Most Relevant Papers

Hepatitis E is an acute, viral hepatitis epidemic in developing regions, but which is detected with increasing frequency in sporadic form in developed regions. Pigs and possibly some other mammals are considered reservoirs of zoonotic infection with hepatitis E virus (HEV). However, whilst the relative significance of potential transmission routes from pigs to people is still unclear, the consumption of raw or undercooked pig meat has been implicated as a source of HEV infection. The lack of information about HEV zoonotic transmission is due in part to the difficulties of in vitro propagation of HEV. The Rotating Wall Vessel (RVW) has been described as a useful tool for the culture of cell lines in a 3-dimensional (3D) configuration. The aim of this work was to develop a 3D cell culture system for HEV to facilitate studies into the viability of virions contaminating pig tissues. This study, demonstrated that HEV can replicate efficiently in the RWV in human hepatoblastoma PLC/PRF/5 cells for up to 5 months not only by real time RT-PCR but also by detection of complete virions via electron microscopy. Furthermore, the replication of HEV progeny was observed by detecting HEV RNA by RT-PCR. The progeny were able to infect fresh 3D cultures, showing that this method is able to produce infectious hepatitis E virions.

Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Molecular techniques and virus concentration methods have shown that previously unknown viruses are shed by humans and animals, and may be transmitted by sewage-contaminated water. In the present study, 10-L river-water samples from urban areas in Barcelona, Spain and Rio Janeiro, Brazil, have been analyzed to evaluate the viral dissemination of human viruses, validating also a low-cost concentration method for virus quantification in fresh water. Three viral groups were analyzed: (i) recently reported viruses, klassevirus (KV), asfarvirus-like virus (ASFLV), and the polyomaviruses Merkel cell (MCPyV), KI (KIPyV) and WU (WUPyV); (ii) the gastroenteritis agents noroviruses (NoV) and rotaviruses (RV); and (iii) the human fecal viral indicators in water, human adenoviruses (HAdV) and JC polyomaviruses (JCPyV). Virus detection was based on nested and quantitative PCR assays. For KV and ASFLV, nested PCR assays were developed for the present study. The method applied for virus concentration in fresh water samples is a one-step procedure based on a skimmed-milk flocculation procedure described previously for seawater. Using spiked river water samples, inter- and intra-laboratory assays showed a viral recovery rate of about 50% (20-95%) for HAdV, JCPyV, NoV and RV with a coefficient of variation ≤ 50%. HAdV and JCPyV were detected in 100% (12/12) of the river samples from Barcelona and Rio de Janeiro. Moreover, NoV GGII was detected in 83% (5/6) and MCPyV in 50% (3/6) of the samples from Barcelona, whereas none of the other viruses tested were detected. NoV GGII was detected in 33% (2/6), KV in 33% (2/6), ASFLV in 17% (1/6) and MCPyV in 50% (3/6) of the samples from Rio de Janeiro, whereas KIPyV and WUPyV were not detected. RV were only analyzed in Rio de Janeiro and resulted positive in 67% (4/6) of the samples. The procedure applied here to river water represents a useful, straightforward and cost-effective method that could be applied in routine water quality testing. The results of the assays expand our understanding of the global distribution of the viral pathogens studied here and their persistence in the environment.

Copyright © 2013 Elsevier Ltd. All rights reserved

Determining the stability, or persistence in an infectious state, of foodborne viral pathogens attached to surfaces of soft fruits and salad vegetables is essential to underpin risk assessment studies in food safety. Here, we evaluate the effect of temperature and sunlight on the stability of infectious human adenoviruses type 2 and MS2 bacteriophages on lettuce and strawberry surfaces as representative fresh products. Human adenoviruses have been selected because of their double role as viral pathogens and viral indicators of human fecal contamination. Stability assays were performed with artificially contaminated fresh samples kept in the dark or under sunlight exposure at 4 and 30°C over 24h. The results indicate that temperature is the major factor affecting HAdV stability in fresh produce surfaces, effecting decay between 3 and 4 log after 24h at 30°C. The inactivation times to achieve a reduction between 1 and 4-log are calculated for each experimental condition. This work provides useful information to be considered for improving food safety regarding the transmission of foodborne viruses through supply chains.

Copyright © 2013 Elsevier B.V. All rights reserved.

