viruses Article Analysis of the Immune Responses in the Ileum of Gnotobiotic Pigs Infected with the Recombinant GII.p12_GII.3 Human Norovirus by mRNA Sequencing Byung-Joo Park 1, Hee-Seop Ahn 1, Sang-Hoon Han 1, Hyeon-Jeong Go 1, Dong-Hwi Kim 1, Changsun Choi 2 , Soontag Jung 2 , Jinjong Myoung 3, Joong-Bok Lee 1, Seung-Yong Park 1, Chang-Seon Song 1 , Sang-Won Lee 1, Hoon-Taek Lee 4 and In-Soo Choi 1,* 1 Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, Gwangjin-gu, Seoul 05029, Korea; [email protected] (B.-J.P.); [email protected] (H.-S.A.); [email protected] (S.-H.H.); [email protected] (H.-J.G.); [email protected] (D.-H.K.); [email protected] (J.-B.L.); [email protected] (S.-Y.P.); [email protected] (C.-S.S.); [email protected] (S.-W.L.) 2 Department of Food and Nutrition, College of Biotechnology and Natural Resources, Chung-Ang University, Anseong, Gyeonggi 17546, Korea; [email protected] (C.C.); [email protected] (S.J.) 3 Korea Zoonosis Research Institute, Chonbuk National University, Jeonju, Jeollabuk-do 54896, Korea; [email protected] 4 Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-2049-6228 Abstract: 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 Citation: Park, B.-J.; Ahn, H.-S.; Han, remain poorly understood. Recently, mRNA sequencing analysis (RNA-Seq) has been used to S.-H.; Go, H.-J.; Kim, D.-H.; Choi, C.; identify cellular responses to viruses. In this study, we investigated the host immune response Jung, S.; Myoung, J.; Lee, J.-B.; Park, S.-Y.; et al. Analysis of the Immune and possible mechanisms involved in virus evasion in the ileum of gnotobiotic pigs infected with Responses in the Ileum of Gnotobiotic HuNoV by RNA-Seq. HuNoV was detected in the feces, blood, and tissues of the jejunum, ileum, Pigs Infected with the Recombinant colon, mesenteric lymph node, and spleen of pigs infected with HuNoV. In analysis of mRNA GII.p12_GII.3 Human Norovirus by sequencing, expression of anti-viral protein genes such as OAS1, MX1, and MX2 were largely mRNA Sequencing. Viruses 2021, 13, decreased, whereas type I IFN was increased in pigs infected with HuNoV. In addition, expression of 92. https://doi.org/10.3390/ TNF and associated anti-inflammatory cytokine genes such as IL10 was increased in HuNoV-infected v13010092 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 Academic Editor: Lennart Svensson genes were further confirmed by qRT-PCR and Western blot. These results suggest that infection with Received: 7 December 2020 HuNoV induces NK cell-mediated cytotoxicity but suppresses type I IFN- and CD8+ T cell-mediated Accepted: 8 January 2021 antiviral responses. Published: 11 January 2021 Keywords: human norovirus; gnotobiotic pig; immune response; high-throughput mRNA sequencing Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- ms in published maps and institutio- nal affiliations. 1. Introduction Norovirus (NoV) constitutes an important pathogen of food-borne illnesses in humans. Human norovirus (HuNoV) is transmitted by the fecal–oral route and generally causes Copyright: © 2021 by the authors. Li- self-limiting acute gastroenteritis characterized by diarrhea, vomiting, stomach pain, and censee MDPI, Basel, Switzerland. fever [1]. HuNoV infection leads to approximately 20 million illnesses, 60,000 hospitaliza- This article is an open access article tions, and 700 deaths annually in the United States [2,3]. NoVs comprise non-enveloped distributed under the terms and con- single-stranded positive-sense RNA viruses belonging to the family Caliciviridae [1]. They ditions of the Creative Commons At- tribution (CC BY) license (https:// are putatively classified into ten genogroups and further divided into at least 49 geno- creativecommons.org/licenses/by/ types [4], with NoVs of genogroups I (GI), II (GII), and IV (GIV) representing the main 4.0/). causative agents of most human infections [5]. Viruses 2021, 13, 92. https://doi.org/10.3390/v13010092 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 92 2 of 21 However, although the clinical features of NoV gastroenteritis are well established, the target cells of HuNoV infection and associated host immune responses are poorly understood because of previous difficulties in identifying suitable cell culture systems and establishing animal models [6]. Initially, human B cells were proposed to constitute a major target of HuNoV, with reproductive viral replication being demonstrated in these cells [7]. Human enterocytes, macrophages, T cells, and dendritic cells (DCs) of the small intestine have also been suggested as HuNoV targets [8]. In addition, the production of T helper type 1 (Th1) cytokines and acute chemokines is observed in patients infected with HuNoV [9]. Gnotobiotic pigs have been used as an alternative animal model to study the viral pathogenesis and immune responses to