Virion Concentration Method for the Detection of Human Enteric Viruses in Extracts of Hard-Shelled Clams†

1998 ◽  
Vol 61 (4) ◽  
pp. 458-465 ◽  
Author(s):  
ALISSA B. DIX ◽  
LEE-ANN JAYKUS
Keyword(s):  
Cell Culture ◽  
Hepatitis A Virus ◽  
Direct Detection ◽  
Enteric Viruses ◽  
Precipitation Method ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Genomic Amplification ◽  
Initial Inoculum ◽  
Human Enteric Viruses

A method to extract and concentrate intact human enteric viruses from oyster extracts for detection using reverse transcription-polymerase chain reaction (RT-PCR) was applied to hard-shelled clams (Mercenaria mercenaria). Fifty-gram clam samples were processed by an adsorption-elution-precipitation method and then seeded with 101 to 105 PFU of poliovirus 1 (PV1) and/or hepatitis A virus (HAV). Seeded viruses in extracts were purified by fluorocarbon (Freon) extraction and concentrated by polyethylene glycol (PEG) precipitation and elution. Efficiency of virion recovery from PEG precipitates was dependent upon PEG concentration and elution buffer volume, with optimized variables yielding recoveries as high as 99% for PV1 and 45% for FIAV, as evaluated by cell culture infectivity assay. To further concentrate viruses, remove inhibitors, and reduce sample volumes, the protein-precipitating agent Pro-Cipitate was used in an adsorption-elution-precipitation scheme. The final concentrate was of low volume (<1 ml) and directly compatible with viral genomic amplification using RT-PCR. When extracts from 50-g clam samples were seeded and processed by the combined concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 103 PFU for PV1 and HAV. Corresponding virus recoveries based on cell culture infectivity were 7 to 50% and 0.3 to 8% for PV1 and HAV, respectively. When extracts of clams were artificially contaminated with the Norwalk virus, direct detection of virion RNA using RT-PCR and subsequent oligoprobe hybridization was possible at levels as low as 450 RT-PCR amplifiable units of the Norwalk virus per extract of 50-g clam sample.

Detection Methods for Human Enteric Viruses in Representative Foods†

2000 ◽  
Vol 63 (12) ◽  
pp. 1738-1744 ◽  
Author(s):  
PARIS R. LEGGITT ◽  
LEE-ANN JAYKUS
Keyword(s):  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Food Sample ◽  
Viral Rna ◽  
Detection Methods ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Viral Contamination ◽  
Initial Inoculum ◽  
Human Enteric Viruses

Although viral foodborne disease is a significant problem, foods are rarely tested for viral contamination, and when done, testing is limited to shellfish commodities. In this work, we report a method to extract and detect human enteric viruses from alternative food commodities using an elution-concentration approach followed by detection using reverse transcription-polymerase chain reaction (RT-PCR). Fifty-gram lettuce or hamburger samples were artificially inoculated with poliovirus type 1 (PV1), hepatitis A virus (HAV), or the Norwalk virus and processed by the sequential steps of homogenization, filtration, Freon extraction (hamburger), and polyethylene glycol (PEG) precipitation. To reduce volumes further and remove RT-PCR inhibitors, a secondary PEG precipitation was necessary, resulting in an overall 10- to 20-fold sample size reduction from 50 g to 3 to 5 ml. Virus recoveries in secondary PEG concentrates ranged from 10 to 70% for PV1 and 2 to 4% for HAV as evaluated by mammalian cell culture infectivity assay. Total RNA from PEG concentrates was extracted to a small volume (30 to 40 μl) and subjected to RT-PCR amplification of viral RNA sequences. Detection limit studies indicated that viral RNA was consistently detected by RT-PCR at initial inoculum levels ≥102 PFU/50-g food sample for PV1 and ≥103 PFU/50-g food sample for HAV. In similar studies with the Norwalk virus, detection at inoculum levels ≥1.5 × 103 PCR-amplifiable units/50-g sample for both food products was possible. All RT-PCR amplicons were confirmed by subsequent Southern hybridization. The procedure reported represents progress toward the development of methods to detect human enteric viral contamination in foods other than shellfish.

scholarly journals A Multiplex Reverse Transcription-PCR Method for Detection of Human Enteric Viruses in Groundwater