Viral pathogens, such as norovirus (NoV), are frequently associated with foodborne gastroenteritis worldwide, and the detection of NoV in food requires appropriate methods and the use of process controls. In this study, an adsorption-elution concentration method using negatively charged membranes was optimized to recover NoV from lettuce, using murine norovirus 1 (MNV-1) as a human NoV (HuNoV) surrogate. Initially, three elution buffers were evaluated by direct elution using a Stomacher® apparatus with a filter bag and different concentrations of MNV-1 genomic copies. The eluates were filtered in a Stericup® and concentrated by a Centriprep Concentrator®, and the viral RNA was quantified by real-time PCR that was preceded by reverse transcription. The MNV-1 recovery efficiency varied based on the buffers used, ranging from 5.2 to 9.8 % for PBS pH 7.2, 0.2-18 % for glycine NaCl pH 9.5 and 10.8-33.3 % for glycine Tris-HCl pH 9.5. Further analysis of the glycine Tris-HCl pH 9.5 buffer revealed that gentle-shaking, direct elution could replace the use of a Stomacher®, with recovery rates reaching 66 and 32 % for MNV-1 and HuNoV, respectively, all of which suggested that this procedure is a quick and efficient method for recovering NoV from lettuce.

SUMMARY: This study aimed to assess norovirus (NoV) contamination and genotype diversity in surface water in Gauteng, South Africa. Between January 2008 and December 2010, three rivers, namely Klip, Suikerbosrant, and Rietspruit were monitored for NoV genogroup (G)I and GII. Viruses were recovered using the glass wool adsorption-elution technique and detected by real-time reverse transcription-polymerase chain reaction. From 2008 to 2010, NoVs were detected in 66% (70/106) of Klip river samples. The Rietspruit and Suikerbosrant rivers were contaminated with NoV in 95% (20/21) and 21% (5/24) of samples, respectively. NoV-positive samples comprised of 33% GI, 29% GII and 38% of both GI and GII strains. Based on partial capsid gene analysis (region C), 16 NoV genotypes (6 GI, 10 GII) were identified. The major genotypes detected were GI.4, GI.5 and GII.4. These rivers could be a potential source of NoV infection for communities using the water for domestic or recreational purposes.

Over a 2-year period, from January 2009 to December 2010, water samples were collected from three rivers (Klip, Rietspruit and Suikerbosrand) in the Vaal River System, South Africa. Enteric viruses were recovered by a glass wool adsorption-elution method and concentrated using polyethylene glycol/sodium chloride precipitation. Sapoviruses (SaVs) were detected using published sapovirus (SaV)-specific primers and Taqman probes in a two-step real-time reverse transcription-polymerase chain reaction assay. Based on sequence analysis of the 5'-end of the capsid gene, SaVs were genotyped. In 2009, SaVs were detected in 39% (15/38) of samples from the Klip river, 83% (5/6) from the Rietspruit and 14% (1/7) of samples from the Suikerbosrand river. In 2010, SaVs were detected in 54% (14/26) of Klip river samples, 92% (11/12) from the Rietspruit and 20% (2/10) of samples from the Suikerbosrand river. SaV strains identified in the water samples were characterised into several GI and GII genotypes. The presence of SaVs in these rivers indicates human faecal contamination which may pose a potential health risk to persons exposed to these water sources during domestic or recreational activities.

AIM:

To investigate the diversity of human caliciviruses (HuCVs) in wastewater from small- to medium-sized communities in five provinces of South Africa (SA).

METHODS AND RESULTS:

Wastewater samples (51) were screened for norovirus (NoV) GI, GII, GIV and sapovirus (SaV) using real-time reverse transcription (RT)-PCR. Partial capsid nucleotide sequences were analysed for genotyping. At least one HuCV was detected in 42 samples (82%) with NoV GI being detected in 15 (29%), NoV GII in 32 (63%) and SaV in 37 (73%) samples. NoV GIV was not detected. Five NoV GI genotypes (GI.1, GI.3, GI.4, GI.8 and GI.unassigned), eight NoV GII genotypes (GII.2, GII.3, GII.4, GII.6, GII.7, GII.12, GII.13 and GII.17) and six SaV genotypes (GI.2, GI.3, GI.6, GI.7, GII.1 and GII.2) were characterized.

CONCLUSIONS:

Many NoV and SaV genotypes were detected in wastewater, demonstrating a high genetic diversity of HuCVs in the surrounding communities. Caliciviruses were characterized from several provinces in SA, indicating widespread occurrence in the country.

SIGNIFICANCE AND IMPACT OF THE STUDY:

This study provides valuable new data on CVs circulating in SA, including the first data on SaV strains from wastewater in Africa. Environmental surveillance is especially important in countries like SA where outbreak reporting systems or routine HuCV surveillance is lacking.

© 2013 The Society for Applied Microbiology.

