AEM Accepts, published online ahead of print on 23 October 2009

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Appl. Environ. Microbiol. doi:10.1128/AEM.01986-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Development of a Fluorescent in situ Method for Visualization of Enteric Viruses

Helen Rawsthorne^*, Trevor G Phister, and Lee-Ann Jaykus

Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695

^* To whom correspondence should be addressed. Email: hrawsth{at}ncsu.edu.

Studying the interactions between enteric pathogens and their environment is important in improving our understanding of their persistence and transmission. However, this remains challenging in large part because of difficulties associated with tracking pathogens in their natural environment(s). In this study, we report a fluorescent labeling strategy which was applied to murine norovirus (MNV-1), a human norovirus surrogate and hepatitis A virus (HAV). Specifically, streptavidin-labeled Quantum dots (Q-Dots) were bound to biotinylated capsids of MNV-1 and HAV (bio-MNV-1 and bio-HAV) which was confirmed using a sandwich- type approach in which streptavidin-bound plates were reacted with biotinylated virus followed by a secondary binding to Q-Dots 655. The assay demonstrated a relative fluorescence of 528 ± 48.1 and 112 ± 8.6 for bio-MNV-1 and control MNV-1, respectively. The biotinylation process did not impact virus infectivity nor did it interfere with the interactions between the virus and host cells or model produce items. Using fluorescent microscopy, it was possible to visualize both bio-HAV and bio-MNV-1 attached to the surface of permissive mammalian cells and green onion tissue. The method provides a powerful tool for the labeling and detection of enteric viruses (and their surrogates) which can be used to track virus behavior in situ.