Enteric Viruses In Environmental Samples Research Articles

Bioaccumulation Dynamic by Crassostrea gigas Oysters of Viruses That Are Proposed as Surrogates for Enteric Virus Contamination in Environmental Samples.

Oysters are filter-feeders and retain sewage-derived pathogens in their organs or tissues. Since most enteric viruses involved in outbreaks cannot grow in cell culture, studies using viral surrogate models are essential. Some species are proposed as surrogates for enteric viruses in environmental samples, including in bivalve mollusk samples, such as murine norovirus type 1 (MNV-1) and somatic (as φX) or F-specific coliphages (as MS2) bacteriophages. This study evaluated the tissue distribution of viral surrogates for enteric virus contamination after their bioaccumulation by Crassostrea gigas. Oyster tissues were analyzed for the distribution of viral surrogates (MNV-1, φX-174, and MS2) in digestive tissue (DT), gills (GL), and mantle (MT) after 4, 6, and 24h of experimental bioaccumulation. MNV-1 had higher counts at 6h in DT (1.2 × 103 PFU/g), followed by GL and MT (9.5 × 102 and 3.8 × 102 PFU/g, respectively). The bacteriophage φX-174 had a higher concentration in the MT at 4 and 6h (3.0 × 102 PFU/g, in both) and MS2 in the GL after 24h (2.2 × 102 PFU/g). The bioaccumulation pattern of MNV-1 by oysters was similar to the other enteric viruses (more in DT), while that of phages followed distinct patterns from these. Since the MNV-1 is bioaccumulated by C. gigas and is adapted to grow in cell culture, it is an important tool for bioaccumulation and viral inactivation tests in oysters. Although bacteriophage bioaccumulation was not similar to enteric viruses, they can be indicated for viral bioaccumulation analysis, analyzing MT and GL, since they do not bioaccumulate in DT.

Detection of potentially pathogenic enteric viruses in environmental samples from Kenya using the bag-mediated filtration system.

Enteric virus environmental surveillance via a highly sensitive method is critical, as many enteric viruses have low infectious doses and can persist in the environment for extended periods. This study determined the potential of the novel bag-mediated filtration system (BMFS) to recover human enteric viruses and pepper mild mottle virus (PMMoV) from wastewater and wastewater-impacted surface waters, examined PMMoV use as a fecal contamination indicator in Kenya, and identified potential BMFS process controls. From April 2015 to April 2016, BMFS samples were collected from seven sites in Kenya (n = 59). Enteroviruses and PMMoV were detected in 100% of samples, and human adenovirus, human astrovirus, hepatitis A virus, norovirus GI, norovirus GII, sapovirus, and human rotavirus were detected in the majority of samples. The consistent detection of enteroviruses and PMMoV suggests that these viruses could be used as indicators in similarly fecally contaminated sites and BMFS process controls. As contamination of surface water sources remains a global issue, enteric virus environmental surveillance is necessary. This study demonstrates an effective way to sample large volumes of wastewater and wastewater-impacted surface waters for the detection of multiple enteric viruses simultaneously.

Evaluation of Two Triplex One-Step qRT-PCR Assays for the Quantification of Human Enteric Viruses in Environmental Samples

Human enteric viruses are responsible for waterborne and shellfish-associated disease outbreaks worldwide. Quantitative reverse transcription PCR (qRT-PCR) is often used to assess the health risks associated with shellfish and environmental water, but viral titres in sediments are less commonly investigated. In this study, we developed and validated two multiplex qRT-PCR assays for aquatic sediment and shellfish samples targeting viruses that are a common cause of gastroenteritis (norovirus GI, GII and hepatitis A virus), two emerging viruses (sapovirus and hepatitis E virus), along with mengovirus (MgV), which is often used as a sample process control for the assessment of RNA extraction efficiency. Singleplex and multiplex assays demonstrated comparable PCR efficiencies and gave reliable results over a wide concentration range. The multiplex assays showed remarkable sensitivity with a limit of detection of 1 RNA copy/µL nucleic acid extract for all target viruses and limits of quantification of 3–18 RNA copies/µL for the targeted human pathogenic viruses and 20–40 RNA copies/µL for MgV. The results demonstrated the veracity of multiplex qRT-PCR for the estimation of viral titres in sediment and shellfish, allowing the rapid assessment of viral infection risks associated with environments exposed to wastewater contamination.

Adsorption characteristics of an enteric virus-binding protein to norovirus, rotavirus and poliovirus.

