Application of Long-Range and Binding Reverse Transcription-Quantitative PCR To Indicate the Viral Integrities of Noroviruses

Abstract

This study intends to establish and apply methods evaluating both viral capsid and genome integrities of human noroviruses (NoVs), which thus far remain nonculturable. Murine norovirus 1 (MNV-1) and human NoV GII.4 in phosphate-buffered saline suspensions were treated with heat, UV light, or ethanol and detected by reverse transcription-quantitative PCR (RT-qPCR), long-range RT-qPCR, binding RT-qPCR, and binding long-range RT-qPCR. For MNV-1 heated at 60°C for 2 and 30 min, limited reductions of genomic copies (<0.3-log) were obtained by RT-qPCR and long-range RT-qPCR, while the cell-binding pretreatments obtained higher reductions (>1.89-log reduction after 60°C for 30 min by binding long-range RT-qPCR). The human NoV GII.4 was found to be more heat resistant than MNV-1. N oroviruses (NoVs) are a group of important food-borne viruses and the leading cause of human gastroenteritis worldwide (1, 2). Methods for the detection of NoVs have been progressing over a number of years. Due to the inability to culture NoVs in vitro (3, 4), reverse transcription-quantitative PCR (RTqPCR) is still recognized as the gold standard for virus detection, although this method cannot differentiate between infectious and noninfectious viruses (5, 6). Thus far, prediction of NoV infectivity has been attempted from the integrities and/or functions of viral RNA molecules and capsid proteins (5), which are the two essential parts for an intact and infectious virus particle.As for the genome integrity evaluation, although it is possible to amplify nearly full-length human NoV genomes (7), the amplification efficiency decreases with fragment size, thus making amplification of full-length genomic RNA relatively insensitive. Wolf et al. (8) suggested that one important factor for assessing the genomic integrities of RNA viruses is the dependency of the reverse transcription (RT) reaction on high-quality, nonfragmented template RNA. This fact can be exploited by separating the PCR amplification site and RT priming site within the virus genome. This approach has been used to examine the integrity of the human NoV genome after high-temperature (72°C) and UV treatment (8).Multiple studies have been conducted to assess the number of infectious NoVs based on the capsid integrity or function (9-11). Recently, binding-based RT-PCRs were developed in our laboratory and were able to decrease the detection of noninfectious NoVs by approximately 1 to 3 log, whereas all infectious viral particles were detected (12). For murine norovirus 1 (MNV-1), the cell line RAW 264.7 and the ganglioside GD1a were used as binding receptors and, for human NoVs, differentiated Caco-2 cells and pig gastric mucin were tested as the binding receptors (12).Our aim here was to apply these methods (RT-qPCR, longrange RT-qPCR, and binding RT-qPCR) and the combination method (binding long-range RT-qPCR) in order to indicate the viral integrities of NoVs. First, murine norovirus 1 (MNV-1, a surrogate of human NoVs) and human NoV GII.4 were treated in phosphate-buffered salin...

A systematic review of human norovirus survival reveals a greater persistence of human norovirus RT-qPCR signals compared to those of cultivable surrogate viruses

“…It is generally recognized that surrogates and hNoV survive in a variety of raw, processed, and/or stored foods, including berries, herbs, and molluscan shellfish (Baert et al, 2009;Verhaelen et al, 2012;Richards, 2012;Richards et al, 2012;Poschetto et al, 2007;Butot et al, 2008;Li et al, 2014;Sánchez et al, 2011;Lou et al, 2012). Studies have also shown that the food matrix and environmental factors have a significant impact on virus survival (Le Guyader and Atmar, 2008;Sobsey and Meschke, 2003).…”

Persistence and Elimination of Human Norovirus in Food and on Food Contact Surfaces: A Critical Review

“…Using a combination of RNase pretreatment and binding to cultured mammalian cells (Caco-2), with the hypothesis that only undamaged virus particles will bind to the cells, Li et al (45) observed a~1.5log reduction in detectable NoV GII.4 GE at 708C for 2 min and a 2.3-log reduction at 858C for 2 min. Li et al (47) extended their study using a combination of binding to Caco-2 cells with subsequent long-range RT-qPCR. Following heat treatment at 608C for both 2 and 30 min, they observed~3-log reductions in detectable NoV GII.4 GE.…”

“…Dancho et al (16), using the PGM-MB binding assay, observed that treatment with 0.5 JÁcm À2 caused a 1.8-log reduction in NoV GI detectable by RT-PCR, whereas after a treatment with 2 JÁcm À2 , a 3.8-log reduction in binding was observed. Li et al (47) used a combination of binding to Caco-2 cells with subsequent long-range RT-qPCR. Following exposure of NoV GII.4 to 20 mJÁcm À2 and 200 mJÁcm À2 ,~3 log-and .4-log reductions in detectable GE were observed, respectively.…”

“…Researchers have sought methods for predicting infectivity that include ligand ''receptor'' binding combined with RT-qPCR (16) and ''long-range PCR'' to better determine genomic integrity (47,108). Human NoVs are known to bind to histo-blood group antigens (HBGA) and to porcine gastric mucin (16,105).…”

In Situ Capture RT-qPCR: A New Simple and Sensitive Method to Detect Human Norovirus in Oysters

“…So far, RT-PCR and quantitative RT-PCR (RT-qPCR) have been widely used for the detection of HuNoVs (Kageyama et al, 2003; Trujillo et al, 2006; Yu et al, 2015). However, these molecular approaches have limited value in distinguishing infectious viruses from non-infectious viruses or free viral RNA (Li et al, 2014; Wang and Tian, 2014). …”