The influence of temperature, pH, and water immersion on the high hydrostatic pressure inactivation of GI.1 and GII.4 human noroviruses

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Highlights

  • Both GI.1 and GII.4 strains showed increasing pressure sensitivity as sample temperature decreased.

  • Both GI.1 and GII.4 strains were more resistant to pressure at pH 4 than at neutral pH.

  • The GI.1 strain was more resistant to pressure than the GII.4 strain.

  • Presence of water greatly enhanced pressure inactivation of the GI.1 strain.

Abstract

Detection of human norovirus (HuNoV) usually relies on molecular biology techniques, such as qRT-PCR. Since histo-blood group antigens (HBGAs) are the functional receptors for HuNoV, HuNoV can bind to porcine gastric mucin (PGM), which contains HBGA-like antigens. In this study, PGM-conjugated magnetic beads were used to collect and quantify potentially infectious HuNoV strains GI.1 and GII.4 treated by high hydrostatic pressure (HHP). Both GI.1 and GII.4 strains used in this study showed increasing pressure sensitivity as judged by loss of PGM binding with decreasing temperature over a range of 1 to 35 °C. Both GI.1 and GII.4 were more resistant to pressure at pH 4 than at neutral pH. Because GI.1 was significantly more resistant to pressure than GII.4, it was used to evaluate HuNoV pressure inactivation in blueberries. GI.1 on dry blueberries was very resistant to pressure while immersion of blueberries in water during pressure treatments substantially enhanced the inactivation. For example, a 2 min-600 MPa treatment of dry blueberries at 1 and 21 °C resulted in < 1-log reductions while a 2.7-log reduction of GI.1 was achieved by a treatment at 500 MPa for 2 min at 1 °C when blueberries were immersed in water. In total, this novel study provides unique information for designing pressure processing parameters (pressure, temperature, and time) and product formulations (such as pH) to inactivate HuNoV in high-risk foods such as berries.

Introduction

Human norovirus (HuNoV) causes the majority of foodborne illness and is primarily transmitted through the fecal–oral route (Li et al., 2012, Scallan et al., 2011). Foods consumed raw such as berries, have frequently been involved in HuNoV outbreaks because they can be contaminated by irrigation water, liquid fertilizer and pesticide application, or by human harvesters and food handlers (Carter, 2005, Cotterelle et al., 2005, Falkenhorst et al., 2005, Le Guyader et al., 2004, Potera, 2013, Rodriguez-Lazaro et al., 2012). The main difficulty hindering research of HuNoV inactivation method research has been the lack of suitable cell culture systems or practical small animal models (Duizer et al., 2004, Herbst-Kralovetz et al., 2013, Li et al., 2012). Therefore, HuNoV detection mostly depends on molecular methods, such as RT-PCR. However, RT-PCR only detects the presence of HuNoV RNA, and does not provide information about whether the virus from which the RNA was derived was infectious or inactivated. What complicates this issue is the fact that in many cases, viruses inactivated by capsid damage can retain virion integrity, protecting viral RNA from enzymatic degradation by environmentally-ubiquitous RNAses (Diez-Valcarce et al., 2011, Kingsley, 2013).

The obligatory first step for any virus infection is binding to the appropriate cell receptor. The HuNoV receptors are histo-blood group antigens (HBGAs) in the human intestinal tract (Marionneau et al., 2002). Subsequent work by Tian and coworkers showed that norovirus and norovirus-like particles can bind to porcine gastric mucin (PGM) because porcine mucins are chemically- and antigenically-similar to human histo-blood group antigens (Tian et al., 2007, Tian et al., 2008, Tian et al., 2010). Of particular note, it was shown that all GI strains tested, and 85% of GII HuNoVs tested, bound to PGM (Tian et al., 2010). When the porcine gastric mucin was conjugated to magnetic beads (PGM-MB), HuNoV could be extracted and purified from complex food matrices such as fresh produce, salad, and sewage, for subsequent quantification using qRT-PCR (Pan et al., 2012, Tian et al., 2008, Tian et al., 2011, Tian et al., 2012). The utility of the PGM-MB binding followed by qRT-PCR assay for discriminating potentially infectious human norovirus was further demonstrated by Dancho et al. (2012) who showed that thermal, UV, and high hydrostatic pressure (HHP) treatments resulted in loss of PGM-MB binding consistent with inactivation of the virus by damage to its capsid.