SUMMARY: This study aimed to assess norovirus (NoV) contamination and genotype diversity in surface water in Gauteng, South Africa. Between January 2008 and December 2010, three rivers, namely Klip, Suikerbosrant, and Rietspruit were monitored for NoV genogroup (G)I and GII. Viruses were recovered using the glass wool adsorption-elution technique and detected by real-time reverse transcription-polymerase chain reaction. From 2008 to 2010, NoVs were detected in 66% (70/106) of Klip river samples. The Rietspruit and Suikerbosrant rivers were contaminated with NoV in 95% (20/21) and 21% (5/24) of samples, respectively. NoV-positive samples comprised of 33% GI, 29% GII and 38% of both GI and GII strains. Based on partial capsid gene analysis (region C), 16 NoV genotypes (6 GI, 10 GII) were identified. The major genotypes detected were GI.4, GI.5 and GII.4. These rivers could be a potential source of NoV infection for communities using the water for domestic or recreational purposes.

Hepatitis E is a viral disease that causes serious concerns for public health. Hepatitis E virus (HEV) genotype 3 is endemic in commercial pig farms worldwide that act as a reservoir. Pig-to-human transmission may occur when infectious animals enter the food chain at slaughter, through consumption of contaminated meat, direct exposure or use of by-products. To reduce the fraction of infectious animals at slaughter age and thus the risk for public health, it is important to understand the transmission dynamics of HEV in pig populations. In this study, we estimate the transmission rate parameter and mean infectious period of HEV in pigs from field data, using a Bayesian analysis. The data were collected in ten commercial pig herds that are each divided into three different age groups. Two transmission models were compared, assuming that animals are infected either locally by their group mates or globally by any infectious animal regardless of its group. For local and global transmission, the transmission rate parameters were 0.11 (posterior median with 95% credible interval: 0.092-0.14 day(-1)) and 0.16 (0.082-0.29 day(-1)), the mean infectious periods were 24 (18-33) days and 27 (20-39) days and the reproduction numbers were 2.7 (2.2-3.6) and 4.3 (2.8-6.9). Based on these results, global transmission is considered to be the more conservative model. Three effects of vaccination were explored separately. When vaccination is not sufficient to eliminate the virus, a shorter mean infectious period decreases the fraction of infectious animals at slaughter age, whereas a reduced transmission rate parameter adversely increases it. With a reduced susceptibility, vaccination of animals at a later age can be a better strategy than early vaccination. These effects should be taken into account in vaccine development.

Copyright © 2012 Elsevier B.V. All rights reserved.

BACKGROUND:

Hepatitis E virus (HEV) genotype 3 and 4 can cause liver disease in human and has its main reservoir in pigs. HEV investigations in pigs worldwide have been performed but there is still a lack of information on the infection dynamics in pig populations.

FINDINGS:

The HEV transmission dynamics in commercial pig farms in six different European countries was studied. The data collected show prevalence in weaners ranging from 8% to 30%. The average HEV prevalence in growers was between 20% and 44%. The fatteners prevalence ranged between 8% and 73%. Sows prevalence was similar in all countries. Boar faeces were tested for HEV only in Spain and Czech Republic, and the prevalence was 4.3% and 3.5% respectively. The collected data sets were analyzed using a recently developed model to estimate the transmission dynamics of HEV in the different countries confirming that HEV is endemic in pig farms.

CONCLUSIONS:

This study has been performed using similar detection methods (real time RT-PCR) for all samples and the same model (SIR model) to analyse the data. Furthermore, it describes HEV prevalence and within-herd transmission dynamics in European Countries (EU): Czech Republic, Italy, Portugal, Spain, The Netherlands and United Kingdom, confirming that HEV is circulating in pig farms from weaners to fatteners and that the reproductive number mathematical defined as R0 is in the same range for all countries studied.

We investigated contamination by hepatitis E virus (HEV) in the pork production chain in the United Kingdom. We detected HEV in pig liver samples in a slaughterhouse, in surface samples from a processing plant, and in pork sausages and surface samples at point of sale. Our findings provide evidence for possible foodborne transmission of HEV during pork production.

The qualitative performance characteristics of a qPCR-based method to detect human adenoviruses in raspberries were determined through a collaborative trial involving 11 European laboratories. The method incorporated a sample process control (murine norovirus) and an internal amplification control. Trial sensitivity or correct identification of 25-g raspberry samples artificially contaminated with between 5 × 10^2 and 5 × 10^4 PFU was 98.5%; the accordance and concordance were 97.0%. The positive predictive value was 94.2%. The trial specificity or percentage correct identification of non-artificially contaminated samples was 69.7%; the accordance was 80.0% and the concordance was 61.7%. The negative predictive value was 100%. Application of a method for the detection of human adenoviruses in food samples could be useful for routine monitoring for food safety management. It would help to determine if a route of contamination exists from human source to food supply chain which pathogenic viruses such as norovirus and hepatitis A virus could follow.

