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Application of genetically engineered (GE) large animals carrying multi-allelic modifications has been hampered by low efficiency in production and extended gestation period compared to rodents. Here, we rapidly generated RAG2/IL2RG double knockout pigs using direct injection of CRISPR/Cas9 system into developing embryos. RAG2/IL2RG deficient pigs were immunodeficient, characterized by depletion of lymphocytes and either absence of or structurally abnormal immune organs. Pigs were maintained in gnotobiotic facility and evaluated for human norovirus (HuNoV) infection. HuNoV shedding lasted for 16 days in wild type pigs, compared to 27 days (until the end of trials) in RAG2/IL2RG deficient pigs. Additionally, higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs and the importance of lymphocytes in HuNoV clearance. These results suggest that GE immunodeficient gnotobiotic pigs serve as a novel model for biomedical research and will facilitate HuNoV studies.

Human noroviruses (HuNoVs) are the leading cause of epidemic gastroenteritis worldwide. Study of HuNoV biology has been hampered by the lack of an efficient cell culture system. Recently, enteric commensal bacteria Enterobacter cloacae has been recognized as a helper in HuNoV infection of B cells in vitro. To test the influences of E. cloacae on HuNoV infectivity and to determine whether HuNoV infects B cells in vivo, we colonized gnotobiotic pigs with E. cloacae and inoculated pigs with 2.74 × 10(4) genome copies of HuNoV. Compared to control pigs, reduced HuNoV shedding was observed in E. cloacae colonized pigs, characterized by significantly shorter duration of shedding in post-inoculation day 10 subgroup and lower cumulative shedding and peak shedding in individual pigs. Colonization of E. cloacae also reduced HuNoV titers in intestinal tissues and in blood. In both control and E. cloacae colonized pigs, HuNoV infection of enterocytes was confirmed, however infection of B cells was not observed in ileum, and the entire lamina propria in sections of duodenum, jejunum, and ileum were HuNoV-negative. In summary, E. cloacae inhibited HuNoV infectivity, and B cells were not a target cell type for HuNoV in gnotobiotic pigs, with or without E. cloacae colonization.

Previously, we showed that rice bran (RB) was able to reduce human rotavirus (HRV) diarrhea in gnotobiotic pigs. Here, we investigated its effect on the growth of diarrhea-reducing probiotic Lactobacillus rhamnosus GG (LGG) and Escherichia coli Nissle (EcN), and the resulting effects on HRV diarrhea, gut epithelial health, permeability and innate immune responses during virulent HRV challenge. On 3, 5, and 7 days of age pigs were inoculated with 2 × 10(4) colony-forming-units LGG+EcN to initiate colonization. Daily RB supplementation (replacing 10% calorie intake) was started at 5 days of age and continued until euthanasia. A subset of pigs in each group was challenged orally with 10(5) focus-forming-units of virulent HRV at 33 days of age. RB completely prevented HRV diarrhea in LGG+EcN colonized pigs. RB significantly promoted the growth of both probiotic strains in the gut (~5 logs) and increased the body-weight-gain at 4-5 weeks of age compared to non-RB group. After HRV challenge, RB-fed pigs had significantly lower ileal mitotic index and villus width, and significantly increased intestinal IFN-γ and total IgA levels compared to non-RB group. Therefore, RB plus LGG+EcN colonization may represent a highly effective therapeutic approach against HRV and potentially a variety of other diarrhea-inducing enteric pathogens.