Binding of Escherichia coli Does Not Protect Tulane Virus from Heat-Inactivation Regardless the Expression of HBGA-Like Molecules.

Qianqian Li,David Yang,Peng Tian,Lei Shan,Dapeng Wang

doi:10.3389/fmicb.2017.01746

Abstract

Histo-blood group antigens (HBGAs) are considered as receptors/co-receptors for human norovirus (HuNoV). It has been reported that binding of HuNoV-derived virus-like particles (VLPs) to HBGA-like molecules-expressing bacteria increased the stability of VLPs to heat-denaturation (HD). In this study, we tested for HBGA-like-binding-conveyed protection against HD on viral replication using Tulane virus (TV) and Escherichia coli O86:H2 (O86:H2), with E. coli K-12 (K-12) used as a control. Expression of HBGA type B was confirmed by ELISA in O86:H2 but not in K-12. Binding of TV was confirmed by ELISA in O86:H2 (P/N = 2.23) but not in K-12 (P/N = 1.90). Pre-incubation of TV with free HBGA could completely inhibit its ability to bind to O86:H2 (p = 0.004), while producing no significant change in its ability to bind K-12 (p = 0.635). We utilized a bacterial-capture-RT-qPCR procedure to confirm that both bacterial strains were capable of binding TV, and that O86:H2 exhibited fivefold greater binding capacity than K-12. Pre-incubation of TV with free HBGA would partially inhibit the binding of TV to O86:H2 (p = 0.047). In contrast, not only did pre-incubation of TV with free HBGA not inhibit the binding of TV to K-12, binding was slightly enhanced (p = 0.13). The viral infectivity assay allowed us to conduct a direct evaluation of the ability of HBGA-like-bound bacteria to confer HD protection to TV. Prior to inoculate to LLC-MK2 cells, TV was incubated with each bacterial strain at ratios of 1:0, 1:1 and 100:1, then both partially and fully HD. The viral amplification was quantitated by RT-qPCR 48 h later. The binding of bacteria to TV reduced viral replication in a dose-dependent matter. We found that neither bound O86:H2 nor K-12 conferred protection of TV against partial or full HD conditions. Partial HD reduction of viral replication was not significantly impacted by the binding of either bacterial strain, with infectivity losses of 99.03, 99.42, 96.32, 96.10, and 98.88% for TV w/o bacteria, TV w/O86:H2 (1:1), TV w/O86:H2 (100:1), TV w/K-12 (1:1), and TV w/K-12 (100:1), respectively. Full HD reduction of viral replication was not impacted by the binding of either bacterial strain, as full loss of infectivity was observed in all cases.

Highlights

Human noroviruses (HuNoVs) are the major cause of outbreaks of acute non-bacterial gastroenteritis
No other HBGAlike molecules were detected in E. coli O86:H2 and K-12 by using Monoclonal antibodies (MAbs) listed in Section “Materials and Methods”
histo-blood group antigens (HBGAs)-like moieties are present on the surface of some enteric bacteria (Springer et al, 1961; Yi et al, 2006; Miura et al, 2013; Li et al, 2015)

Summary

Introduction

Human noroviruses (HuNoVs) are the major cause of outbreaks of acute non-bacterial gastroenteritis. HuNoVs are difficult to grow in cell culture, and culturable viruses such as Tulane virus (TV), murine norovirus (MNV), and feline calicivirus (FCV) are often utilized as surrogates for studying the fundamental biology of HuNoV, such as viral replication pattern, and mechanism of infection (Cannon et al, 2006; Wobus et al, 2006; Farkas et al, 2008; Tian et al, 2013). HuNoVs were found to interact with cell-surfacedisplayed HBGAs, and were thought to be important for viral infection (Harrington et al, 2002; Huang et al, 2005). Surrogate viruses bind to molecules other than HBGAs. MNV-1 and FCV bind to sialic acid (Stuart and Brown, 2007; Taube et al, 2009)

Methods

Results

Conclusion