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: Molecular amplification using Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) is currently considered as the gold standard to detect enteric human pathogenic viruses such as norovirus and hepatitis A virus in food and water. However, the molecular-based detection requires an adequate sampling strategy and a sample preparation specific for viruses. Sampling for enteric human viruses in water and food should not necessarily follow bacterial sampling plans. The development of a reference detection method including sample preparation as proposed in ISO/TS 15216 represents a milestone to facilitate the evaluation of the performance and eventually validation of future virus detection methods. The potential viral infectivity linked to a positive PCR result is a remaining issue and pretreatments allowing the differentiation of infectious viruses would be useful for future risk assessments.Current opinion in virology. 01/2014; 4C:66-70.
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ABSTRACT: Swine production is an important economic activity in Brazil, and there is interest in the development of clean production mechanisms to support sustainable agro-industrial activities. The biomass derived from swine manure has good potential to be used as a biofertilizer due to its high nutrient concentration. However, the land application of manure should be based on safety parameters such as the presence of pathogens that can potentially infect animals and people. This study was designed to assess the presence of porcine circovirus-2 (PCV2), porcine adenovirus (PAdV), rotavirus-A (RV-A) and Salmonella spp. in liquid manure, as well the infectivity of two genotypes of circovirus-2 (PCV2a and PCV2b) present in liquid manure. Three swine farms were evaluated: 1) a nursery production farm (manure analyzed before and after anaerobic biodigestion), 2) a grow-finish production farm (analyzed before and after anaerobic biodigestion), and 3) a second grow-finish production farm (raw manure-affluent). PCV2, PAdV and RV-A were present before and after anaerobic biodigestion (either affluent or effluent) at all farms. Salmonella spp. were detected at farm 1 (affluent and effluent) and farm 3 (raw manure-affluent) but not farm 2 (affluent and effluent). When the ability of the anaerobic biodigestion process to reduce viral concentration was evaluated, no significant reduction was observed (P>0.05). Both the PCV2a and PCV2b genotypes were detected, suggesting viral co-infection in swine production. The results revealed infectious PCV2 even after anaerobic biodigestion treatment. The presence of Salmonella spp. and enteric viruses, especially infectious PCV2, in the final effluent from the anaerobic biodigester system suggests that the process is inefficient for pathogen inactivation. Due to the prevalence and infectivity of PCV2 and considering the successful use of molecular methods coupled to cell culture for detecting infectious PCV2, we suggest that this virus can be used as a bioindicator in swine manure treatment systems to check the efficiency of pathogen inactivation and ensure the production of safe biofertilizers from swine manure.Science of The Total Environment 01/2014; s 479–480:277–283. · 3.26 Impact Factor
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ABSTRACT: Pathogenic viruses are emerging contaminants in water which should be analyzed for water safety to preserve public health. A strategy was developed to quantify RNA and DNA viruses in parallel on chemiluminescence flow-through oligonucleotide microarrays. In order to show the proof of principle, bacteriophage MS2, ΦX174, and the human pathogenic adenovirus type 2 (hAdV2) were analyzed in spiked tap water samples on the analysis platform MCR 3. The chemiluminescence microarray imaging unit was equipped with a Peltier heater for a controlled heating of the flow cell. The efficiency and selectivity of DNA hybridization could be increased resulting in higher signal intensities and lower cross-reactivities of polymerase chain reaction (PCR) products from other viruses. The total analysis time for DNA/RNA extraction, cDNA synthesis for RNA viruses, polymerase chain reaction, single-strand separation, and oligonucleotide microarray analysis was performed in 4-4.5 h. The parallel quantification was possible in a concentration range of 9.6 × 10(5)-1.4 × 10(10) genomic units (GU)/mL for bacteriophage MS2, 1.4 × 10(5)-3.7 × 10(8) GU/mL for bacteriophage ΦX174, and 6.5 × 10(3)-1.2 × 10(5) for hAdV2, respectively, by using a measuring temperature of 40 °C. Detection limits could be calculated to 6.6 × 10(5) GU/mL for MS2, 5.3 × 10(3) GU/mL for ΦX174, and 1.5 × 10(2) GU/mL for hAdV2, respectively. Real samples of surface water and treated wastewater were tested. Generally, found concentrations of hAdV2, bacteriophage MS2, and ΦX174 were at the detection limit. Nevertheless, bacteriophages could be identified with similar results by means of quantitative PCR and oligonucleotide microarray analysis on the MCR 3.Analytical and Bioanalytical Chemistry 02/2014; · 3.66 Impact Factor