Evaluation of the Porcine Gastric Mucin Binding Assay for High-Pressure-Inactivation Studies Using Murine Norovirus and Tulane Virus

Abstract

We compared the results of high-hydrostatic-pressure (HHP) inactivation of murine norovirus type 1 (MNV-1) and Tulane virus (TV) obtained by a porcine gastric mucin binding assay followed by quantitative reverse transcription-PCR (referred to here as the PGM-MB/PCR assay) and a plaque assay and evaluated HHP inactivation of a human norovirus (HuNoV) genogroup I genotype 1 (GI.1) strain and a HuNoV GII.4 strain by using the PGM-MB/PCR assay. Viruses were treated at different pressure levels for 2 min at 4 or 21°C in culture medium of neutral pH and in culture medium of pH 4 at 21°C. The log reductions of infectious MNV-1 and TV particles caused by HHP were assessed using the PGM-MB/PCR and plaque assays, while the log reductions of HuNoVs were assessed by the PGM-MB/PCR assay only. For TV and MNV-1, the two pressure inactivation curves obtained using the plaque and PGM-MB/PCR assays were almost identical at <2-log-reduction levels regardless of the treatment temperature and pH. Further increasing the pressure over the 2-log-reduction level resulted in higher log reductions of TV and MNV-1, as assessed by the plaque assay, but did not increase the log reductions, as assessed by the PGM-MB/PCR assay. HHP treatments could achieve maximum reductions of ϳ3 and 3.5 log units for GI.1 and GII.4, respectively, as assessed by the PGM-MB/PCR assay. On the basis of these results, it can reasonably be concluded that the PGM-MB/PCR assay would very likely be able to estimate HHP inactivation of HuNoV at <2-log-reduction levels. It would also likely conservatively quantify HHP inactivation of the GI.1 strain at 2-to 3-log-reduction levels and the GII.4 strain at 2-to 3.5-log-reduction levels.H uman norovirus (HuNoV) is the leading cause of foodborne illnesses in the United States (1). It has frequently been associated with a variety of ready-to-eat foods, including berries, salsa, and guacamole, and raw shellfish, such as oysters (2-4). Currently, the lack of suitable cell culture systems and practical small-animal models has been hindering research for developing and/or identifying effective processing methods to inactivate HuNoV (1, 5). Therefore, evaluation of the efficacy of processing treatments must rely on HuNoV surrogates. The accuracy of methods that use these surrogates is questionable since direct comparison of methods that use surrogates with methods that use HuNoV is not possible. Molecular biology methods, mostly quantitative reverse transcription-PCR (qRT-PCR), can be used for HuNoV quantification. However, these methods can detect only the presence of HuNoV RNA and cannot distinguish between infectious and noninfectious virus particles.HuNoVs are reported to bind to histo-blood group antigens (HBGAs) in the human intestinal tract, and HBGAs are considered the attachment factors necessary for infection (6, 7). To initiate infection, HuNoVs need to be able to bind to HBGAs to enter the host cells. Porcine gastric mucin (PGM) contains HBGAs and has been shown to be able to bind to genogroup I (GI) and genogroup II...

Effects of High‐Hydrostatic Pressure on Inactivation of Human Norovirus and Physical and Sensory Characteristics of Oysters

“…The 2 pressure inactivation curves for MNV‐1 and TV obtained using the plaque and PGM‐MB/PCR assays were almost identical at less than approximately 2 log 10 reduction levels. We concluded that the PGM‐MB binding assay would be very likely able to correctly quantify HHP inactivation of HuNoV at less than 2 log 10 ‐reduction levels and also likely to correctly or conservatively quantify HHP inactivation of HuNoV at 2 to 3.5 log 10 ‐reduction levels (Li and Chen ). Taking together, the PGM‐MB binding method could potentially reflect the infectivity of HuNoV in oysters after HHP and thus, it was adopted in this study.…”

Human Norovirus Aptamer Exhibits High Degree of Target Conformation-Dependent Binding Similar to That of Receptors and Discriminates Particle Functionality

“…When PGM was used to evaluate the inactivation of GI.1 Norwalk virus by high-pressure processing, reductions corresponded to those observed in a human feeding study with the same virus (11, 12). On the other hand, Li and Chen (9) found that their PGM binding assay correlated with infectivity assay results for Tulane virus and murine norovirus treated with high pressure at a ≤2-log 10 reduction, but the infectious virus titer was overestimated at higher degrees of inactivation. Another recent report found that PGM binding was more conservative than other in vitro infectivity assays for Tulane virus (29).…”

“…One method that is widely used is viral receptor binding, most commonly done with histo-blood group antigens (HBGAs). HBGA (or porcine gastric mucin [PGM], which contains some HBGAs) binding has performed well in comparison with the plaque assay when murine norovirus and Tulane virus surrogates were subjected to a variety of physical and chemical stresses (8–10). In the most compelling work yet, GI.1 Norwalk virus inactivation estimated by a combined PGM binding RT-qPCR method compared favorably to results from a parallel human challenge study evaluating the efficacy of high-pressure processing on norovirus in oysters (11, 12).…”

Detection of Infectious Noroviruses from Wastewater and Seawater Using PEMAXTM Treatment Combined with RT-qPCR

“…In addition, different inactivation mechanisms (UV treatment, chlorination, etc.) should be explored because the effectiveness of photoactivable dye-based methods can provide different infectivity profiles [28].…”