Molecular diagnosis of norovirus and its control by vitamin A

Abstract

Norovirus is known as the pathogenic microorganism that causes gastroenteritis throughout the world, and the cause of more than 90% of nonbacterial enteritis. Recently, according to the Center for Disease Control and Prevention (CDC) of the United States, more than 70% of patients with waterborne enteritis and more than 90% of patients with enteritis by foodborne also has appeared by norovirus. Infection of norovirus usually stops at 2 to 3 day level of acute gastroenteritis, but it can be fatal to the elderly or children, patients with low resistance. Due to the importance of health and sanitation of norovirus, norovirus has been designated as contaminant candidate list (CCL) of Environmental Protection Agency [1] of USA. And, as the importance of norovirus increasingly has become higher since 2007, current infection cases by norovirus are being reported by food poisoning statistical system of the Korea Food and Drug Administration (KFDA). Norovirus as an ssRNA virus of 7.6 kilo-base is known as genetically and immunologically various. So far there are 5 different genogroups, and among them GI, GII, and GIV type of norovirus can cause acute enteritis in humans, and particularly GI and GII type of norovirus are the major genogroups. Despite a very important pathogen in public health, as there are no culture methods or animal models in infecting people by the norovirus, compared to the its importance, molecular base research, especially immunology research has been hardly performed. In doctoral dissertation study, three kinds of the subject have applied to the norovirus. Firstly, this study is a research of genetic detection and diagnosis of norovirus in clinical and environmental samples by developing duplex realtime qRT-PCR. Secondly, this is for immunological detection by manufacturing norovirus virus like particles (VLP) and peptide antibodies. Finally, this study carried out research for norovirus infection control of vitamin A using norovirus replicon bearing cells (HG23 cells), Raw 264.7 cells and murine norovirus and then analyzed the result by microarray assay to study the mechanisms and confirmed the degree of influence of selected genes by realtime RT-PCR. In the first study, the duplex realtime qRT-PCR convenient to detect norovirus that outbreaks across the worldwide, also adopted norovirus sequencing outbreaks in Korea, has been developed to substitute to the clinical and environmental samples, and its excellence was confirmed by comparing its influence of newly developed one to existing conventional RT-PCR set. In the second study, VLP production system of norovirus that has not been cultured in the laboratory was built, and peptide antibodies were produced by using the sequence of parts of the norovirus antigen to promote the understanding of immunological research of norovirus through performing detection experiment. Finally, this study identified control of norovirus replication under the condition of HG23 cells and Raw 264.7 cells bases that were for a research in the molecular base against restraint of surrogates of norovirus infection by vitamin A confirmed in the clinical test and examined its mechanism by identifying inducible genes through microarray assay. Through the doctoral thesis, an overall understanding of norovirus that has not been cultured in the lab yet has been promoted and provides very useful research results that can be linked by genetically and immunologically various approaches to norovirus in the future.