epa-method-1615-measurement-enterovirus-norovirus-occurrence-water

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

U.S Environmental Protection Agency

U.S. Environmental Protection Agency

In February 2001, episodes of acute gastroenteritis were reported to the Wyoming Department of Health from persons who had recently vacationed at a snowmobile lodge in Wyoming. A retrospective cohort study found a significant association between water consumption and illness, and testing identified Norwalk-like virus (NLV) in 8 of 13 stool samples and 1 well. Nucleotide sequences from the positive well-water specimen and 6 of the positive stool samples were identical. This multistrain NLV outbreak investigation illustrates the importance of NLV as a cause of waterborne illness and should encourage monitoring for NLVs in drinking water.

A waterborne outbreak of Norwalk-like virus among snowmobilers-Wyoming, 2001.

Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries. Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water. We have developed and optimized a reverse transcription - polymerase chain reaction (RT-PCR) method that was able to amplify all eight astrovirus serotypes in a single reaction. In addition, a positive control construct was designed so that any inhibitors of this astrovirus assay could be detected. The assay was adapted for use in a real-time PCR assay and the sensitivity of these two methods was compared. The real-time assay was then combined with CaCo2 cell culture to produce an integrated cell culture/RT-PCR (ICC/RT-PCR) assay that was able to detect low levels of astrovirus after an incubation of 7 days or less. Also, the sensitivity of the ICC/RT-PCR assay was compared with RT-PCR alone. The methods were used to detect astrovirus in acute phase illness stool samples as well as in a water sample spiked with astrovirus.

Development of an astrovirus RT-PCR detection assay for use with conventional, real-time, and integrated cell culture/RT-PCR.

Electropositively charged filters are frequently used for concentrating enteric viruses from large volumes of water. A major disadvantage to the use of these filters, however, is that they are not cost-effective. At US$ 150-180 per filter, routine viral monitoring of water is cost-prohibitive. This study describes the development of a method which allows a filter to be used up to three times, achieving comparable recoveries to new filters. Zetapor 1MDS and N66 Posidyne electropositive filters were tested. The method was analyzed using tap water and Ohio River water that was spiked with poliovirus. Tap water recoveries averaged 32% for new filters, 30% for filters used twice, and 38% for filters used three times. River water recoveries averaged 68% for new filters, 83% for filters used twice, and 100% for filters used three times. RT-PCR and dot-blot hybridization were performed on sample concentrates to ensure that all viral nucleic acid from the previous test had been removed from the filters by the treatment process.

Evaluation of a method to re-use electropositive cartridge filters for concentrating viruses from tap and river water.

A survey of enteric viruses and indicator bacteria was carried out in eight community water supply sources (four wells and four springs) in East Tennessee. Seven sites derived their water from carbonate aquifers and one from fractured sandstone. Four of the sites were deemed "low-risk" based on prior monitoring of fecal indicators and factors such as presence of thick layers of overlying sediments. The remaining sites were deemed "high-risk." Enteric viruses (enterovirus and reovirus) were detected by cell culture at least once in seven of the eight wells or springs including all but one of the four low-risk sites. Viral RNA, however, was not detected in any of the samples by reverse transcription-polymerase chain reaction. Conventional indicators of microbial contamination (Escherichia coli and total coliform bacteria) were detected together with culturable viruses in seven of nine virus positive samples. Bacteroides, an alternative fecal indicator which has not previously been used in groundwater investigations, was also detected in all but one of the samples containing E. coli or total coliform bacteria, as well as in one sample where viruses were present in the absence of other bacterial indicators. The study highlights some of the challenges involved in surveys of virus occurrence and indicates that culturable enteric viruses in East Tennessee karst aquifers may be more widespread than previously observed in studies of karst aquifers in Pennsylvania (8%), the Ozark region of Missouri (< 1%), or several other states covered in a national microbial water quality survey conducted by the U.S. Environmental Protection Agency (43%).

Viruses and bacteria in karst and fractured rock aquifers in East Tennessee, USA.

