Characterizing Human Norovirus Bacterial Interactions for Exploring Methods to Improve Food and Water Safety

The major goal of this project is to characterize and understand interactions between strains of human norovirus and bacteria and for ulitmate application to the development of improved detection/capture methodologies and intervention strategies to improve overal health and food safety.The first objective of this project is to characterize the molecular nature of interactions between human noroviruses and bacteria. This objective will be acheived by first determining the degree to which endemic and sporadic strains of human norovirus bind to commensal and environmental bacteria. These experiments will then be followed by using optimal attachment conditions in combination with electron microscopy to visually determine the bacterial surface structures to which the viruses bind. These results will be confirmed by generating bacterial mutant strains deficient in the observed attachment structures to confirm loss of norovirus binding. If the virus binds to carbohydrate structures (e.g. lipopolysaccharide, peptidoglycan, lipoteichoic acid, etc), then, as a complimentary approach, competitive inhibition assays will be employed to examine the ability of synthetic versions of these compounds to prevent norovirus-bacterial binding and thereby confirm both visual and mutational experiments.The second objective of this project is to determine the contribution of bacterial interactions on human norovirus stability. Human noroviruses are able to unexplicably survive for weeks up to years on surfaces and environmental samples and is highly resistant to commonly used sanitizers and disinfectants. We propose that bacterial interactions may be responsible for this extreme stability. To test this hypothesis, we will examine the stability of norovirus particles the presence and absence of bacteria and measure the stability using thermal scanning. Conditions that will be explored using this approach include temperature (ranging from -20C to 65C), resistance to disinfectants (using bleach, alcohol and sanitizer treatments), desiccation and humidity (1% - 85%). Understanding the role bacteria play in norovirus stability will provide the foundational knowledge needed to begin development of improved intervention or mitigation technologies to reduce the presence of the virus in soil, water and food.The third objective is to determine the feasibility of exploiting HuNoV-bacterial interactions to reduce viral stability or improve current capture methods. If enhanced stability is observed, we will perform MALDI-Mass Spectrometry to determine if enhanced stability is due to conformational changes in virus structure which are common after virus-receptor interactions. Furthermore, if interactions with bacteria enhance viral stability, we will also determine if removing bacteria reduces/restores viral stability. Some enteric viruses retain their stable phenotype even after bacteria have been removed, therefore it is critical to downstream applications to understand if this same phenotype exists for human noroviruses. To acheive this, first interactions between human nororviruses and bacteria will be disrupted using chloroform and stability studies performed above will be repeated to determine if changes in stability occur. In addition, using the information gathered in objectives 1 and 2 we will explore the possibility of using bacterial ligands to improve current capture methods for human norovirus.