HHP is a non-thermal processing technique that has been successfully applied in the food industry for different food products, such as oysters, guacamole, fruit jams, ready-to-eat meats, salsa, and orange juice. Previously, most studies evaluating the potential for HHP to inactivate noroviruses relied on surrogates such as feline calicivirus (FCV), murine norovirus (MNV), and Tulane virus (TV) (Kingsley, 2013, Kovač et al., 2010, Kovač et al., 2012, Li et al., 2013). Until recently, direct evaluation of HuNoV inactivation by HPP has been restricted due to lack of practical assays to assess HuNoV inactivation. One study used human volunteers for direct assessment of HuNoV inactivation, using the GI.1 8FIIb Norwalk strain (Leon et al., 2011). The volunteer study evaluated HHP's potential to inactivate 4-log RT-PCR units within oysters showing that a 5 min-600 MPa treatment at room temperature did protect human volunteers, but a 5 min-400 MPa treatment at room temperature was not protective. A 5-min-400 MPa treatment performed at 6 °C suggested that cooler temperatures might enhance HuNoV inactivation since most volunteers were protected by this treatment.

Recent application of the PGM-MB binding assay to HHP-treated norovirus confirmed that HHP inactivated virus does not bind to PGMs. In Dancho et al. (2012), a GI.1 strain 8FIIa in DMEM plus 10% FBS was treated from 300 to 600 MPa at 5 °C for 5 min. When GI.1 was treated at 400–600 MPa, additional 2–3 log reductions of qRT-PCR detectable RNA with prior PGB-MB binding were noticed compared to qRT-PCR detectable RNA without prior binding, suggesting that PGM-MB binding probably could extract infectious virus particles for subsequent qRT-PCR quantification.

Based on research with HuNoV surrogates FCV, MNV-1, TV and other viruses (Chen et al., 2005, Kingsley, 2013, Li et al., 2013, Lou et al., 2011), the temperature at which HPP is performed, the food matrix pH, and the presence of water would be predicted to affect HuNoV inactivation by HPP. Given the nascent availability of the PGM-MB binding assay, it is now possible to confirm and characterize these effects, as well as to determine if different HuNoV strains responded differently to pressure. The objectives of this study were to investigate the effects of temperature and pH on HHP inactivation of HuNoVs GI.1 and GII.4 strains and the effect of the presence of water on HHP inactivation of GI.1 on blueberries.

Section snippets

HuNoV stock preparation

Fecal suspensions containing a HuNoV GII.4 strain was generously provided by Dr. Xi Jiang at Cincinnati Children's Hospital Medical Center. GI.1 8FIIb norovirus sample was from patient #34-9 of a human volunteer study (Leon et al., 2011). Fecal suspensions were centrifuged at 4000 ×g for 20 min, filtered through a 0.22-μm filter, aliquoted, and stored at − 80 °C until use.

HHP treatment of GI.1 and GII.4 at different temperatures

HuNoV GII.4 and GI.1 stocks were diluted using phosphate-buffered saline (PBS, pH 7.2, KeraFAST, Boston, MA). Each virus (150 μl)

Pressure inactivation of GI.1 and GII.4 at different temperatures

The effect of temperature on HHP inactivation of GI.1 and GII.4 strains is shown in Table 1. The detection limits for GI.1 and GII.4 determined by serial diluted virus samples for temperature and pH studies were 10 3 and 10 4 dilution levels, respectively. When reduction calculated by qRT-PCR results was > 3 logs (GI.1) or > 4 logs (GII.4) or qRT-PCR showed no amplification of the treated sample, the result was considered as below detection limit and the 3-log reduction (GI.1) or 4-log reduction

Discussion

Although a few HuNoV strains have been observed to be unable to bind to PGM (Tian et al., 2010), our results clearly indicat that untreated GI.1 and GII. 4 strains used in this study bound to PGM, and with sufficient pressure application, these strains lost the ability to bind to PGM. It is important to note that circulating GII.4 strains are known to be dynamically-evolving by changing specificity for the HBGA antigen types utilized as receptors to initiate infection (Donaldson et al., 2008).

Acknowledgment

This project was supported by the Agriculture and Food Research Initiative Competitive Grants Program of the USDA National Institute of Food and Agriculture, NIFA Award No: 2011-68003-30005.

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