Performing Department
Bioengineering
Non Technical Summary
Human norovirus has been reported to be the leading cause of viral gastroenteritis and foodborne illness worldwide. Each year, norovirus is responsible for almost 20 million illnesses in the United States, which contributes nearly $2.7-3.9 billion in economic losses. Human norovirus is extremely transmissible and can be found in a full range of food products such as leafy vegetables, soft fruits, and shellfish. A variety of analytical methods have been reported to detect emerging viruses, including immunoassays (e.g., ELISA), mass spectrometry-based tests (e.g., MALDI-TOF), nucleic acid-based detection (e.g., reverse transcription- quantitative polymerase chain reaction, RT-qPCR), and sequencing-based techniques. However, these detection methods can provide reliable, sensitive, and specific detectiononly ifhuman noroviruses have been concentrated and purified from complex samples. The lack of practical methods for the concentration and purification of human noroviruses from complex samples remains a hindrance to the successful development of rapid detection assays.
Animal Health Component
40%
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Goals / Objectives
In this proposal, we aim to genetically engineer yeast to display specific virus-binding single-domain antibodies (called nanobodies) with high capture efficiency on the cell surface, facilitating the concentration and purification of human noroviruses in food matrices. The binding nanobodies on yeast surfaces can be programmed to achieve a relatively high capture efficiency and/or serve as anchors to capture different norovirus genogroups. In this study, food-grade yeasts (Saccharomyces cerevisiae,EBY100) will be engineered to display specific virus-binding nanobodies that can specifically bind to human noroviruses. After binding, human noroviruses in complex food samples can be easily concentrated and purified by filtration or centrifugation of the much more massive yeast cells. RT-qPCR will then be used to estimate capture efficiency and detect human norovirus. Herein, thehypothesis of this workis that engineered yeasts can serve as the next generation of foodborne contaminant-specific substrates for the easy concentration and rapid detection of human noroviruses in food samples. This study would not only be a revolutionary novel strategy to detect foodborne viruses but will also be amongthe most efficient and cost-effectivetechniques for norovirus detection.??
Project Methods
Aim 1: Engineer yeast strains displaying various norovirus-binding nanobodies on their cell surfaces and evaluate their ability to bind different human norovirus genogroups.Five nanobodies (two for norovirus GI.1 Norwalk and three for GII.4 Sydney) will be expressed on the surface of food-grade yeast (S. cerevisiae EBY100), respectively. The resulting engineered yeasts (EY-GI and EY-GII) will be tested for their ability to bind norovirus virus-like particles (VLPs) using various methods, including flow cytometry, confocal microscopy, and transmission electron microscopy (TEM). In addition, the detection limit of norovirus VLPs after concentration with EY-GI or EY-GII strains will be quantified using ELISA. Strains expressing nanobodies with the highest affinity for GI.1 Norwalk and GII.4 Sydney will be used in Aim 2.Aim 2: Engineer yeast strains displaying repeating norovirus-binding nanobodies that can capture both the representative human noroviruses (GI.1 Norwalk and GII.4 Sydney).Repeated multimers of nanobodies on yeast surfaces can provide more norovirus-binding sites (i.e., each nanobody unit can bind a separate capsid protein on the same viral particle), increasing their capture efficiency of noroviruses. Nanobodies capable of concentrating GI and GII VLPs from Aim 1 will be used to create multimeric fusions of 2-5 repeated nanobodies separated by linker peptides (EY-GI-Rx and EY-GII-Rx). The norovirus capture efficiency of these nanobody fusions will be tested to identify the best fusion repeat number using flow cytometry, confocal microscopy, TEM, and ELISA. In addition, the optimal nanobody fusions will be simultaneously expressed on the yeast surface (EY-GI/GII-Rx), and their ability to capture mixed norovirus VLPs will be tested.Aim 3: Validate the use of engineered yeasts displaying human norovirus-binding nanobodies to concentrate and detect human noroviruses in human stool and food matrices.Human stool containing infectious human norovirus and real food matrices (raspberry and oyster) will be used to evaluate the detection performance of human noroviruses using engineered yeasts combined with RT-qPCR. Noroviruses in human stool and food matrices will be concentrated using engineered yeasts and quantified using RT-qPCR. The detection sensitivity (as low as 10 genomic copies) and specificity (against various viruses that are enterically or genetically similar to human norovirus) will be investigated. The detection performance will be compared with the conventional technology (antibody-conjugated magnetic beads combined with RT-qPCR). In addition, the degree to which the proposed detection strategy can tolerate inhibitors (pectin and hemocyanin that have been identified to inhibit PCR in foods) will be investigated.