Replication of norovirus in cultured cells

• Main Author: Chia, Pei Shan
• Other Authors: Liu Ding Xiang
• Format: Final Year Project
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/59590
• Institution: Nanyang Technological University

Human Norovirus (NoV), a positive-sense RNA virus, is well-known for causing epidemic gastroenteritis worldwide. Over the past decades, studies on NoV have been restricted due to a lack of appropriate cell culture system. Based on the hypothesis that NoV could be propagated by circumventing the viral entry step, this study aims to deliver the naked NoV genome into different cell lines, in order to develop a reliable cell culture system. However, in the absence of a full length NoV genome, the construction of the full length cDNA genome was firstly conducted. Molecular cloning was selected as the main approach, which involved steps such asrestriction enzyme digestion, ligation and transformation. As a result, NoV nucleotide 1-5456 fragments were successfully isolated and cloned in CMV/pBR322(-) plasmid vector. However, subsequent attempts in joining NoV nucleotide 5421-7559 fragments with the upstream nucleotides were unsuccessful. Challenges had surfaced from the three-part ligations performed, which could lead to various combinations of products, therefore further reducing the cloning efficiency. Besides, the cloned full length genome might be toxic or incompatible to the transformed bacteria, causing deleterious mutation effects or death of cells harboring the genome. In short, this study provides reference for future relevant studies and suggestions for improvement of the strategies employed.