The effective recovery of adenovirus from water is a critical first step in developing a virus occurrence method able to provide accurate data for risk assessments and other applications. During virus concentration, electropositive filters are typically eluted with beef extract, undergo secondary concentration using either an organic flocculation or polyethylene glycol (PEG) precipitation technique and are ultimately resuspended in sodium phosphate buffer. In this study, an alternative secondary concentration procedure using celite was optimized by identifying the optimal celite and elution buffer to use. Two elution buffers, sodium phosphate and 1× PBS, were evaluated for their impact on real-time PCR. Sodium phosphate produced high levels of PCR inhibition compared to 1× PBS and so 1× PBS was used in subsequent experiments. The two secondary concentration techniques that were tested with adenovirus 40 and 41 gave recoveries of 69% and 65% for the optimized celite method and 75% and 109% for the organic flocculation method, respectively. Fine particle, calcinated celites in combination with 1× PBS elution buffer were shown to be effective at concentrating adenovirus 40 and 41 during secondary concentration and their subsequent detection using PCR. Heat extraction efficiencies were compared to samples processed using a DNA extraction kit to address possible virus aggregation issues. Samples processed through DNA extraction were found to produce realistic adenovirus recoveries compared to exaggerated recoveries using heat extraction.

Evaluation of the celite secondary concentration procedure and an alternate elution buffer for the recovery of enteric adenoviruses 40 and 41.

The U.S. EPA developed a sample concentration and preparation assay in conjunction with the total culturable virus assay for concentrating and measuring culturable viruses in source and drinking waters as part of the Information Collection Rule (ICR) promulgated in 1996. In an effort to improve upon this method, the U.S. EPA recently developed Method 1615: Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Method 1615 uses a culturable virus assay with reduced equipment and labor costs compared to the costs associated with the ICR virus method and introduces a new molecular assay for the detection of enteroviruses and noroviruses by reverse transcription-quantitative PCR. In this study, we describe the optimization of several new components of the molecular assay and examine virus recovery from ground, reagent-grade, and surface water samples seeded with poliovirus type 3 and murine norovirus. For the culturable virus and molecular assays, mean poliovirus recovery using the complete method was 58% and 20% in groundwater samples, 122% and 39% using low-titer spikes in reagent-grade water, 42% and 48% using high-titer spikes in reagent-grade water, and 11% and 10% in surface water with high turbidity, respectively. Murine norovirus recovery by the molecular assay was 30% in groundwater samples, less than 8% in both low- and high-titer spikes in reagent-grade water, and 6% in surface water with high turbidity. This study demonstrates the effectiveness of Method 1615 for use with groundwater samples and highlights the need for further research into its effectiveness with surface water.

Development and evaluation of EPA method 1615 for detection of enterovirus and norovirus in water.

Enteroviruses, noroviruses and adenoviruses are among the most common viruses infecting humans worldwide. These viruses are shed in the feces of infected individuals and can accumulate in wastewater, making wastewater a source of a potentially diverse group of enteric viruses. In this study, two procedures were evaluated to concentrate noroviruses, adenoviruses and enteroviruses from primary effluent of wastewater. In the first procedure, indigenous enteroviruses, noroviruses and adenoviruses were concentrated using celite (diatomaceous earth) followed by centrifugation through a 30K MWCO filter and nucleic acid extraction. The second procedure used celite concentration followed by nucleic acid extraction only. Virus quantities were measured using qPCR. A second set of primary effluent samples were seeded with Coxsackievirus A7, Coxsackievirus B1, poliovirus 1 or enterovirus 70 before concentration and processed through both procedures for recovery evaluation of enterovirus species representatives. The pairing of the single step extraction procedure with the celite concentration process resulted in 47-98% recovery of examined viruses, while the celite concentration process plus additional centrifugal concentration before nucleic acid extraction showed reduced recovery (14-47%). The celite concentration process followed by a large volume nucleic acid extraction technique proved to be an effective procedure for recovering these important human pathogens from wastewater.

Evaluation of methods using celite to concentrate norovirus, adenovirus and enterovirus from wastewater.

Practical difficulties of the traditional adenovirus infectivity assay such as intensive labor requirements and longer turnaround period limit the direct use of adenovirus as a testing microorganism for systematic, comprehensive disinfection studies. In this study, we attempted to validate the applicability of integrated cell culture quantitative PCR (ICC-qPCR) as an alternative to the traditional cell culture method with human adenovirus type 2 (HAdV2) in a low-pressure UV disinfection study and to further optimize the procedures of ICC-qPCR for 24-well plate format. The relatively high stability of the hexon gene of HAdV2 was observed after exposure to UV radiation, resulting in a maximum gene copy reduction of 0.5 log10 at 280 mJ cm(-2). Two-day post-inoculation incubation period and a maximum spiking level of 10(5) MPN mL(-1) were selected as optimum conditions of ICC-qPCR with the tested HAdV2. An approximate 1:1 correlation of virus quantities by the traditional and ICC-qPCR cell culture based methods suggested that ICC-qPCR is a satisfactory alternative for practical application in HAdV2 disinfection studies. ICC-qPCR results, coupled with a first-order kinetic model (i.e., the inactivation rate constant of 0.0232 cm(2) mJ(-1)), showed that an UV dose of 172 mJ cm(-2) achieved a 4-log inactivation credit for HAdV2. This estimate is comparable to other studies with HAdV2 and other adenovirus respiratory types. The newly optimized ICC-qPCR shows much promise for further study on its applicability of other slow replicating viruses in disinfection studies.