Viruses excreted by humans affect the commercial and recreational use of coastal water. Shellfish produced in contaminated waters have been linked to many episodes and outbreaks of viral gastroenteritis, as well as other food-borne diseases worldwide. The risk can be reduced by appropriate treatment following harvesting and by depuration. The kinetics of inactivation of murine norovirus 1 and human adenovirus 2 in natural and artificial seawater by free available chlorine was studied by quantifying genomic copies (GC) using quantitative PCR and infectious viral particles (PFU). Human JC polyomavirus Mad4 kinetics were evaluated by quantitative PCR. DNase or RNase were used to eliminate free genomes and assess potential viral infectivity when molecular detection was performed. At 30 min of assay, human adenovirus 2 showed 2.6- and 2.7-log(10) GC reductions and a 2.3- and 2.4-log(10) PFU reductions in natural and artificial seawater, respectively, and infectious viral particles were still observed at the end of the assay. When DNase was used prior to the nucleic acid extraction the kinetic of inactivation obtained by quantitative PCR was statistically equivalent to the one observed by infectivity assays. For murine norovirus 1, 2.5, and 3.5-log(10) GC reductions were observed in natural and artificial seawater, respectively, while no viruses remained infectious after 30 min of contact with chlorine. Regarding JC polyomavirus Mad4, 1.5- and 1.1-log(10) GC reductions were observed after 30 min of contact time. No infectivity assays were conducted for this virus. The results obtained provide data that might be applicable to seawater used in shellfish depuration.

We evaluated the prevalence of hepatitis E virus (HEV) in the pork production chain in Czech Republic, Italy, and Spain during 2010. A total of 337 fecal, liver, and meat samples from animals at slaughterhouses were tested for HEV by real-time quantitative PCR. Overall, HEV was higher in Italy (53%) and Spain (39%) than in Czech Republic (7.5%). HEV was detected most frequently in feces in Italy (41%) and Spain (39%) and in liver (5%) and meat (2.5%) in Czech Republic. Of 313 sausages sampled at processing and point of sale, HEV was detected only in Spain (6%). HEV sequencing confirmed only g3 HEV strains. Indicator virus (porcine adenovirus) was ubiquitous in fecal samples and absent in liver samples and was detected in 1 slaughterhouse meat sample. At point of sale, we found porcine adenovirus in sausages (1%-2%). The possible dissemination of HEV and other fecal viruses through pork production demands containment measures.

BACKGROUND:

Environmental surfaces can play a role in the spread of pathogens, such as enteric viruses, within a hospital. This study assessed the level of contamination of group A rotavirus (RV-A) on environmental surfaces samples from an adult intensive care unit in a hospital in Rio de Janeiro, Brazil.

METHODS:

A total of 504 environmental surface samples were obtained from multiple sites in the intensive care unit, including flushing buttons, telephones, and alcohol gel supports. Nested and quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) were used to detect and quantify RV-A levels through partial amplification of VP6 and NSP3 genes, respectively, and the viability of the viruses detected was assessed by MA-104 cell integrated cell culture/RT-PCR.

RESULTS:

RV-A was detected by nested RT-PCR in 14% of the samples (73 of 504), with viral loads ranging from 3.4 genomic copies/mL to 2.9 × 10(3) genomic copies/mL. The nucleotide sequence of the amplicons obtained from nested RT-PCR confirmed that the positive samples were RV-A. Moreover, 3 of 10 strains investigated demonstrated viability by integrated cell culture/RT-PCR.

CONCLUSION:

The detection of RV-A on environmental surface samples indicates a need for improvements to hospital cleaning procedures to reduce viral contamination, and suggests, as reported previously, that RV-A can be used as a biomarker to assess contamination in hospitals.

Copyright © 2012 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

Numerous outbreaks have been attributed to the consumption of raw or minimally processed leafy green vegetables contaminated with enteric viral pathogens. The aim of the present study was an integrated virological monitoring of the salad vegetables supply chain in Europe, from production, processing and point-of-sale. Samples were collected and analysed in Greece, Serbia and Poland, from 'general' and 'ad hoc' sampling points, which were perceived as critical points for virus contamination. General sampling points were identified through the analysis of background information questionnaires based on HACCP audit principles, and they were sampled during each sampling occasion where as-ad hoc sampling points were identified during food safety fact-finding visits and samples were only collected during the fact-finding visits. Human (hAdV) and porcine (pAdV) adenovirus, hepatitis A (HAV) and E (HEV) virus, norovirus GI and GII (NoV) and bovine polyomavirus (bPyV) were detected by means of real-time (RT-) PCR-based protocols. General samples were positive for hAdV, pAdV, HAV, HEV, NoV GI, NoV GII and bPyV at 20.09 % (134/667), 5.53 % (13/235), 1.32 % (4/304), 3.42 % (5/146), 2 % (6/299), 2.95 % (8/271) and 0.82 % (2/245), respectively. Ad hoc samples were positive for hAdV, pAdV, bPyV and NoV GI at 9 % (3/33), 9 % (2/22), 4.54 % (1/22) and 7.14 % (1/14), respectively. These results demonstrate the existence of viral contamination routes from human and animal sources to the salad vegetable supply chain and more specifically indicate the potential for public health risks due to the virus contamination of leafy green vegetables at primary production.

