Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Summary

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

Abstract

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

Introduction

Human noroviruses (HuNoVs) are the leading cause of gastrointestinal illness worldwide, responsible for 685 million infections and over 200,000 deaths each year¹. As with other enteric viruses, the presence of commensal bacteria has been shown to enhance infection of this pathogen as well as its surrogate virus, murine norovirus^2,3. There are also conflicting reports that bacteria may inhibit infection by human norovirus^4,5,6. For several viruses, direct interaction between the virus and bacter....

Protocol

NOTE: The bacterial growth conditions outlined in the protocol are standard culture conditions for Enterobacter cloacae and Lactobacillus gasseri. To perform the virus:bacteria attachment assay with other bacterial species, the chosen bacteria should be cultured under standard conditions appropriate for the bacterium.

1. Preparing Bacterial Growth Medium

1. Enterobacter cloacae growth media
   1. Prepare liquid medium by dissolving 10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride (NaCl) in 1 L of de-ionized (DI) water (see Table of Materials). Mix all media thoroug....

Results

The gating strategies used to quantify human norovirus VLP binding to commensal bacteria are shown in Figure 1. Representative density dot provides an overview of how samples were gated to eliminate cellular debris and cell clumps so VLP attachment was determined on singlet populations (Figure 1A). Representative histograms demonstrate low levels of anti-norovirus antibody signal in bacteria only samples lacking .......

Discussion

The ability to quantify binding of enteric viruses to bacteria is a critical first step for elucidating the mechanisms by which these bacteria alter viral infection. The methods described herein have been optimized to measure human norovirus VLP interactions with both E. cloacae (gram-negative bacterium) and L. gasseri (a gram-positive bacterium), but can be adapted for use with any mammalian virus and bacterium of interest. While VLPs are an ideal alternative to live vi.......

Materials

Name	Company	Catalog Number	Comments
5ml Polystrene Round-Bottom Tubes with Cell-Strainer Cap	Corning	352235	After antibody staining, sample are transferred into tubes for flow cytometry analysis.
Agar	Sigma	A7002	Used for media preparation
AnaeroPack	Thermo Scientific	R681001	Anaerobic gas pack used for culture of Lactobacillus gasseri
BD FacsDiva software
BD LSR Fortessa flow cytometer
Bovine Serum Albumin	Fisher Bioreagents	BP1605	Used for flow cytometry
Flow Cytometry Stain Buffer (FCSB)	BD Biosciences	554657	Used for flow cytometry
Mouse IgG2b kappa Isotype Control (eBMG2b), PE, eBioscience	Thermo Fisher Scientific	12473281	Isotype control. This antibody is purchased in the conjugated form from the manufacturer.
MRS Powder	BD Biosciences	288130	Used for media preparation and to culture Lactobacillus gasseri.
Norovirus capsid G2 Monoclonal Antibody (L34D)	Thermo Fisher Scientific	MA5-18241	Norovirus GII antibody. This antibody is only available in the unconjugated form and thus must be fluorescently conjugated prior to use in the outlined flow cytometry assays. In this protocol, PE was the chosen fluor, however, other fluorescent molecules can be chosen as best suits the flow cytometer being used by the researcher.
Norovirus GII.4 VLP	Creative Biostructure	CBS-V700	human norovirus virus like particle, VLPs were generated using the baculovirus system and resuspended in phosphate buffered saline with 10% glycerol. The authors performed independent nanosight tracking analysis to determine the particle concentration of the VLPs. The concentration is approximately 1011 VLPs per milliliter. Based on the protein concentration of the VLPs, approximately 200 particles are added per bacterium in VLP attachment assays.
PBS 10X	Fisher Bioreagents	BP665	Dilute to 1X prior to use.
SiteClick R-PE Antibody Labeling Kit	Thermo Fisher Scientific	S10467	Conjugation kit used for labling of unconjugated antibody.
Sodium Chloride	Fisher Scientific	S271	Used for media preparation
Tryptone	Oxoid	LP0042	Used for media preparation
Tube Revolver	ThermoFisher Scientific	88881001	Used in virus:bacterium attachment assay. Set to max speed (40 rpm).
Yeast Extract	BD Biosciences	212750	Used for media preparation