BackgroundWater contamination with human enteric viruses has posed human health risks all over the world. Reasonable and facile methodologies for recovering and quantifying infectious enteric viruses in environmental samples are needed to address the issues of waterborne viral infectious diseases. In this study, a bacterial protein that has a binding capability with several enteric viruses is discovered, and its binding characteristics were investigated for utilizing it as a viral adsorbent in virus recovery and detection technologies.ResultsA gene of an enteric virus-binding protein (EVBP), derived from a monomer of a bacterial chaperon protein GroEL, was successfully acquired from a genomic DNA library of activated sludge microorganisms with nested PCR. Equilibrium dissociation constants between EVBP and norovirus-like particles (NoVLPs) of genotypes GI.7 and GII.4, estimated with quartz crystal microbalance method, were 240 and 210 nM, respectively. These values of equilibrium dissociation constant imply that the binding affinity between EVBP and NoVLPs is 1 to 3-log weaker than that in general antigen-antibody interactions, but about 2-log stronger than that in weak specific interactions of proteins with cations and organic polymers. The adsorptions of EVBP to norovirus, group A rotavirus and poliovirus type 1 were found to be significant in enzyme-linked immunosorbent assay. Meanwhile, the binding of native GroEL tetradecamer to viral particles was weaker than that of EVBP, presumably because of a steric hindrance. The small molecule of EVBP could have an advantage in the access to the surface of viral particles with rugged structure.ConclusionsEVBP that has a broad binding spectrum to enteric viruses was newly discovered. The broad binding characteristic of EVBP would allow us to utilize it as a novel adsorbent for detecting diverse enteric viruses in clinical and environmental samples.

Detection and molecular characterization of enteric viruses in environmental samples in Monastir, Tunisia between January 2003 and April 2007

A prospective study was performed to characterize the main human enteric viruses able to persist in sewage samples and in shellfish tissues, and to establish the correlation between environmental strains and viral infantile diarrhoea observed in the same area during the same period. A total of 250 sewage (raw and treated) and 60 shellfish samples were collected between January 2003 and April 2007 in Monastir region, Tunisia. Group A rotavirus (RVA) was detected in 80 (32%) sewage samples, norovirus (NoV) in 11 (4·4%) and enteric adenovirus (AdV) in 1 (0·4%). Among 60 shellfish samples collected near sewage effluents, one was contaminated by NoV (1·6%). Our data represent the first documentation in Tunisia, combining gastroenteritis viruses circulating in the environment and in clinical isolates. We observed a correlation between environmental strains and those found in children suffering from gastroenteritis during the same period study. This suggests the existence of a relationship between water contamination and paediatric diarrhoea. Our results address the potential health risks associated with transmission of human enteric viruses through water-related environmental routes. The research findings will aid in elucidating the molecular epidemiology and circulation of enteric viruses in Tunisia and in Africa, where data are rare.

Hepatitis E virus-based evaluation of a virion concentration method and detection of enteric viruses in environmental samples by multiplex nested RT-PCR

The prevalence of enteric viruses in drinking and river water samples collected from Pune, India was assessed. During an outbreak of HEV in a small town near pune, water samples were screened for enteric viruses. The water samples were subjected to adsorption-elution-based virus concentration protocol followed by multiplex nested PCR. Among 64 Mutha river samples, 49 (76.56%) were positive for Hepatitis A Virus, 36 (56.25%) were positive for Rotavirus, 33 (51.56%) were positive for Enterovirus and 16 (25%) were positive for Hepatitis E Virus RNA. Only enterovirus RNA was detected in 2/662 (0.3%) drinking water samples, and the samples from the city's water reservoir tested negative for all four viruses. HEV RNA was detected in three out of four river water samples during HEV outbreak and partial sequences from patients and water sample were identical. The study suggests absence of enteric viruses both in the source and in the purified water samples from Pune city, not allowing evaluation of the purification system and documents high prevalence of enteric viruses in river water, posing threat to the community. The rapid, sensitive and relatively inexpensive protocol developed for virological evaluation of water seems extremely useful and should be adapted for evaluating viral contamination of water for human consumption. This will lead to development of adequate control measures thereby reducing disease burden because of enteric viruses.

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

The advent of the PCR has greatly enhanced our ability to detect human enteric viral pathogens in the environment, including water, municipal wastes, sewage, food, air, and fomites ([2][1], [3][2], [59][3], [69][4], [79][5]). This is especially true for those viruses which do not grow in cell

Integrated cell culture/PCR for detection of enteric viruses in environmental samples.