Human noroviruses (hNoV) have been detected on soft fruits. Especially raspberries have been found to be associated with outbreaks of gastroenteritis suggesting persistence of hNoV on these fruits. Therefore, the persistence of hNoV GII.4 and GI.4, murine norovirus (MNV-1, a culturable surrogate for hNoV), and human adenovirus (hAdV, an indicator for human fecal contamination), on raspberries, strawberries and in phosphate buffered saline (PBS) at 4°C, 10°C and 21°C, mimicking commonly applied storage conditions was studied by molecular and cell culture techniques. Monophasic, biphasic and Weibull models were fitted to virus counts with maximum likelihood estimation. The tested viruses were persistent (≤0.5 log(10)-unit reduction in viral titer) under all studied conditions in PBS, at 4°C and 10°C on raspberries, and at 4°C on strawberries. The difference in viral persistence on raspberries and strawberries was most pronounced at 21°C. Here, infectious MNV-1 and hAdV particles decayed rapidly on strawberries with TFL-values (time for the first log(10)-unit reduction) of only 1day (95% CI of 0.6-1 and 0.8-1days, respectively). On raspberries, however, the TFL-value of infectious MNV-1 was found to likely exceed the shelf life of the berries with 3days (95% CI of 2.8-3.1days); hAdV remained infectious with only 0.3 log(10)-unit reduction (95% CI of 0.2-0.4) in viral titer. For hNoV GI, a TFL-value of 2days (95% CI 1-4days) was determined based on the targeted genome fragment, whereas the TFL-value of hNoV GII exceeded the shelf life of strawberries at 21°C. The greater viral persistence on raspberries as compared to strawberries, especially at 21°C, may at least in part explain why raspberries are more frequently associated with hNoV outbreaks than strawberries. Moreover, our results show that due to the high persistence of the virus already low contamination levels of the highly infectious hNoV may be associated with an infection risk of humans after consumption of raspberries. The estimated decay parameters and uncertainties of this study serve as important input requirements in the quantitative assessment of public health risks from the consumption of soft fruits.

Copyright © 2012 Elsevier B.V. All rights reserved.

The consumption of fresh produce is frequently associated with outbreaks of human norovirus (hNoV) disease. To prevent the contamination of fresh produce with hNoV, knowledge of the possible introduction sources of the viruses, such as water, is needed to be able to implement appropriate and efficient preventive measures. Contaminated water used to reconstitute pesticides could be a relevant source of infectious hNoV, determined by the initial level of virus contamination and the persistence of these viruses in reconstituted pesticides. We studied the persistence of hNoV GI.4, hNoV GII.4 and murine norovirus (MNV-1), the only culturable norovirus, in eight different pesticides after 0 and 2h. Virus concentrations were determined by reverse transcriptase PCR, and infectivity of MNV-1 was determined by endpoint dilutions followed by maximum likelihood estimations. MNV-1 was found to remain infectious in seven of the eight tested pesticides at the highest concentration applied in practice. In the presence of the insecticide Vertimec, MNV-1 infectivity decreased rapidly with a 1.9 log(10)-unit reduction at timepoint T(0). Also, the concentration of NoV GI.4 RNA decreased considerably with a 1.7 log(10)-unit reduction; whereas the detected PCR fragment of hNoV GII.4 remained stable. Assuming a similar persistence of infectious MNV-1 and hNoV we can conclude that water containing hNoV used to dilute pesticides may be an important source of infectious hNoV in fresh produce chains. The application of pesticides may therefore not only be a chemical hazard, but also a microbiological hazard for public health. The inclusion of antiviral substances in reconstituted pesticides may be appropriate to reduce the virological health risk posed by the application of pesticides.

Copyright © 2012 Elsevier B.V. All rights reserved.

Public concerns relating to food safety remain high, with most attention focused on manufactured foods and those served in catering operations. The viral contamination of food can occur anywhere in the food supply chain from farm to fork, but most food-borne viral infections can be traced back to infected persons who handle food that is neither heated nor otherwise treated. Regard to the increasing incidence of food-borne viral infections, the Codex Alimentarius Committee on Food Hygiene issued an international draft on a Code of Hygienic Practice for the control of viruses in foods. Using SWOT analysis as a methodological tool, the main results of the analysis revealed limitations of the document regarding language terminology, detection methodology and transparency.

