Analysis of the Immune Responses in the Ileum of Gnotobiotic Pigs Infected with the Recombinant GII.p12_GII.3 Human Norovirus by mRNA Sequencing

Norovirus genogroup II (NoV GII) induces acute gastrointestinal food-borne illness in humans. Because gnotobiotic pigs can be infected with human norovirus (HuNoV) GII, they are frequently used to analyze the associated pathogenic mechanisms and immune responses, which remain poorly understood. Recently, mRNA sequencing analysis (RNA-Seq) has been used to identify cellular responses to viruses. In this study, we investigated the host immune response and possible mechanisms involved in virus evasion in the ileum of gnotobiotic pigs infected with HuNoV by RNA-Seq. HuNoV was detected in the feces, blood, and tissues of the jejunum, ileum, colon, mesenteric lymph node, and spleen of pigs infected with HuNoV. In analysis of mRNA sequencing, expression of anti-viral protein genes such as OAS1, MX1, and MX2 were largely decreased, whereas type I IFN was increased in pigs infected with HuNoV. In addition, expression of TNF and associated anti-inflammatory cytokine genes such as IL10 was increased in HuNoV-infected pigs. Expression of genes related to natural killer (NK) cell cytotoxicity and CD8+ T cell exhaustion was increased, whereas that of MHC class I genes was decreased. Expression profiles of selected genes were further confirmed by qRT-PCR and Western blot. These results suggest that infection with HuNoV induces NK cell-mediated cytotoxicity but suppresses type I IFN- and CD8+ T cell-mediated antiviral responses.

Evaluation of the 50% Infectious Dose of Human Norovirus Cin-2 in Gnotobiotic Pigs: A Comparison of Classical and Contemporary Methods for Endpoint Estimation

Human noroviruses (HuNoVs) are the leading causative agents of epidemic and sporadic acute gastroenteritis that affect people of all ages worldwide. However, very few dose–response studies have been carried out to determine the median infectious dose of HuNoVs. In this study, we evaluated the median infectious dose (ID50) and diarrhea dose (DD50) of the GII.4/2003 variant of HuNoV (Cin-2) in the gnotobiotic pig model of HuNoV infection and disease. Using various mathematical approaches (Reed–Muench, Dragstedt–Behrens, Spearman–Karber, logistic regression, and exponential and approximate beta-Poisson dose–response models), we estimated the ID50 and DD50 to be between 2400–3400 RNA copies, and 21,000–38,000 RNA copies, respectively. Contemporary dose–response models offer greater flexibility and accuracy in estimating ID50. In contrast to classical methods of endpoint estimation, dose–response modelling allows seamless analyses of data that may include inconsistent dilution factors between doses or numbers of subjects per dose group, or small numbers of subjects. Although this investigation is consistent with state-of-the-art ID50 determinations and offers an advancement in clinical data analysis, it is important to underscore that such analyses remain confounded by pathogen aggregation. Regardless, challenging virus strain ID50 determination is crucial for identifying the true infectiousness of HuNoVs and for the accurate evaluation of protective efficacies in pre-clinical studies of therapeutics, vaccines and other prophylactics using this reliable animal model.

Infection Dynamics of Hepatitis E Virus in Wild-Type and Immunoglobulin Heavy Chain Knockout JH−/−Gnotobiotic Piglets

ABSTRACTHepatitis E virus (HEV), the causative agent of hepatitis E, is an important but incompletely understood pathogen causing high mortality during pregnancy and leading to chronic hepatitis in immunocompromised individuals. The underlying mechanisms leading to hepatic damage remain unknown; however, the humoral immune response is implicated. In this study, immunoglobulin (Ig) heavy chain JH−/−knockout gnotobiotic pigs were generated using CRISPR/Cas9 technology to deplete the B-lymphocyte population, resulting in an inability to generate a humoral immune response to genotype 3 HEV infection. Compared to wild-type gnotobiotic piglets, the frequencies of B lymphocytes in the Ig heavy chain JH−/−knockouts were significantly lower, despite similar levels of other innate and adaptive T-lymphocyte cell populations. The dynamic of acute HEV infection was subsequently determined in heavy chain JH−/−knockout and wild-type gnotobiotic pigs. The data showed that wild-type piglets had higher viral RNA loads in feces and sera compared to the JH−/−knockout pigs, suggesting that the Ig heavy chain JH−/−knockout in pigs actually decreased the level of HEV replication. Both HEV-infected wild-type and JH−/−knockout gnotobiotic piglets developed more pronounced lymphoplasmacytic hepatitis and hepatocellular necrosis lesions than other studies with conventional pigs. The HEV-infected JH−/−knockout pigs also had significantly enlarged livers both grossly and as a ratio of liver/body weight compared to phosphate-buffered saline-inoculated groups. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems.IMPORTANCEAccording to the World Health Organization, approximately 20 million HEV infections occur annually, resulting in 3.3 million cases of hepatitis E and >44,000 deaths. The lack of an efficient animal model that can mimic the full-spectrum of infection outcomes hinders our ability to delineate the mechanism of HEV pathogenesis. Here, we successfully generated immunoglobulin heavy chain JH−/−knockout gnotobiotic pigs using CRISPR/Cas9 technology, established a novel JH−/−knockout and wild-type gnotobiotic pig model for HEV, and systematically determined the dynamic of acute HEV infection in gnotobiotic pigs. It was demonstrated that knockout of the Ig heavy chain in pigs decreased the level of HEV replication. Infected wild-type and JH−/−knockout gnotobiotic piglets developed more pronounced HEV-specific lesions than other studies using conventional pigs, and the infected JH−/−knockout pigs had significantly enlarged livers. The availability of this novel model will facilitate future studies of HEV pathogenicity.

Protein Malnutrition Alters Tryptophan and Angiotensin-Converting Enzyme 2 Homeostasis and Adaptive Immune Responses in Human Rotavirus-Infected Gnotobiotic Pigs with Human Infant Fecal Microbiota Transplant

ABSTRACT Malnutrition leads to increased morbidity and is evident in almost half of all deaths in children under the age of 5 years. Mortality due to rotavirus diarrhea is common in developing countries where malnutrition is prevalent; however, the relationship between malnutrition and rotavirus infection remains unclear. In this study, gnotobiotic pigs transplanted with the fecal microbiota of a healthy 2-month-old infant were fed protein-sufficient or -deficient diets and infected with virulent human rotavirus (HRV). After human rotavirus infection, protein-deficient pigs had decreased human rotavirus antibody titers and total IgA concentrations, systemic T helper (CD3+ CD4+) and cytotoxic T (CD3+ CD8+) lymphocyte frequencies, and serum tryptophan and angiotensin I-converting enzyme 2. Additionally, deficient-diet pigs had impaired tryptophan catabolism postinfection compared with sufficient-diet pigs. Tryptophan supplementation was tested as an intervention in additional groups of fecal microbiota-transplanted, rotavirus-infected, sufficient- and deficient-diet pigs. Tryptophan supplementation increased the frequencies of regulatory (CD4+ or CD8+ CD25+ FoxP3+) T cells in pigs on both the sufficient and the deficient diets. These results suggest that a protein-deficient diet impairs activation of the adaptive immune response following HRV infection and alters tryptophan homeostasis.