Application of PCR-Based Methods To Assess the Infectivity of Enteric Viruses in Environmental Samples

Rodríguez, Roberto; Pepper, Ian L.; Gerba, Charles P.

doi:10.1128/aem.01150-08

Cited by 174 publications

(164 citation statements)

References 90 publications

(101 reference statements)

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“…Various approaches to overcome this limitation have been evaluated. Of them, integrated systems based on the molecular detection of viruses after cell culture infection are the most promising techniques (Pintó et al 1994;Reynolds et al 2001); a detailed overview of these approaches can be found elsewhere (Rodriguez et al 2009). The integrate cell culture (ICC)-PCR assay is based on a selective enumeration of infectious viruses in combination with a rapid molecular detection, circumventing long incubation periods for cytopathic effect formation.…”

Section: Virus Detectionmentioning

confidence: 99%

Analytical Methods for Virus Detection in Water and Food

et al. 2010

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Potential ways to address the issues that relate to the techniques for analyzing food and environmental samples for the presence of enteric viruses are discussed. It is not the authors' remit to produce or recommend standard or reference methods but to address specific issues in the analytical procedures. Foods of primary importance are bivalve molluscs, particularly, oysters, clams, and mussels; salad crops such as lettuce, green onions and other greens; and soft fruits such as raspberries and strawberries. All types of water, not only drinking water but also recreational water (fresh, marine, and swimming pool), river water (irrigation water), raw and treated sewage are potential vehicles for virus transmission. Well over 100 different enteric viruses could be food or water contaminants; however, with few exceptions, most well-characterized foodborne or waterborne viral outbreaks are restricted to hepatitis A virus (HAV) and calicivirus, essentially norovirus (NoV). Target viruses for analytical methods include, in addition to NoV and HAV, hepatitis E virus (HEV), enteroviruses (e.g., poliovirus), adenovirus, rotavirus, astrovirus, and any other relevant virus likely to be transmitted by food or water. A survey of the currently available methods for detection of viruses in food and environmental matrices was conducted, gathering information on protocols for extraction of viruses from various matrices and on the various specific detection techniques for each virus type.

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Section: Virus Detectionmentioning

confidence: 99%

Analytical Methods for Virus Detection in Water and Food

et al. 2010

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“…Virus detected by PCR may have been rendered noninfectious by natural die-off or by disinfection, and studies which have followed viral nucleic acids and infectivity in water environments have shown that nucleic acids last longer than infectivity (13). Therefore, for investigations of outbreaks and for monitoring recreational and drinking water, there is interest in a rapid approach that can distinguish between infectious and noninfectious virus particles (4,37). Nuanualsuwan and Cliver (28) have demonstrated the feasibility of this type of approach for RNA viruses under certain conditions.…”

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confidence: 99%

Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples

Parshionikar

Laseke

Fout

2010

Appl Environ Microbiol

200

136

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Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72°C, 37°C, and 19°C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivatedby treatment at 72°C and 37°C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19°C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37°C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.

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“…Reverse transcription (RT)-PCR is the most common detection method for NoVs because of its rapidity and high sensitivity (19); however, since it only detects the presence of NoV RNA and cannot distinguish between infectious and noninfectious viral particles, the number of infectious viral units in foods that have been treated is potentially overestimated (20)(21)(22). Attachment to a receptor on a cell surface, the first step of a viral life cycle, is essential to initiate the infection.…”

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confidence: 99%

Inactivation of Human Norovirus in Contaminated Oysters and Clams by High Hydrostatic Pressure

Kingsley

et al. 2014

Appl Environ Microbiol

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cHuman norovirus (NoV) is the most frequent causative agent of food-borne disease associated with shellfish consumption. In this study, the effect of high hydrostatic pressure (HHP) on inactivation of NoV was determined. Genogroup I.1 (GI.1) or genogroup II.4 (GII.4) NoV was inoculated into oyster homogenates and treated at 300 to 600 MPa at 25, 6, and 1°C for 5 min. After HHP, samples were treated with RNase and viral particles were extracted with porcine gastric mucin (PGM)-conjugated magnetic beads (PGM-MBs). Viral RNA was then quantified by real-time reverse transcription (RT)-PCR. Since PGM contains histoblood group-like antigens, which can act as receptors for NoV, deficiency for binding to PGM is an indication of loss of infectivity of NoV. After binding to PGM-MBs, RT-PCR-detectable NoV RNA in oysters was reduced by 0.4 to >4 log 10 by HHP at 300 to 600 MPa. The GI.1 NoV was more resistant to HHP than the GII.4 NoV (P < 0.05). HHP at lower temperatures significantly enhanced the inactivation of NoV in oysters (P < 0.05). Pressure treatment was also conducted for clam homogenates. Treatment at 450 MPa at 1°C achieved a >4 log 10 reduction of GI.1 NoV in both oyster and clam homogenates. It is therefore concluded that HHP could be applied as a potential intervention for inactivating NoV in raw shellfish. The method of pretreatment of samples with RNase, extraction of viral particles using PGM-MB binding, and quantification of viral RNA using RT-PCR can be explored as a practical means of distinguishing between infectious and noninfectious NoV.

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Application of PCR-Based Methods To Assess the Infectivity of Enteric Viruses in Environmental Samples

Cited by 174 publications

References 90 publications

Analytical Methods for Virus Detection in Water and Food

Analytical Methods for Virus Detection in Water and Food

Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples

Inactivation of Human Norovirus in Contaminated Oysters and Clams by High Hydrostatic Pressure

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