Potential ways to address the issues that relate to the techniques for analyzing food and environmental samples for the presence of enteric viruses are discussed. It is not the authors’ remit to produce or recommend standard or reference methods but to address specific issues in the analytical procedures. Foods of primary importance are bivalve molluscs, particularly, oysters, clams, and mussels; salad crops such as lettuce, green onions and other greens; and soft fruits such as raspberries and strawberries. All types of water, not only drinking water but also recreational water (fresh, marine, and swimming pool), river water (irrigation water), raw and treated sewage are potential vehicles for virus transmission. Well over 100 different enteric viruses could be food or water contaminants; however, with few exceptions, most well-characterized foodborne or waterborne viral outbreaks are restricted to hepatitis A virus (HAV) and calicivirus, essentially norovirus (NoV). Target viruses for analytical methods include, in addition to NoV and HAV, hepatitis E virus (HEV), enteroviruses (e.g., poliovirus), adenovirus, rotavirus, astrovirus, and any other relevant virus likely to be transmitted by food or water. A survey of the currently available methods for detection of viruses in food and environmental matrices was conducted, gathering information on protocols for extraction of viruses from various matrices and on the various specific detection techniques for each virus type.

The most effective methods for virus detection in food and environmental samples are those based on nucleic acid amplification. Complex methods must be applied by the analyst in order to control for false negative results of virus detection assays in those samples that may be contaminated by virus concentrations above the detection level. The verification of analytical results is a necessity and this depends on using an appropriate suite of controls to monitor the efficacy of the critical steps in the method and allow correct result interpretations to be made. We describe the suite of controls necessary for analysing food and environmental samples for the presence of enteric viruses by nucleic acid amplification-based methods. To exclude false negative and positive interpretations of results, the inclusion of this suite of controls will be essential when considering incorporating monitoring of viruses in food or environmental safety management plans.

Internal amplification controls (IACs) were constructed for incorporation into real-time nucleic acid amplification assays for bovine polyomavirus, hepatitis A virus, hepatitis E virus, human adenovirus, human norovirus genogroup I, human norovirus genogroup II, murine norovirus and porcine adenovirus. The addition of optimised amounts of IAC into the assays did not affect the limits of detection for each specific target virus. A poorly performed extraction of viral nucleic acids was simulated, and the effectiveness of IACs in identifying failed assays was demonstrated. The IACs constructed in this study can be reliably used in their specific assays to provide a robust control that can be routinely applied in the analysis of foods for viruses.

Rotavirus specie A (RVA) infection is the leading cause of severe acute diarrhea among young children worldwide. To reduce this major RVA health impact, the Rotarix® vaccine (GlaxoSmithKline, Rixensart, Belgium) was introduced in the Brazilian Expanded Immunization Program in March 2006 and became available to the entire birth cohort. The aim of this study was to evaluate the spread of RVA in the environment after the introduction of Rotarix® in Brazil. For this purpose, a Wastewater Treatment Plant (WTP) in Rio de Janeiro was monitored for one year to detect, characterize and discriminate RVA genotypes and identify possible circulation of vaccine strains. Using TaqMan® quantitative PCR (qPCR), RVA was detected in 100% (mean viral loads from 2.40×10(5) to 1.16×10(7) genome copies (GC)/L) of sewage influent samples and 71% (mean viral loads from 1.35×10(3) to 1.64×10(5)GC/L) of sewage effluent samples. The most prevalent RVA genotypes were P[4], P[6] and G2, based on VP4 and VP7 classification. Direct nucleotide sequencing (NSP4 fragment) and restriction enzyme digestion (NSP3) analysis did not detect RVA vaccine-like strains from the sewage samples. These data on RVA detection, quantification and molecular characterization highlight the importance of environmental monitoring as a tool to study RVA epidemiology in the surrounding human population and may be useful on ongoing vaccine monitoring programs, since sewage may be a good screening option for a rapid and economical overview of the circulating genotypes.

Copyright © 2011 Elsevier Ltd. All rights reserved.