Applicability of integrated cell culture quantitative PCR (ICC-qPCR) for the detection of infectious adenovirus type 2 in UV disinfection studies.

Recent studies have demonstrated the potential to use Bacillus pumilus endospores as a surrogate for human adenovirus (HAdV) in UV disinfection studies. The use of endospores has been limited by observations of batch-to-batch variation in UV sensitivity. This study reports on a propagation method that utilizes a commercially available medium to produce UV tolerant B. pumilus endospores with a consistent UV sensitivity. It is further demonstrated that the endospores of B. pumilus strain (ATCC 27142), produced using this protocol (half strength Columbia broth, 5 days incubation, with 0.1mM MnSO4), display a UV dose-response that is similar to that of HAdV. Endospore stocks could be stored in ethanol for up to two months at 4 °C without a significant change in UV sensitivity. Synergistic endospore damage was observed by pre-heat treatment of water samples followed by UV irradiation. UV tolerant B. pumilus endospores are a potential surrogate of HAdV for UV treatment performance tests in water utilities which do not have in-house research virology laboratories.

Applicability of UV resistant Bacillus pumilus endospores as a human adenovirus surrogate for evaluating the effectiveness of virus inactivation in low-pressure UV treatment systems.

Cell culture based assays used to detect waterborne viruses typically call for incubating the sample for at least two weeks in order to ensure that all the culturable virus present is detected. Historically, this estimate was based, at least in part, on the length of time used for detecting poliovirus. In this study, we have examined A549 cells infected with human adenovirus type 2, and have found that a three week incubation of virus infected cells results in a higher number of detected viruses by quantal assay than what is seen after two weeks of incubation, with an average 955% increase in Most Probable Number (MPN) from 2 weeks to 3 weeks. This increase suggests that the extended incubation time is essential for accurately estimating viral titer, particularly for slow-growing viruses, UV treated samples, or samples with low titers of virus. In addition, we found that for some UV-treated samples, there was no detectable MPN at 2 weeks, but after 3 weeks, MPN values were obtained. For UV-treated samples, the average increase in MPN from 2 weeks to 3 weeks was 1401%, while untreated samples averaged a change in MPN of 674%, leading us to believe that the UV-damaged viral DNA may be able to be repaired such that viral replication then occurs.

ArticleTotal : 3	Year
Publication title Cited by 26	2015
Publication title Cited by 9	2016
Publication title Cited by 13	2016

Article	Year
A waterborne outbreak of Norwalk-like virus among snowmobilers-Wyoming, 2001. The Journal of infectious diseases\| PubMed ID: 12552455	2003
Development of an astrovirus RT-PCR detection assay for use with conventional, real-time, and integrated cell culture/RT-PCR. Canadian journal of microbiology\| PubMed ID: 15213751	2004
Evaluation of a method to re-use electropositive cartridge filters for concentrating viruses from tap and river water. Journal of virological methods\| PubMed ID: 16194574	2006
Viruses and bacteria in karst and fractured rock aquifers in East Tennessee, USA. Ground water\| PubMed ID: 20331750	2011
Evaluation of the celite secondary concentration procedure and an alternate elution buffer for the recovery of enteric adenoviruses 40 and 41. Journal of virological methods\| PubMed ID: 22138439	2012
Development and evaluation of EPA method 1615 for detection of enterovirus and norovirus in water. Applied and environmental microbiology\| PubMed ID: 23087037	2013
Evaluation of methods using celite to concentrate norovirus, adenovirus and enterovirus from wastewater. Journal of virological methods\| PubMed ID: 23727118	2013
Applicability of integrated cell culture quantitative PCR (ICC-qPCR) for the detection of infectious adenovirus type 2 in UV disinfection studies. Journal of environmental science and health. Part A, Toxic/hazardous substances & environmental engineering\| PubMed ID: 26030683	2015
Applicability of UV resistant Bacillus pumilus endospores as a human adenovirus surrogate for evaluating the effectiveness of virus inactivation in low-pressure UV treatment systems. Journal of microbiological methods\| PubMed ID: 26825005	2016
The Influence of Incubation Time on Adenovirus Quantitation in A549 Cells by Most Probable Number. Journal of virological methods\| PubMed ID: 27596270	2016

Jennifer L. Cashdollar

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