2003 ◽  
Vol 69 (6) ◽  
pp. 3158-3164 ◽  
Author(s):  
G. Shay Fout ◽  
Beth C. Martinson ◽  
Michael W. N. Moyer ◽  
Daniel R. Dahling
Keyword(s):  
Reverse Transcription ◽  
Hepatitis A Virus ◽  
Disease Outbreaks ◽  
Enteric Viruses ◽  
The United States ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Groundwater Samples ◽  
Human Enteric Viruses

ABSTRACT Untreated groundwater is responsible for about half of the waterborne disease outbreaks in the United States. Human enteric viruses are thought to be leading etiological agents of many of these outbreaks, but there is relatively little information on the types and levels of viruses found in groundwater. To address this problem, monthly samples from 29 groundwater sites were analyzed for 1 year for enteroviruses, hepatitis A virus, Norwalk virus, reoviruses, and rotaviruses by multiplex reverse transcription-PCR (RT-PCR). A procedure with which to remove environmental RT-PCR inhibitors from groundwater samples was developed. The procedure allowed an average of 71 liters of the original groundwater to be assayed per RT-PCR, with an average virus recovery rate of 74%, based on seeded samples. Human enteric viruses were detected in 16% of the groundwater samples analyzed, with reoviruses being the most frequently detected virus group.

scholarly journals Three-Year Study To Assess Human Enteric Viruses in Shellfish

2000 ◽  
Vol 66 (8) ◽  
pp. 3241-3248 ◽  
Author(s):  
F. Le Guyader ◽  
L. Haugarreau ◽  
L. Miossec ◽  
E. Dubois ◽  
M. Pommepuy
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Virus Detection ◽  
Enteric Viruses ◽  
Molecular Technique ◽  
Norwalk Virus ◽  
Viral Contamination ◽  
Long Period ◽  
Reverse Transcription Pcr ◽  
Human Enteric Viruses

ABSTRACT The main pathogenic enteric viruses able to persist in the environment, such as hepatitis A virus (HAV), Norwalk-like virus (NLV), enterovirus (EV), rotavirus (RV), and astrovirus (AV), were detected by reverse transcription-PCR and hybridization in shellfish during a 3-year study. Oyster samples (n = 108), occasionally containing bacteria, were less frequently contaminated, showing positivity for AV (17%), NLV (23%), EV (19%), and RV (27%), whereas mussel samples, collected in areas routinely impacted by human sewage, were more highly contaminated: AV (50%), HAV (13%), NLV (35%), EV (45%), and RV (52%). Sequences obtained from HAV and NLV amplicons showed a great variety of strains, especially for NLV (strains close to Mexico, Snow Mountain Agent, or Norwalk virus). Viral contamination was mainly observed during winter months, although there were some seasonal differences among the viruses. This first study of virus detection over a fairly long period of time suggests that routine analysis of shellfish by a molecular technique is feasible.

Development of a Molecular Method for the Detection of Enteric Viruses in Oysters

1995 ◽  
Vol 58 (12) ◽  
pp. 1357-1362 ◽  
Author(s):  
LEE-ANN JAYKUS ◽  
RICARDO DE LEON ◽  
MARK D. SOBSEY
Keyword(s):  
Cell Culture ◽  
Nucleic Acid ◽  
Hepatitis A Virus ◽  
Enteric Virus ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Ammonium Bromide ◽  
Rt Pcr ◽  
Initial Inoculum ◽  
Ctab Precipitation

Detection of enteric virus contamination of shellfish is limited by current methodology, which is time-consuming, tedious, and lacking in sensitivity due to reliance on cell culture infectivity. Alternative detection methods based on nucleic acid amplification have been hampered by high sample volumes and the presence of enzymatic inhibitors. The goal of this study was to develop methods to purify and concentrate intact virions from oyster extracts to a volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase-polymerase chain reaction (RT-PCR). Fifty-gram oyster samples were homogenized and processed by standard adsorption-elution precipitation methodology and then seeded with 105 PFU of poliovirus 1 (PV1) or hepatitis A virus (HAV). Seeded viruses were concentrated by fluorocarbon extraction, polyethylene glycol (PEG) precipitation, chloroform extraction, and cetyltrimethyl ammonium bromide (CTAB) precipitation to a volume of 100 μl with removal of RT-PCR inhibitors. Virus recovery after elution of PEG precipitates was 50% for PVI and IS to 20% for HAV as evaluted by cell culture infectivity. The CTAB precipitation step yielded a concentrated sample which was directly compatible with RT-PCR reactions and capable of detecting about 100 placque=forming units (PFU) of PVl or HAV. When 50-g oyster extracts were seeded and processed by the entire concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 104 PFU of HAV and 103 PFU of PV1, with recoveries of 1 to 5% of seeded viruses.

