Methods to Detect Infectious Human Enteric Viruses in Environmental Water Samples
Department of Hygiene, Social and Environmental Medicine, Ruhr-University Bochum, Germany.International journal of hygiene and environmental health (Impact Factor: 3.83). 09/2011; 214(6):424-36. DOI: 10.1016/j.ijheh.2011.07.014
Currently, a wide range of analytical methods is available for virus detection in environmental water samples. Molecular methods such as polymerase chain reaction (PCR) and quantitative real time PCR (qPCR) have the highest sensitivity and specificity to investigate virus contamination in water, so they are the most commonly used in environmental virology. Despite great sensitivity of PCR, the main limitation is the lack of the correlation between the detected viral genome and viral infectivity, which limits conclusions regarding the significance for public health. To provide information about the infectivity of the detected viruses, cultivation on animal cell culture is the gold standard. However, cell culture infectivity assays are laborious, time consuming and costly. Also, not all viruses are able to produce cytopathic effect and viruses such as human noroviruses have no available cell line for propagation. In this brief review, we present a summary and critical evaluation of different approaches that have been recently proposed to overcome limitations of the traditional cell culture assay and PCR assay such as integrated cell culture-PCR, detection of genome integrity, detection of capsid integrity, and measurement of oxidative damages on viral capsid protein. Techniques for rapid detection of infectious viruses such as fluorescence microscopy and automated flow cytometry have also been suggested to assess virus infectivity in water samples.
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- "The method of choice for the analysis of enteric viruses in the aquatic environment is real-time PCR. However, in comparison to cell culture, no information on the infectivity of the virus is given (Hamza et al. 2011b). In water hygiene, the use of indicator organisms is discussed for years ( Albinana-Gimenez et al. 2006; Bofill-Mas et al. 2006; Hamza et al. 2011a; Noble et al. 2004). "
ABSTRACT: Enteric viruses enter surface waters through discharge of sewage treatment plants. They have a high environmental resistance and persistence and have low infectious doses. The aim of this study was to investigate the efficiency of polishing pond in the removal of viruses and bacteria. The samples were taken approximately once a week at the influent of secondary treatment (n = 39), effluent of secondary treatment (n = 39), and polishing pond (tertiary treatment, n = 29). Human adenoviruses (HAdV) were detected in 82–100 % of wastewater samples, whereas 62–79 % of the samples were positive for human polyomavirus (HPyV). The median concentrations ranged from 6.8 × 103 genome equivalents/l (HAdV) to 6.0 × 103 genome equivalents/l (HPyV). The concentration of HAdV and HPyV did not change significantly during the wastewater treatment. For somatic coliphages and bacteria an overall reduction of 1.84–2.65 log10 has been detected. Based on the data collected, this type of tertiary treatment achieved a significant reduction in bacteria and phages, but not for viruses.
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- "Fruits and vegetables can be contaminated either at the pre-harvest stage through contact with fecally contaminated irrigation water or during harvesting, packaging, processing, or cooking due to poor hand sanitation (Bitler et al., 2013; Kotwal and Cannon, 2014; Mathijs et al., 2012; Rodriguez-Lazaro et al., 2012). Most foodborne viruses are difficult or currently impossible to cultivate (Hamza et al., 2011) and sensitive molecular methods are therefore used to detect them in food and environmental samples. Today, RTqPCR is widely used for virus detection because it is sensitive, specific, rapid and can deliver quantitative data. "
ABSTRACT: Noroviruses (genogroup I (NoV GI) and genogroup II (NoV GII)) and the hepatitis A virus (HAV) are frequently involved in foodborne infections worldwide. They are mainly transmitted via the fecal-oral route, direct person-to-person contact or consumption of contaminated water and foods. In food virology, detection methods are currently based on identifying viral genomes using real-time reverse transcriptase PCR (RT-qPCR). One of the general requirements for detecting these viruses in food involves the use of a process control virus to monitor the quality of the entire viral extraction procedure as described in the ISO/TS 15216-1 and 15216-2 standards published in 2013. The selected process control virus should have similar morphological and physicochemical properties as the screened pathogenic virus and thus have the potential to provide comparable extraction efficiency. The aim of this study was to determine which virus should be used for process control, murine norovirus (MNV-1) or Mengovirus, when testing for the presence of HAV, NoV GI and NoV GII in bottled water, lettuce and semi-dried tomatoes. Food samples were spiked with HAV, NoV GI or NoV GII alone or in the presence of MNV-1 or Mengovirus. Recovery rates of each pathogenic virus were compared to those of both process control viruses using a multiple comparison procedure. Neither process control virus influenced the recovery of pathogenic virus regardless of the type of food matrix. MNV-1 was the most appropriate virus for validating the detection of HAV and NoV GII in all three food matrices as well as NoV GI in lettuce. Mengovirus proved to be the most appropriate control for NoV GI detection in bottled water and semi-dried tomatoes. The process control virus is essential for validating viral detection in food and the choice of virus depends on food type and the screened pathogenic virus. Copyright © 2015 Elsevier B.V. All rights reserved.
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- "The assay is also not specific for particular viruses; therefore, the viral pathogen should be confirmed with other approaches, such as a molecular or immunological assay. In addition, virus propagation in cell culture is time consuming, labor-intensive and expensive; thus, it cannot be used as a routine and robust detection tool (Hamza et al., 2011b). "
ABSTRACT: Despite the great sensitivity of PCR in monitoring enteric viruses in an aquatic environment, PCR detects viral nucleic acids of both infectious and noninfectious viruses, limiting the conclusions regarding significance for public health. Ethidium monoazide (EMA) and propidium monoazide (PMA) are closely related membrane impermeant dyes that selectively penetrate cells with compromised membranes. Inside the cells, the dye can intercalate into nucleic acids and inhibit PCR amplification. To assess whether EMA and PMA pretreatment is a suitable approach to inhibit DNA amplification from noninfectious viruses upon heat treatment, UV exposure or chlorine treatment, viruses were measured by qPCR, EMA-qPCR, PMA-qPCR and cell culture titration. EMA/PMA-qPCR of UV- and heat-treated viruses did not correlate with the results of the cell culture assay. However, the data from EMA/PMA-qPCR of chlorine-inactivated viruses was consistent with the cell culture infectivity assay. Therefore, a dye treatment approach could be a rapid and inexpensive tool to screen the efficacy of chlorine disinfection, but it is not able to distinguish between infectious and noninfectious viruses inactivated via heat treatment or UV irradiation. Indeed, different viruses may have different trends and mechanisms of inactivation; thus, the assay must be evaluated for each virus separately. Copyright © 2015 Elsevier GmbH. All rights reserved.
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