|Jiang, XI - UNIV. CINCINNATI, OHIO|
|Zhong, Weiming - UNIV. CINCINNATI, OHIO|
|Cannonn, J.L. -|
|Burkhardt, William - FDA/CFSAN DAUPHIN IS. AL|
|Woods, Jackie - FDA/CFSAN DAUPHIN IS. AL|
|Hartman, Gary - FDA/ORA SAN FRANCISCO, CA|
|Lindsmithe, L. -|
|Baric, R.L. -|
Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 19, 2010
Publication Date: October 1, 2010
Citation: Tian, P., Yang, D., Jiang, X., Zhong, W., Cannonn, J., Burkhardt, W., Woods, J., Hartman, G., Lindsmithe, L., Baric, R., Mandrell, R.E. 2010. Specificity and kinetics of norovirus binding to magnetic bead- conjugated histo-blood group antigens. Journal of Applied Microbiology. 109:1753-1762. Interpretive Summary: It has been estimated that human norovirus (NOR) cause 23 million cases of gastrointestinal (GI) disease in the United States annually, accounting for 50 to 67% of food-borne illnesses. It has been a challenge to recover virus from food samples as the concentration of virus in produce is usually insufficient for detection by current methods, and is further hampered by the presence of commonly-present environmental RT-PCR inhibitors. Histo-blood group antigens (HBGA) have been identified as candidate receptors for NOR. HBGA-conjugated magnetic beads have been applied as a means to concentrate human NOR. However, kinetics and optional conditions for binding have never been characterized. In this paper, we characterized the kinetics of binding, effect of pH, ionic strength and methods for viral RNA release. The binding of NOR occurs rapidly. Viral RNA can be recovered from HBGA-conjugated magnetic beads after as short as 5 min. of exposure, and recovery is reduced with extended incubation times. Heat-release recovery of viral RNA from HBGA-conjugated magnetic-bead-bound viral particles produces higher yields than the common viral RNA extraction method. Two types of NOR-binding patterns are present. Specific, HBGA-related binding is prominent at neutral pH, while nonspecific charge-related binding is prominent at lower pH. The specific binding effect contributes to concentrate NOR as much as 100 fold. For certain strains of NOR, binding is increased up to 10-fold under acidic (pH 3.0-4.2) binding conditions.
Technical Abstract: Histo-blood group antigens (HBGA) have been identified as candidate receptors for human norovirus (NOR). Type A, type H1, and Lewis histo-blood group antigens (HBGAs) in humans have been identified as major targets for NOR binding. Pig HBGA-conjugated magnetic beads have been utilized as a means to concentrate human NOR. However, binding as a function of conditions and kinetics have never been characterized. In this paper, we characterized the kinetics of binding, effect of pH, ionic strength and methods for viral RNA release. The binding of NOR occurs rapidly. The virus can be recovered 5 min. after expose to the beads. Less NOR is recovered with extended incubation time. Heat-release of viral RNA results in a better recovery than using Tri-reagent. Two types of NOR binding pattern have been identified. Specific HBGA-related binding occurs at neutral pH and nonspecific ionic related binding occurs at lower pH. The specific binding contributes to concentrating NOR as much as 100 fold. The recovery of NOR is further increased up to another 10 fold when the solution is adjusted to acidic conditions (pH 3.0-4.2) for certain strains of NOR.