NoVs was genetically and antigenically related to human NoVs and replicated in gnotobiotic pigs. These results raise concerns of whether subclinically infected adult swine may be reservoirs of new human NoVs or if porcine/human GII recombinants could emerge.

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-α]; Th1, interleukin-12 [IL-12] and gamma interferon [IFN-γ]; 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-α and IFN-γ 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-α and IL-10 were elevated acutely in the IC of the HS66-inoculated calves. Compared to controls, at PID 28 higher numbers of IFN-γ and TNF-α 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.

Noroviruses (NoVs) and sapoviruses (SaVs) are emerging enteric pathogens that cause diarrhea in humans and animals. Porcine genogroup II (GII) NoVs replicate in pigs, but their pathogenesis is undefined. The porcine SaV/GIII/Cowden/80/US strain causes diarrhea and intestinal lesions in pigs. Recently, genetically diverse porcine NoVs (genotypes 11, 18, and 19 within GII) and SaVs comprising at least two genogroups (GIII and GVI?/JJ681-like) and two unclassified strains (G?/QW19 and G?/LL26) were identified; however, their prevalence has not been reported. To investigate the prevalence of porcine NoVs and SaVs, 621 fecal samples were collected from swine of various ages from seven swine farms and one slaughterhouse in three states in the United States. Fecal samples were tested by reverse transcription-PCR and microwell hybridization assays with porcine NoV- and SaV-specific primers and probes, respectively. Porcine GII NoVs were detected exclusively from finisher pigs with an overall prevalence of 20%. Porcine GIII SaVs were detected in 62% of pigs, with the highest prevalence in postweaning pigs and lowest in nursing pigs. Porcine GVI?/JJ681-like SaVs and the G?/QW19-like SaVs were detected infrequently in pigs. The G?/LL26-like SaVs were detected mainly in younger pigs. Because some porcine NoVs and SaVs are genetically or antigenically related to human strains and recombinants within NoVs or SaVs occur for human and pig strains, the high prevalence and subclinical infection rate of these viruses in pigs raise questions of whether pigs may be reservoirs for human strains or for the emergence of new human and porcine recombinants.

A human norovirus genogroup II.4 strain HS66 (HuNoV-HS66) infects and causes mild diarrhea in gnotobiotic (Gn) pigs (S. Cheetham, M. Souza, T. Meulia, S. Grimes, M. G. Han, and L. J. Saif, J. Virol. 80:10372-10381, 2006). In this study we evaluated systemic and intestinal humoral and cellular immune responses to HuNoV-HS66 in orally inoculated pigs. Antibodies and type I interferon (IFN-I or IFN-α), proinflammatory interleukin-6 (IL-6), Th1 (IL-12 and IFN-γ), Th2 (IL-4), and Th2/regulatory T ([T_reg] IL-10) cytokine profiles in serum and intestinal contents (IC) of the HuNoV-HS66-inoculated pigs and controls were assessed by enzyme-linked immunosorbent assay at selected postinoculation days (0 to 28). Using an enzyme-linked immunospot assay, we evaluated immunoglobulin M (IgM), IgA, and IgG antibody-secreting cells (ASC) and cytokine-secreting cells (CSC) in intestine, spleen, and blood. In the HuNoV-inoculated pigs, antibody titers in serum and IC were generally low, and 65% seroconverted. Pigs with higher diarrhea scores were more likely to seroconvert and developed higher intestinal IgA and IgG antibody titers. The numbers of IgA and IgG ASC were higher systemically than in the gut. In serum, HuNoV induced persistently higher Th1 (low transient IFN-γ and high IL-12) than the other cytokines, but also low Th2 (IL-4) and Th2/T_reg(IL-10) levels; low, transient proinflammatory (IL-6) cytokines; and, notably, a delayed IFN-α response. In contrast, intestinal innate (IFN-α early and late) and Th1 (IL-12 late) cytokines were significantly elevated postinfection. HuNoV-HS66 also elicited higher numbers of Th1 (IL-12 and IFN-γ) CSC than Th2 (IL-4) and proinflammatory (IL-6) CSC, with the latter responses low in blood and intestine, reflecting low intestinal inflammation in the absence of gut lesions. These data provide insights into the kinetics of cytokine secretion in serum and IC of HuNoV-inoculated Gn pigs and new information on intestinal humoral and cellular immune responses to HuNoV that are difficult to assess in human volunteers.

Histo-blood group antigen (HBGA) phenotypes have been associated with susceptibility to human noroviruses (HuNoVs). Our aims were: (i) to determine the patterns of A/H HBGA expression in buccal and intestinal tissues of gnotobiotic (Gn) pigs; (ii) to determine if virus-like particles (VLPs) of HuNoV genogroup I (GI) and GII bind to A- or H-type tissues; (iii) to compare A/H expression and VLP binding patterns and confirm their binding specificities by blocking assays; (iv) to develop a hemagglutination inhibition test using buccal cells from live pigs to determine the Gn pig's A/H phenotype and to match viral strains with previously determined HuNoV VLP binding specificities; and (v) to determine the A/H phenotypes and compare these data to the infection outcomes of a previous study of 65 Gn pigs inoculated with HuNoV GII/4 strain HS66 and expressing A and/or H or neither antigen on their buccal and intestinal tissues (S. Cheetham, M. Souza, T. Meulia, S. Grimes, M. G. Han, and L. J. Saif, J. Virol. 80:10372-10381, 2006). We found that the HuNoV GI/GII VLPs of different clusters bound to tissues from four pigs tested (two A⁺and two H⁺). The GI/1 and GII/4 VLPs bound extensively to duodenal and buccal tissues from either A⁺or H⁺pigs, but surprisingly, GII/1 and GII/3 VLPs bound minimally to the duodenum of an A⁺pig. The VLP binding was partially inhibited by A-, H1-, or H2-specific monoclonal antibodies, but was completely blocked by porcine mucin. Comparing the A/H phenotypes of 65 HS66-inoculated Gn pigs from our previous study, we found that significantly more A⁺and H⁺pigs (51%) than non-A⁺and non-H⁺pigs (12.5%) shed virus. From the 22 convalescent pigs, significantly more A⁺or H⁺pigs (66%) than non-A⁺or H⁺pigs (25%) seroconverted.