Studies On Genome Characterization Of Norovirus GⅡ Epidemic Strains And Evolution Mechanisms

Noroviruses (NoVs) are regarded as the major cause for acute non-bacterial diarrhea.NoVs was first reported in1968, and GⅡ.4NoV became the pandemic strain all over theworld in the mid-1990s. In the new century, new variant strains appeared every two or threeyears and caused large epidemics, which were known as “Gastroenteritis flu”. In recent years,NoV outbreaks became more frequent in China, which proposed a more urgent requirementon anti-NoV studies. But currently there is no suitable in vitro replication system and infectedmodel, increasing the difficulty of NoV researches. The development of virus detectiontechnology, especially the application of PCR in NoV detection, promoted the developmentfor understanding NoV propagation and its distribution. Meanwhile, it’s also contributed tothe accumulation of NoV genome sequences using PCR technology, which not only helped tounderstand the characteristics of NoV genome, but also promoted to study the mechanisms ofNoV evolution. A hypothesis was put forward recently that the host immune response to viralinfection and human blood group antigens as binding receptors were the two key factors forNoV evolution.The accumulation of NoV sequences is the an important tool for studies on mechanismof NoV evolution, so this thesis focuses on the development of NoV detection technology, thesurvey NoV infection for sporadic diarrhea, genome sequences obtained from the NoV strainsdetected during the survey, and phylogenetic analysis of viral sequences. Specific contents asfollows:1. Technology standardization of NoV detection. Based on the constructed universalDNA/RNA quality control, different types of RT-PCR methods with different primers forNoV detection were optimized and evaluated, and the correspondence of the detection limitsof these methods were also established, which could be used for selection of appropriateddetection methods in actual work as a reference. Besides, different methods for NoV RNAextraction were compared, of which the membrane purification kit showed the best quality;meanwhile, the viral recovery efficiency of different pre-treatment methods, which based onmembrane chromatography, ultrafiltration,and EIA plate, respectively, were also evaluated.2. Survey of NoVs associated with sporadic gastroenteritis during winter in Guangzhou.The genotypes of circulating norovirus strains associated with sporadic diarrhea cases inGuangzhou from November2010to January2011were characterized. Among fecalspecimens collected from89patients with acute diarrhea,9samples (10.11%) werenorovirus-positive and32samples (35.96%) were rotavirus-positive. The partial polymerase and the capsid regions of these norovirus samples were sequenced and phylogeneticallyanalyzed. Three genotypes (GⅡ.4, GⅡ.6, and GⅡ.b/GⅡ.3) were identified, among which GⅡ.4was the most predominant genotype (5/9), followed by GⅡ.6(3/9). A GⅡ.b/GⅡ.3recombinantstrain was also detected.3. Clone and characterization of GⅡ NoV genomes. A simple and novel method for GⅡNoV genome clone with generic primers was established, and three genome sequences ofNoV epidemic strains in South China were obtained. Meanwhile, based on the GⅡ NoVgenome sequences collected from GenBank, the homology and phylogenetic diversity ofdifferent viral protein among NoV genome was analyzed, which showed p22, P2, and VP2asthe most variable regions, and coevolutionary phenomena of VP1and VP2may existed.4. Evolutionary analysis of GⅡ.4NoV capsid protein. All available GⅡ.4NoV capsidprotein VP1sequences were collected from GenBank. Based on multiple sequence alignment,informative sites on the viral capsid were screened and their locations on viral capsid werealso analyzed. Then, the structural differences of P protein of different GⅡ.4NoV epidemicstrains were compared based on homology modeling, and the variation of surface area of eachkey informative site was also calculated to understand the rules of amino acid usage. Finally,six epitopes located on viral capsid were predicted.Virus detection and sequence accumulation are two important tools for NoV research.So, our study was designed around this central topic, including the development of virusdetection, the survey of virus infection, clone and analysis of GⅡ NoV genome sequences,and the evolutionary mechanisms of GⅡ.4NoV capsid. High prevalence and easy-variationcharacteristics raised an urgent claim for NoV researches, and the results of this study wouldhelp to predict NoV evolutionary trend and develop effective vaccines and therapeuticmethods.