scholarly journals Incidence of Enteric Viruses in Groundwater from Household Wells in Wisconsin

2003 ◽  
Vol 69 (2) ◽  
pp. 1172-1180 ◽  
Author(s):  
Mark A. Borchardt ◽  
Phil D. Bertz ◽  
Susan K. Spencer ◽  
David A. Battigelli
Keyword(s):  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
The United States ◽  
Land Application ◽  
Private Household ◽  
Septic Systems ◽  
Rt Pcr ◽  
Sequential Samples ◽  
Human Enteric Viruses ◽  
Public Water Systems

ABSTRACT Recent studies on the contamination of groundwater with human enteric viruses have focused on public water systems, whereas little is known about the occurrence of viruses in private household wells. The objective of the present study was to estimate the incidence of viruses in Wisconsin household wells located near septage land application sites or in rural subdivisions served by septic systems. Fifty wells in seven hydrogeologic districts were sampled four times over a year, once each season. Reverse transcriptase PCR (RT-PCR), followed by Southern hybridization, was used to detect enteroviruses, rotavirus, hepatitis A virus (HAV), and Norwalk-like viruses (NLVs). In addition, cell culture was used to detect culturable enteroviruses. Companion water samples were collected for total coliforms, Escherichia coli, fecal enterococci, F-specific RNA coliphages, nitrate, and chloride analyses. Among the 50 wells, four (8%) were positive for viruses by RT-PCR. Three wells were positive for HAV, and the fourth well was positive for both rotavirus and NLV in one sample and an enterovirus in another sample. Contamination was transient, since none of the wells was virus positive for two sequential samples. Culturable enteroviruses were not detected in any of the wells. Water quality indicators were not statistically associated with virus occurrence, although some concordance was noted for chloride. The present study is the first in the United States to systematically monitor private household wells for virus contamination and, combined with data for public wells, provides further insight on the extent of groundwater contamination with human enteric viruses.

Groundwater microbiological quality in Canadian drinking water municipal wells

2008 ◽  
Vol 54 (6) ◽  
pp. 472-478 ◽  
Author(s):  
Annie Locas ◽  
Christine Barthe ◽  
Aaron B. Margolin ◽  
Pierre Payment
Keyword(s):  
Water Quality ◽  
Cell Culture ◽  
Enteric Viruses ◽  
Contaminated Sites ◽  
Raw Water ◽  
Fecal Indicators ◽  
Rt Pcr ◽  
Total Coliforms ◽  
E Coli ◽  
Human Enteric Viruses

To verify previous conclusions on the use of bacterial indicators suggested in regulations and to investigate virological quality of groundwater, a 1-year study was undertaken on groundwater used as a source of drinking water in 3 provinces in Canada. Raw water from 25 municipal wells was sampled during a 1-year period for a total of 167 samples. Twenty-three sites were selected on the basis of their excellent historical bacteriological water quality data, and 2 sites with known bacteriological contamination were selected as positive controls. Water samples were analyzed for general water quality indicators (aerobic endospores, total coliforms), fecal indicators ( Escherichia coli , enterococci, somatic and male-specific coliphages), total culturable human enteric viruses (determined by cell culture and immunoperoxidase), noroviruses (analyzed by reverse-transcriptase – polymerase chain reaction (RT–PCR)), adenovirus types 40 and 41 (analyzed by integrated cell culture (ICC) – PCR), and enteroviruses and reoviruses types 1, 2, and 3 (analyzed by ICC–RT–PCR). General water quality indicators were found very occasionally at the clean sites but were frequently present at the 2 contaminated sites. Only one of 129 samples from the 23 clean sites was positive for enterococci. These results confirm the value of raw water quality historical data to detect source water contamination affecting wells that are vulnerable. Samples from the 2 contaminated sites confirmed the frequent presence of fecal indicators: E. coli was found in 20/38 samples and enterococci in 12/38 samples. Human enteric viruses were not detected by cell culture on MA-104 cells nor by immunoperoxidase detection in any sample from the clean sites but were found at one contaminated site. By ICC–RT–PCR and ICC–PCR, viruses were found by cytopathic effect in one sample from a clean site and they were found in 3 samples from contaminated sites. The viruses were not detected by the molecular methods but were confirmed as picornaviruses by electron microscopy. Noroviruses were not detected in any samples. The results obtained reinforce the value of frequent sampling of raw water using simple parameters: sampling for total coliforms and E. coli remains the best approach to detect contamination of source water by fecal pollutants and accompanying pathogens. The absence of total coliforms at a site appears to be a good indication of the absence of human enteric viruses.

