Methods to detect infectious human enteric viruses in environmental water samples.
ABSTRACT 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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ABSTRACT: It is important to consider viruses in water quality because of their incidence as causal agents for diarrheal disease, andCLEAN - Soil Air Water 04/2013; · 2.05 Impact Factor
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ABSTRACT: In order to provide a more suitable response to public health concerns, we improved the detection of infectious human adenoviruses in water by optimising the commonly used integrated cell culture-PCR method. Risk evaluation studies seek for rapid detection of infectious adenoviruses, including the enteric types 40 and 41 that are considered as the second most common agents of gastroenteritis in children next to rotaviruses. The here-employed 293A cell line used for infectious status assessment showed its ability to multiply adenoviruses including type 41. Two modifications were moreover applied to the workflow for viral detection. The first occurred at the nucleic acid extraction step performed directly on all infected cells, while the second was the application of real-time quantitative PCR as detection tool. All adaptations led to a 3-day reduction of the response delay and an improved sensitivity especially for the enteric adenoviral types. The infectious status of laboratory strain types 2 and 41 was demonstrated by a more than 2-log10 increase in genome quantity. These conclusions were confirmed and reinforced by the analysis of water samples applying the improved assay. Naturally occurring infectious adenoviruses were detected in wastewater and river water, within 2 days. Types belonging to the species human adenoviruses C and type 31 were observed, but the most frequently identified type was 41 (71 % of identified sequences, n = 34). This highlights the usefulness of our method for a wide range of types, and especially for the most prevalent and public health-relevant enteric adenoviruses.Applied Microbiology and Biotechnology 04/2013; · 3.69 Impact Factor
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ABSTRACT: Human enteric viruses are major agents of foodborne diseases. Because of the absence of a reliable cell culture method for most of the enteric viruses involved in outbreaks, real-time reverse transcriptase PCR is now widely used for the detection of RNA viruses in food samples. However this approach detects viral nucleic acids of both infectious and non infectious viruses, which limits the impact of conclusions with regard to public health concern. The aim of the study was to develop a method to discriminate between infectious and non-infectious particles of hepatitis A virus (HAV) and two strains of rotavirus (RV) following thermal inactivation by using intercalating dyes combined with RT-qPCR. Once the binding of propidium monoazide (PMA) or ethidium monoazide (EMA) was shown to be effective on the viral ssRNA of HAV and dsRNA of two strains of RV (SA11 and Wa), their use in conjunction with three surfactants (IGEPAL CA-630, Tween 20, Triton X-100) prior to RT-qPCR assays was evaluated to quantify the infectious particles remaining following heat treatment. The most promising conditions were EMA (20 muM) and IGEPAL CA-630 (0.5%) for HAV, EMA (20 muM) for RV (WA) and PMA (50 muM) for RV (SA11). The effectiveness of the pre-treatment RT-qPCR developed for each virus was evaluated with three RT-qPCR assays (A, B, C) during thermal inactivation kinetics (at 37[degree sign]C, 68 C, 72[degree sign]C, 80[degree sign]C) through comparison with data obtained by RT-qPCR and by infectious titration in cell culture. At 37[degree sign]C, the quantity of virus (RV, HAV) remained constant regardless of the method used. The genomic titers following heat treatment at 68[degree sign]C to 80[degree sign]C became similar to the infectious titers only when a pre-treatment RT-qPCR was used. Moreover, the most effective decrease was obtained by RT-qPCR assay A or B for HAV and RT-qPCR assay B or C for RV. We concluded that effectiveness of the pre-treatment RT-qPCR is influenced by the viral target and by the choice of the RT-qPCR assay. Currently, it would be appropriate to further develop this approach under specific conditions of inactivation for the identification of infectious viruses in food and environmental samples.BMC Microbiology 10/2013; 13(1):216. · 3.10 Impact Factor