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, *   Citation: Park, B.-J.; Ahn, H.-S.; Han, S.-H.; Go, H.-J.; Kim, D.-H.; Choi, C.; Jung, S.; Myoung, J.; Lee, J.-B.; Park, S.-Y.; et al. Analysis of the Immune Responses in the Ileum of Gnotobiotic Pigs Infected with the Recombinant GII.p12_GII.3 Human Norovirus by mRNA Sequencing. Viruses 2021, 13, 92. https://doi.org/10.3390/ v13010092 Academic Editor: Lennart Svensson Received: 7 December 2020 Accepted: 8 January 2021 Published: 11 January 2021 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- ms in published maps and institutio- nal affiliations. Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, Gwangjin-gu, Seoul 05029, Korea; twilightsd@naver.com (B.-J.P.); heesuob2@naver.com (H.-S.A.); hansh11@naver.com (S.-H.H.); misilseju@naver.com (H.-J.G.); opeean0@naver.com (D.-H.K.); virus@konkuk.ac.kr(J.-B.L.); paseyo@konkuk.ac.kr (S.-Y.P.); songcs@konkuk.ac.kr (C.-S.S.); odssey@konkuk.ac.kr (S.-W.L.) 2 Department of Food and Nutrition, College of Biotechnology and Natural Resources, Chung-Ang University, Anseong, Gyeonggi 17546, Korea; cchoi@cau.ac.kr (C.C.); amazing2257@gmail.com (S.J.) 3 Korea Zoonosis Research Institute, Chonbuk National University, Jeonju, Jeollabuk-do 54896, Korea; jinjong.myoung@jbnu.ac.kr 4 Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Korea; htl3675@konkuk.ac.kr * Correspondence: ischoi@konkuk.ac.kr; 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 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. Keywords: human norovirus; gnotobiotic pig; immune response; high-throughput mRNA sequencing 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 self-limiting acute gastroenteritis characterized by diarrhea, vomiting, stomach pain, and fever [1]. HuNoV infection leads to approximately 20 million illnesses, 60,000 hospitaliza- tions, and 700 deaths annually in the United States [2,3]. NoVs comprise non-enveloped single-stranded positive-sense RNA viruses belonging to the family Caliciviridae [1]. They are putatively classified into ten genogroups and further divided into at least 49 geno- types [4], with NoVs of genogroups I (GI), II (GII), and IV (GIV) representing the main causative agents of most human infections [5]. Viruses 2021, 13, 92. https://doi.org/10.3390/v13010092 https://www.mdpi.com/journal/viruses