We report an in-house protocol for extraction and purification of nucleic acids of enteric viruses, which gives more consistent results than representative commercial methods. The protocol uses 4 M guanidine thiocyanate, 0.5% N-lauroylsarcosine sodium salt, and 25 mM sodium citrate pH 7.0 supplemented with 0.14 M β-mercaptoethanol for lysis of virus particles. The addition of TRIzol followed by chloroform-based separation of the aqueous phase is used to purify nucleic acids from the lysate. RNA precipitation is performed using lithium chloride. This protocol was compared with QIAGEN’s RNeasy Kit and bioMérieux’s NucliSens method, by evaluating the ability of each to detect enteric viruses in a complex food matrix. Three different pork products, i.e., cooked ham, liver, and Spanish fermented sausage (“chorizo”) were artificially contaminated with decreasing numbers of murine norovirus 1 and human adenovirus 2. The extracted and purified viral nucleic acids were detected by real-time polymerase chain reaction (PCR) assays. Whereas the two commercial extraction methods did not facilitate robust results (quantification was only possible with some viruses and/or some matrices), when coupled with the in-house protocol the linearity and the efficiency of the quantitative reverse transcription-PCR (qRT-PCR) assays were close to 1 in all the food matrices, independent of the virus. Scalability of the in-house method was evaluated by analysis of 1 and 2.5 g of spiked pig liver samples, and quantification was possible on 1 g samples contaminated with any of the two model viruses. Therefore, the in-house protocol facilitates robust qRT-PCR-based quantitative detection of viruses in pork products, and is moreover relatively cheap and simple to perform.

Hospital wastewater has been described as an important source of spreading pathogenic microorganisms in the environment. However, there are few studies reporting the presence and concentrations of gastroenteric viruses and hepatitis A viruses in these environmental matrices. The aim of this study was to assess the contamination by viruses responsible for acute gastroenteritis and hepatitis derived from hospital wastewater treatment plants (WWTPs). Rotavirus A (RV-A), human adenoviruses (HAdV), norovirus genogroup I and II (NoV GI/GII) and hepatitis A viruses (HAV) were detected and quantified in sewage samples from two WWTPs located in Rio de Janeiro (Brazil) that operates different sewage treatments. WWTP-1 uses an Upflow Anaerobic Sludge Blanket (UASB reactor) and three serial anaerobic filters while WWTP-2 uses aerobic processes, activated sludge with extended aeration and final chlorination of the effluents. Viruses' detection was investigated by using conventional PCR/RT-PCR, quantitative real-time PCR (qPCR) and partial sequencing of the genome of the viruses detected. Rate of viruses detection ranged from 7% (NoV GI in WWTP-1) to 95% (RV-A in WWTP-2) and genome from all viruses were detected. The most prevalent genotypes were RV-A SG I, HAdV species D and F, NoV GII/4 and HAV subgenotype IA. Mean values of viral loads (genome copies (GC)/ml) obtained in filtered effluents from anaerobic process was 1.9 × 10(3) (RV-A), 2.8 × 10(3) (HAdV) and 2.4 × 10(3) (NoV GII). For chlorinated effluents from activated sludge process, the mean values of viral loads (GC/ml) was 1.2 × 10(5) (RV-A), 1.4 × 10(3) (HAdV), 8.1 × 10(2) (NoV GII) and 2.8 × 10(4) (HAV). Data on viral detection in treated effluents of hospital WWTPs confirmed the potential for environmental contamination by viruses and could be useful to establish standards for policies on wastewater management.

Copyright © 2010 Elsevier Ltd. All rights reserved.

AIM:

To determine the occurrence of eight human enteric viruses in surface water and sewage samples from different geographical areas in Kenya.

METHODS AND RESULTS:

Enteric viruses were recovered from the water and sewage sources by glass-wool adsorption elution and/or polyethylene glycol/NaCl precipitation and detected by singleplex real-time and conventional PCR and reverse transcriptase-PCR assays. One or more enteric viruses were detected in nearly all sewage and river water samples except the urban Mbagathi River. The VP7 (G types) and the VP4 (P types) of the rotaviruses (RV) were characterized by multiplex nested PCR methods. The G and P types could be determined in 95•5% of the RV strains, respectively. Mixed G types were detected with G12 and G1 predominating, and unusual G types, G5 and G10, were present. P[4] predominated in the urban Karen sewage samples, while P[8] predominated in the urban and rural streams.

CONCLUSIONS:

The high prevalence of RVs in surface water highlights the importance of assessing the water sources used for domestic purposes for viral contamination.
SIGNIFICANCE AND IMPACT OF THE STUDY:

This study demonstrates the benefit of environmental surveillance as an additional tool to determine the epidemiology of RVs and other enteric viruses circulating in a given community.