Recently, an integrated cell culture/polymerase chain reaction (ICC/PCR) technique has been developed for the detection of viruses in environmental samples providing a reliable method for practical analysis and direct monitoring of environmental samples for viral pathogens. CC/PCR allows for detection of infectious viruses in hours to days compared with the days or weeks necessary with cell culture alone. Bacterial indicator organisms are commonly used to evaluate environmental samples with respect to fecal contamination and potential public health impacts. These organisms do not correlate well with the presence of viruses, but a rapid, reliable method was not previously available for direct virus testing. Using ICC/PCR, environmental samples may be directly surveyed for pathogenic viruses, in a timely manner. Direct virus analysis will lead to better assessment of the presence and risk of human enteric viruses in the environment, so that control measures may be developed with true virus occurrence data. The ICC/PCR approach combines two previously applied virus detection methods, conventional cell culture and PCR amplification, utilizing the major advantages and overcoming the major limitations of each methodology when used alone. Cell culture assay is the standard method for the detection of viable human viruses (i.e., poliovirus, coxsackievirus, echovirus, adenovirus, hepatitis A virus, reovirus, and rotavirus) in environmental samples, serving as the method against which all newer technologies are evaluated. Although cell culture is theoretically capable of detecting a single viable virus in relatively large volumes of sample, the time required for confirmed results with conventional cell culture makes it an impractical method for routine monitoring of environmental samples. Furthermore, cell culture does not detect noncytopathogenic viruses (viruses that are viable, infecting cells, and continually spreading to neighboring cells but that do not cause a visible cytopathogenic effect [CPE] on the cell monolayer). Rotavirus and most wild-type hepatitis A viruses (HAV) are infectious to cell cultures but do not produce a clear CPE.

Improvements in the RT-PCR detection of enteric viruses in environmental samples

Current methods for the detection of nucleic acid from enteric viruses in environmental samples usually involve extensive concentration and purification of target viruses followed by RT-PCR amplification using two enzymes, reverse transcriptase and Taq polymerase. We have developed a modified method that improves RT-PCR assays by: (i) the use of an RT-PCR internal standard control RNA to identify potential false negative results caused by inhibition of RT-PCR enzymes; (ii) the use of rTth (Perkin-Elmer, Foster City, CA), a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase in a single-tube, single-buffer, elevated-temperature reaction; and (iii) the use of thermolabile uracil N-glycosylase (HK-UNG) (Epicentre Technologies, Madison, WI) to prevent PCR product carryover contamination. The new method was compared to the traditional two-enzyme, RT-PCR method for detection of Norwalk virus (NV) and hepatitis A virus (HAV) in buffer, stool, clam and oyster samples. The new method was at least as sensitive in NV and HAV detection compared to the traditional two-enzyme method. The internal standard control successfully detected inhibitors to RT-PCR amplification. NV and HAV PCR products generated with dUTP replacing dTTP during amplification were seeded into subsequent samples to test the prevention of PCR product carryover contamination by HK-UNG. The new method successfully eliminated PCR product carryover contamination in contrast to the traditional two-enzyme method. These improvements to viral nucleic acid detection have the potential to improve sensitivity, specificity and confidence in RT-PCR results.

Improvements in the RT-PCR detection of enteric viruses in environmental samples

Current methods for the detection of nucleic acid from enteric viruses in environmental samples usually involve extensive concentration and purification of target viruses followed by RT-PCR amplification using two enzymes, reverse transcriptase and Taq polymerase. We have developed a modified method that improves RT-PCR assays by: (i) the use of an RT-PCR internal standard control RNA to identify potential false negative results caused by inhibition of RT-PCR enzymes; (ii) the use of rTth (Perkin-Elmer, Foster City, CA), a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase in a single-tube, single-buffer, elevated-temperature reaction; and (iii) the use of thermolabile uracil N-glycosylase (HK-UNG) (Epicentre Technologies, Madison, WI) to prevent PCR product carryover contamination. The new method was compared to the traditional two-enzyme, RT-PCR method for detection of Norwalk virus (NV) and hepatitis A virus (HAV) in buffer, stool, clam and oyster samples. The new method was at least as sensitive in NV and HAV detection compared to the traditional two-enzyme method. The internal standard control successfully detected inhibitors to RT-PCR amplification. NV and HAV PCR products generated with dUTP replacing dTTP during amplification were seeded into subsequent samples to test the prevention of PCR product carryover contamination by HK-UNG. The new method successfully eliminated PCR product carryover contamination in contrast to the traditional two-enzyme method. These improvements to viral nucleic acid detection have the potential to improve sensitivity, specificity and confidence in RT-PCR results.

Quantification of poliovirus in seawater and sewage by competitive reverse transcriptase - polymerase chain reaction

Reverse transcriptase-polymerase chain reaction (RT-PCR) has been used extensively to detect enteric viruses in environmental samples. Advantages of RT-PCR include its high detection sensitivity and rapid turn-around time. However, unlike traditional cell culture, RT-PCR has not provided quantitation and infectivity information. In this study, we have developed a quantitative RT-PCR method that can be used to determine the amount of poliovirus RNA in environmental samples. An RNA internal standard for poliovirus RT-PCR was designed and obtained through genetic engineering. Serial dilutions of RNA internal standard templates were amplified with a 5'-carboxyfluorescein-labeled poliovirus downstream primer and a nonlabeled poliovirus upstream primer in the RT-PCR. The fluorescent light intensity of labeled RT-PCR products was quantified using an ABI DNA sequencer with GeneScan software. The internal standard was coamplified with poliovirus in the RT-PCR, allowing for enumeration of the poliovirus RNA present in the seawater and sewage samples. This method, using a cloned internal standard and specified primers in the PCR, may be applied to quantify other microorganisms in environmental samples. Although quantitative RT-PCR has begun to be used more extensively for detecting pathogens in clinical samples, the complex nature of many environmental samples has limited the sample range of the effectiveness of quantitative RT-PCR.