HuNoV infection because they harbor the histo- blood group of antigens shown to function as the receptor for the virus [10]. Newborn gnotobiotic pigs infected with HuNoV demonstrate mild diarrhea, virus replication, and histopathologic lesions in the small intestinal tract [11]. Post-weaning gnotobiotic pigs are also used to study viral pathogenesis of HuNoV infection and target tissues of HuNoV in the pig [12,13]. Those studies identified experimental infectious doses 50 of HuNoV, such as 2.74 × 103 and 6.43 × 104 genomic equivalent (GE) copies in neonatal piglets and 33–34 days old pigs, respectively. They also found that the administration of simvastatin into pigs produced the increased infectivity of HuNoV. Notably, gnotobiotic pigs infected with HuNoV produce high levels of Th1 cytokines, albeit low levels of Th2 and proinflammatory cytokines, similar to that observed in human patients [9,14]. Additionally, a longer period of viral shedding is exhibited in RAG2/IL2RG-deficient pigs, indicating the importance of lymphocyte-mediated adaptive immune responses for elimination of HuNoV [15]. Understanding of NoV viral pathogenesis and immune responses has been expanded since the discovery of murine norovirus (MuNoV) [16]. Whereas HuNoV only induces acute infection in immunocompetent individuals, different MuNoV strains may cause persistent or acute infection in wild-type mice [17,18]. MuNoV induces systemic infec- tion, which leads to dissemination of the virus to various tissues, such as the intestine, spleen, liver, and lung [19]. Moreover, infection by MuNoV has been identified in intestinal macrophages, DCs, B lymphocytes, and T lymphocytes as well [16,20–22]. Although wild- type mice infected with MNV do not experience diarrhea, severe diarrhea is induced in interferon-deficient mice [19]. MNV infection is also controlled by the innate and adaptive immune systems [23–25]. Recently, a gene expression profiling study was conducted via a high-throughput mRNA sequencing (RNA-Seq) method to determine up- and downreg- ulated genes in MuNoV-infected mouse macrophage cell lines [26]. However, it remains unclear how the innate and adaptive immune systems are induced or suppressed in the small intestine during the acute period of HuNoV or MuNoV infection. In this study, we ascertained the comprehensive immune responses induced in the small intestine of gnotobiotic pigs that had been infected with HuNoV. The mRNA expres- sion profiles of the ileum were investigated by using high-throughput sequencing and confirmed by quantitative reverse-transcription-PCR (qRT-PCR) and Western blotting anal- yses. Generally, infection with HuNoV appeared to induce the activation of natural killer (NK) cells and suppression of CD8+ T lymphocytes. This new information, provided in this study, will substantively contribute to an improved understanding of NoV pathogenesis and immunology. 2. Materials and Methods 2.1. Virus Preparation HuNoV strain CAU140599 (GenBank accession no. MN199033), HNV GII.p12_GII.3, obtained from a patient suffering from acute gastroenteritis, was used as the infectious agent [27,28]. The virus stock was diluted 10 fold with minimum essential medium (Wellgene, Gyeongsan, Korea). The diluted solutions were centrifuged at 3000× g for 30 min and the supernatant was collected. RNA was extracted from the supernatant using Viruses 2021, 13, 92 3 of 21 a QIAamp viral RNA extraction kit (Qiagen, Valencia, CA, USA), and the viral RNA was stored at −70 ◦C. 2.2. Animals and Experimental Design A total of 15, four-week-old post-weaning gnotobiotic pigs were provided by the Bio-organ Research Center, Konkuk University, Seoul, Korea. The experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC), Konkuk University, Korea (approval number KU14073). The pigs were acclimatized for 3 days at a biosecurity level 2 grade facility by providing commercial feed prior to conducting experiments. All infection experiments were performed at the biosecurity level 2 grade- facility located at the College of Veterinary Medicine, Konkuk University. Pigs were divided into two experimental groups: a mock-infected negative control group and a HuNoV-infected group. Five pigs were used as negative controls. Ten pigs were infected with 1 × 107 genome equivalent (GE) copies of HuNoV. Blood and rectal swab samples were collected every day for 3 days after viral infection. All pigs were euthanized at day 3 with potassium chloride after administering zolazepam. Gross pathogenic legions of the small and large intestines, mesenteric lymph nodes, and spleen were examined. The collected organs were stored at –70 ◦C and fixed in 4% formaldehyde. 2.3. Preparation of Fecal and Serum Samples The rectal swab samples collected from the gnotobiotic pigs were diluted 1:10 (w/v) with phosphate-buffered saline (PBS).