scholarly journals Development of a Tetraplex qPCR for the Molecular Identification and Quantification of Human Enteric Viruses, NoV and HAV, in Fish Samples

2021 ◽  
Vol 9 (6) ◽  
pp. 1149
Author(s):  
Andreia Filipa-Silva ◽  
Mónica Nunes ◽  
Ricardo Parreira ◽  
Maria Teresa Barreto Crespo
Keyword(s):  
Fish Consumption ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Gilthead Seabream ◽  
Fish Samples ◽  
Genome Copy Number ◽  
Raw Fish ◽  
Human Enteric Viruses ◽  
Foodborne Infections

Human enteric viruses such as norovirus (NoV) and hepatitis A virus (HAV) are some of the most important causes of foodborne infections worldwide. Usually, infection via fish consumption is not a concern regarding these viruses, since fish are mainly consumed cooked. However, in the last years, raw fish consumption has become increasingly common, especially involving the use of seabass and gilthead seabream in dishes like sushi, sashimi, poke, and carpaccio. Therefore, the risk for viral infection via the consumption of raw fish has also increased. In this study, a virologic screening was performed in 323 fish specimens captured along the Portuguese coast using a tetraplex qPCR optimised for two templates (plasmid and in vitro transcribed RNA) to detect and quantify NoV GI, NoV GII and HAV genomes. A difference of approximately 1-log was found between the use of plasmid or in vitro transcribed RNA for molecular-based quantifications, showing an underestimation of genome copy-number equivalents using plasmid standard-based curves. Additionally, the presence of NoV genomic RNA in a pool of seabass brains was identified, which was shown to cluster with a major group of human norovirus sequences from genogroup I (GI.1) by phylogenetic analysis. None of the analysed fish revealed the presence of NoV GII or HAV. This result corroborates the hypothesis that enteric viruses circulate in seawater or that fish were contaminated during their transportation/handling, representing a potential risk to humans through raw or undercooked fish consumption.

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.

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.

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 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.

Detection of Human Enteric Viruses in Japanese Clams

2008 ◽  
Vol 71 (8) ◽  
pp. 1689-1695 ◽  
Author(s):  
GRANT S. HANSMAN ◽  
TOMOICHIRO OKA ◽  
TIAN-CHENG LI ◽  
OSAMU NISHIO ◽  
MAMORU NODA ◽  
...  
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Aichi Virus ◽  
Human Consumption ◽  
Reverse Transcription Pcr ◽  
Different Types ◽  
Group A ◽  
Human Enteric Viruses ◽  
High Level

A total of 57 clam packages that were collected from supermarkets and fish markets from 11 different sites in western Japan between 8 December 2005 and 6 September 2006 were examined for human enteric viruses (i.e., norovirus, Aichi virus, rotavirus, adenovirus, hepatitis A virus, and astrovirus), using PCR and reverse transcription PCR. Sixty-one percent of the packages were contaminated with one type of virus, 9% had two different types of viruses, 28% had three different types of viruses, and 9% had at least four different types of viruses. Thirty-one (54%) of 57 packages were contaminated with noroviruses. Norovirus genogroup I and genogroup II sequences were detected in 24 and 23 packages, respectively, and these sequences belonged to nine genogroup I and eight genogroup II genotypes. Aichi viruses were found in 19 (33%) of 57 packages, and these belonged to genogroup A. Rotaviruses (group A) were detected in 14 (42%) of 33 of packages and 9 of 14 rotavirus-positive packages contained two or more rotavirus genogroup types. Adenoviruses (Ad40 and Ad41) were detected in 17 (52%) of 33 packages. One of the 57 (2%) packages was positive with hepatitis A virus (subtype IA). Astrovirus was not detected in any of the packages. This is the first study to detect such a high level of contamination in Japanese clams. These results represent an important finding because the Japanese clams were considered suitable for human consumption. Further studies are needed to determine the health risks associated with eating these highly contaminated clams.

scholarly journals Grape Seed Extract for Control of Human Enteric Viruses

2011 ◽  
Vol 77 (12) ◽  
pp. 3982-3987 ◽  
Author(s):  
Xiaowei Su ◽  
Doris H. D'Souza
Keyword(s):  
Room Temperature ◽  
Hepatitis A Virus ◽  
Antimicrobial Properties ◽  
Enteric Viruses ◽  
Grape Seed Extract ◽  
Grape Seed ◽  
Seed Extract ◽  
Dependent Manner ◽  
Murine Norovirus ◽  
Human Enteric Viruses