Quantification of poliovirus in seawater and sewage by competitive reverse transcriptase - polymerase chain reaction

Reverse transcriptase-polymerase chain reaction (RT-PCR) has been used extensively to detect enteric viruses in environmental samples. Advantages of RT-PCR include its high detection sensitivity and rapid turn-around time. However, unlike traditional cell culture, RT-PCR has not provided quantitation and infectivity information. In this study, we have developed a quantitative RT-PCR method that can be used to determine the amount of poliovirus RNA in environmental samples. An RNA internal standard for poliovirus RT-PCR was designed and obtained through genetic engineering. Serial dilutions of RNA internal standard templates were amplified with a 5'-carboxyfluorescein-labeled poliovirus downstream primer and a nonlabeled poliovirus upstream primer in the RT-PCR. The fluorescent light intensity of labeled RT-PCR products was quantified using an ABI DNA sequencer with GeneScan software. The internal standard was coamplified with poliovirus in the RT-PCR, allowing for enumeration of the poliovirus RNA present in the seawater and sewage samples. This method, using a cloned internal standard and specified primers in the PCR, may be applied to quantify other microorganisms in environmental samples. Although quantitative RT-PCR has begun to be used more extensively for detecting pathogens in clinical samples, the complex nature of many environmental samples has limited the sample range of the effectiveness of quantitative RT-PCR.

Detection of Fastidious Infectious Enteric Viruses in Water

A procedure based on the infection of CaCo-2 cells and molecular hybridization with specific cDNA probes has been developed forthe detection of infectious fastidious enteric viruses in environmental samples. CaCo-2 cells, derived from a human colon adenocarcinoma, showed an increased sensitivity when compared to the usual routine host cell line to laboratory strains of rotavirus 3, reovirus 3, astrovirus 1, poliovirus 1, coxsackievirus A 24, enterovirus 70, and adenovirus 5, 40, and 41. Using this methodology, wild-type rotaviruses, enteric adenoviruses, enteroviruses, and for the first time, astroviruses have been detected in freshwater samples. Direct dot-blot hybridization alone was not sufficient for virus detection from environmental samples. CaCo-2 cells may be used as a universal in vivo amplification system for human enteric viruses, enabling the specific monitoring of infectious viral agents in the environment.

Detection and enumeration of enteric viruses in cell culture

Abstract The use of cell cultures remains the most reliable and functional tool for the detection of viable human enteric viruses in environmental samples. There is, however, a definite need for uniformity in cell culture practices. Among the serious concerns repeatedly addressed for virus monitoring have been negative results arising, not from the absence of virus, but from the lack of sensitivity due to significant variations in detection methodology. Over the past 5 years, many cell culture procedures applied to environmental virology have been upgraded, refined, or replaced; thus, the continued use of insensitive methodology may hinder reliable virus monitoring programs. This report provides a singular updated resource, encompassing those changes which have been shown to make a difference in the sensitivity of the cell culture virus detection system.

A method for the enumeration of poliovirus in selected molluscan shellfish

A virus extraction procedure was developed and evaluated on five commercially important molluscan shellfish species: Crassostrea virginica (Eastern oyster), Mya arenaria (softshell clam), Mytilus edulis (blue mussel), Mercenaria mercenaria (hardshell clam), and Crassostrea gigas, (Pacific oyster). Shellfish tissue homogenates were spiked with poliovirus, extracted, and plaque assayed. Mean virus recoveries were: C. virginica, 63.8%; M. arenaria, 42.1%; M. edulis, 67.3%; M. mercenaria, 48.3%; and C. gigas 10.1%. Shellfish were also allowed to accumulate poliovirus from spiked seawater (10 to 20 PFU/ml of water) over 48 to 72 h. The results indicate that poliovirus could be extracted from four shellfish species exposed to near environmental levels of virus. Virus recoveries per gram of tissue were: M. arenaria, 11.7 PFU; M. mercenaria, 26.0 PFU; M. edulis, 21.5 PFU; and C. virginica, 2.0 PFU. The results of this study indicate that the procedure is effective in extracting ingested viruses from several shellfish species. This procedure may have practical application for enumeration of enteric viruses in environmental samples.