Development of RT-PCR Method to Detect Various Human Enteric Viruses

2009 ◽  
Vol 39 (1) ◽  
pp. 41 ◽  
Author(s):  
Sung Ae Oh ◽  
Mi Suk Kim ◽  
So Young Jang ◽  
Sang Jong Kim ◽  
Jae In Lee ◽  
...  
Keyword(s):  
Enteric Viruses ◽  
Rt Pcr ◽  
Pcr Method ◽  
Human Enteric Viruses

Comparative survival of hepatitis A virus, poliovirus and indicator viruses in geographically diverse seawaters

1995 ◽  
Vol 31 (5-6) ◽  
pp. 189-193 ◽  
Author(s):  
Kathleen M. Callahan ◽  
Douglas J. Taylor ◽  
Mark D. Sobsey
Keyword(s):  
North Carolina ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Health Concern ◽  
Log10 Reduction ◽  
Public Health Concern ◽  
Human Enteric Viruses ◽  
Virus Survival

The presence and persistence of enteric viruses in sewage contaminated seawater is an important public health concern for bathing, surfing and shellfishing. In an effort to find suitable indicators of enteric viruses in seawater, we compared the survival of two groups of enteric bacteriophages, F-specific coliphages (FRNA phages) and somatic Salmonella bacteriophages (SS phages), to the survival of two human enteric viruses, hepatitis A virus (HAV) and poliovirus type 1 (PV-1), in coastal seawater from three geographic areas (So. California, Hawaii, and North Carolina) at 20°C. Concentrations of all four viruses decreased over 30 days from their initial titers and there was little difference in the survival of a particular virus among the three seawaters. However, the extent of reduction varied among the four viruses. Survival was greater for the SS phages than for any of the other viruses, with an estimated 4 log10 reduction time of about 10 weeks. FRNA phages and PV-1 were inactivated rapidly, with 4 log10 reductions in ~ 1 week. HAV reductions were intermediate between SS phages and FRNA phages, with 4 log10 reductions in about 4 weeks. The observed differences in virus survival suggest that SS phages are more persistent in seawater than other viruses and hence may be good indicators for enteric viruses in seawater.

A Multiplex Reverse Transcription Polymerase Chain Reaction Method for the Detection of Foodborne Viruses†

1999 ◽  
Vol 62 (10) ◽  
pp. 1210-1214 ◽  
Author(s):  
SORAYA I. ROSENFIELD ◽  
LEE-ANN JAYKUS
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Hepatitis A Virus ◽  
Simultaneous Detection ◽  
Polymerase Chain Reaction Method ◽  
Human Enterovirus ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

A multiplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the simultaneous detection of the human enteroviruses, hepatitis A virus (HAV) and Norwalk virus (NV). Poliovirus type 1 (PV1) was chosen as a model for the human enterovirus group. Three different sets of primers were used to produce three size-specific amplicons of 435 bp, 270 bp, and 192 bp for PV1, NV, and HAV, respectively. RT-PCR products were separated by agarose gel electrophoresis, and amplicon identity was confirmed by Southern transfer followed by DNA hybridization using nonradio-active, digoxigenin-labeled internal probes. When tested on mixed, purified virus suspensions, the multiplex method achieved detection limits of ≤1 infectious unit (PV1 and HAV) or RT-PCR-amplifiable unit (NV) for all viruses. With further streamlining efforts such as single tube amplification and liquid hybridization, multiplex PCR offers advantages over cell culture methodology and monoplex PCR because it allows for rapid and cost-effective detection of several human enteric viruses in a single reaction tube.

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