ABSTRACTGrape seed extract (GSE) is reported to have many pharmacological benefits, including antioxidant, anti-inflammatory, anticarcinogenic, and antimicrobial properties. However, the effect of this inexpensive rich source of natural phenolic compounds on human enteric viruses has not been well documented. In the present study, the effect of commercial GSE, Gravinol-S, on the infectivity of human enteric virus surrogates (feline calicivirus, FCV-F9; murine norovirus, MNV-1; and bacteriophage MS2) and hepatitis A virus (HAV; strain HM175) was evaluated. GSE at concentrations of 0.5, 1, and 2 mg/ml was individually mixed with equal volumes of each virus at titers of ∼7 log10PFU/ml or ∼5 log10PFU/ml and incubated for 2 h at room temperature or 37°C. The infectivity of the recovered viruses after triplicate treatments was evaluated by standardized plaque assays. At high titers (∼7 log10PFU/ml), FCV-F9 was significantly reduced by 3.64, 4.10, and 4.61 log10PFU/ml; MNV-1 by 0.82, 1.35, and 1.73 log10PFU/ml; MS2 by 1.13, 1.43, and 1.60 log10PFU/ml; and HAV by 1.81, 2.66, and 3.20 log10PFU/ml after treatment at 37°C with 0.25, 0.50, and 1 mg/ml GSE, respectively (P< 0.05) in a dose-dependent manner. GSE treatment of low titers (∼5 log10PFU/ml) at 37°C also showed viral reductions. Room-temperature treatments with GSE caused significant reduction of the four viruses, with higher reduction for low-titer FCV-F9, MNV-1, and HAV compared to high titers. Our results indicate that GSE shows promise for application in the food industry as an inexpensive novel natural alternative to reduce viral contamination and enhance food safety.

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.

Thermal Inactivation of Foodborne Enteric Viruses and Their Viral Surrogates in Foods

2015 ◽  
Vol 78 (8) ◽  
pp. 1597-1617 ◽  
Author(s):  
HAYRIYE BOZKURT ◽  
DORIS H. D'SOUZA ◽  
P. MICHAEL DAVIDSON
Keyword(s):  
Thermal Processing ◽  
Hepatitis A ◽  
Thermal Inactivation ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Industrial Applications ◽  
Inactivation Kinetics ◽  
Human Consumption ◽  
Foodborne Viruses

Foodborne viruses, in particular human norovirus and hepatitis A virus, are the most common causes of food-associated infections and foodborne illness outbreaks around the world. Since it is currently not possible to cultivate human noroviruses and the wild-type strain of hepatitis A virus in vitro, the use of a variety of viral surrogates is essential to determine appropriate thermal processing conditions to reduce the risk associated with their contamination of food. Therefore, the objectives of this review are to (i) present pertinent characteristics of enteric foodborne viruses and their viral surrogates, (ii) discuss the viral surrogates currently used in thermal inactivation studies and their significance and value, (iii) summarize available data on thermal inactivation kinetics of enteric viruses, (iv) discuss factors affecting the efficacy of thermal treatment, (v) discuss suggested mechanisms of thermal inactivation, and (vi) provide insights on foodborne enteric viruses and viral surrogates for future studies and industrial applications. The overall goal of this review is to contribute to the development of appropriate thermal processing protocols to ensure safe food for human consumption.

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 (&lt;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.

Inactivation of Hepatitis a Virus and Model Viruses in Water by Free Chlorine and Monochloramine

1988 ◽  
Vol 20 (11-12) ◽  
pp. 385-391 ◽  
Author(s):  
Mark D. Sobsey ◽  
Takashi Fuji ◽  
Patricia A. Shields
Keyword(s):  
Phosphate Buffer ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Low Ph ◽  
Free Chlorine ◽  
High Ph ◽  
Human Enteric Viruses ◽  
Reasonable Model

The kinetics and extent of inactivation of hepatitis A virus (HAV) as well as three other viruses, coxsackievirus B5 (CB5) and coliphages MS2 and ϕX174, by 0.5 mg/l free chlorine, pH 6-10, and 10 mg/l monochloramine, pH 8, at 5°C in 0.01 M phosphate buffer were determined. HAV wae relatively sensitive to 0.5 mg/l free chlorine but relatively resistant to 10 mg/l monochloramine. Compared to HAV, CB5 was quite resistant to inactivation by free chlorine but similar in resistance to inactivation by monochloramine. Inactivation of ϕX174 by free chlorine was rapid at pH 6-9 and intermediate between that of HAV and CB5 at pH 10. ϕX174 was inactivated most rapidly of all viruses tested by 10 mg/l monochloramine. Inactivation of MS2 by free chlorine was somewhat more rapid than HAV at low pH but less rapid than HAV at high pH. MS2 inactivation by 10 mg/l monochloramine was slowset of all viruses tested. These reeslts indicate that HAV is inactivated relatively rapidly by free chlorine but relatively slowly by monochloramine. Coliphage MS2 is a reasonable model to predict inactivation of HAV by free chlorine and inactivation of HAV and CB5 by monochloramine. It is a poor model for predicting free chlorine inactivation of CB5 and perhaps some other human enteric viruses.

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