© 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

Rotaviruses are important enteric pathogens for humans and animals. Group A rotaviruses (RV-A) are the most common agents of severe gastroenteritis in infants and young children and vaccination is the most effective method to reduce RV-A-associated diseases. G1P[8], the most prevalent RV-A genotype worldwide, is included in the RV-A vaccine Rotarix®. The discrimination between wild-type G1P[8] and vaccine G1P[8] strains is an important topic in the study of RV-A epidemiology to manage outbreaks and to define control measures for vaccinated children. In this study, we developed a novel method to segregate the wild-type and vaccine strains using restriction endonucleases. The dsRNA from the Rotarix® vaccine was sequenced and the NSP3 gene was selected as the target gene. The vaccine strain has a restriction pattern that is different than that of wild-type RV-A G1P[8] isolates after digestion with the restriction endonuclease BspHI. This pattern could be used as a marker for the differentiation of wild-type G1P[8] strains from the vaccine strain.

To assess the presence of the four main viruses responsible for human acute gastroenteritis in a hydrographic network impacted by a disordered urbanization process, a 1-year study was performed involving water sample collection from streams in the hydrographic basin surrounding the city of Manaus, Amazonas, Brazil. Thirteen surface water sample collection sites, including different areas of human settlement characterized as urban, rural, and primary forest, located in the Tarumã-Açu, São Raimundo, Educandos, and Puraquequara microbasins, were defined with a global positioning system. At least one virus was detected in 59.6% (31/52) of the water samples analyzed, and rotavirus was the most frequent (44.2%), followed by human adenovirus (30.8%), human astrovirus (15.4%), and norovirus (5.8%). The viral contamination observed mainly in the urban streams reflected the presence of a local high-density population and indicated the gastroenteritis burden from pathogenic viruses in the water, principally due to recreational activities such as bathing. The presence of viral genomes in areas where fecal contamination was not demonstrated by bacterial indicators suggests prolonged virus persistence in aquatic environments and emphasizes the enteric virus group as the most reliable for environmental monitoring.

The aim of this study was to assess the potential risk of infection constituted by HAV to persons using surface dam and river water for domestic and recreational purposes. It estimates the potential risk using a deterministic exponential risk assessment model with mean values and conservative assumptions. Hepatitis A virus was detected in 17.5% of river and 14.9% of dam water samples tested. The number of indicator organisms in these sources exceeded drinking and recreational water quality guidelines set by the United States Environmental Protection Agency (US EPA), indicating possible health risks to recreational water users. Based on the available data and taking all the assumptions into consideration, the probability of infection (Pinf) to the higher socio-economic population using the river water for recreational purposes was 1.1 x 10(-3) per day and 3.3 x 10(-1) per annum if 100 ml was ingested per day. For recreation in the dam water the Pinf value was 1.2 x 10(-4) per day and 4.2 x 10(-2) per annum. For the lower socio-economic population, risk values for drinking purposes (2 L day(-1)) were ten-fold greater. These surface waters therefore did not conform to the US EPA guidelines of 1 infection per 10,000 consumers per year for drinking water or eight gastrointestinal illnesses per 1,000 bathers per day in environmental waters used for recreational purposes. This is the first risk assessment study addressing the risk of infection by HAV in surface water to different socio-economic populations in South Africa.

Environmental factors are highly relevant to the global dissemination of viral pathogens. However, the specific contribution of major effectors such as temperature and sunlight on the inactivation of waterborne viruses is not well characterized. In this study, the effect of temperature (7, 20, and 37 °C), UVB and UVA radiation on viral inactivation was evaluated in phosphate buffered saline (PBS), mineral water, wastewater, 1,000-fold diluted wastewater and seawater. The stability of human adenoviruses infectivity, known as human pathogens and indicators of fecal contamination, was monitored during 24 h, both in the dark and exposed to UV radiation by immunofluorescence assays. In the dark, no Human adenovirus (HAdV) inactivation was observed in PBS and mineral water at any of the temperatures studied, whereas at 37 °C in reactors with higher microbial concentration (wastewater, diluted wastewater, and seawater), decays between 2.5 and 5 log were recorded. UVB radiation showed a dramatic effect on HAdV inactivation and 6-log were achieved in all reactors by the end of the experiments. The effect of UVA showed to be dependent on the water matrix analyzed. At 20 °C, HAdV showed a 2-log decay in all reactors radiation while at 37 °C, results in wastewater, diluted wastewater, and seawater reactors were equivalent to those observed in the dark. These results suggest UVB radiation as the major environmental factor challenging viral inactivation, followed by biotic activity indirectly associated to higher temperatures and finally, by UVA radiation.

Multiple outbreaks of human norovirus (hNoV) have been associated with fresh produce, such as soft berries and lettuce. Even though food handlers are considered an important source for the introduction of hNoV into food chains, their contribution to public health risks associated with hNoV remains unknown. To assess to which extent food handlers contribute to the introduction and spread of hNoV in fresh produce chains quantitative virus transfer data are needed. We estimated transfer proportions of hNoV GI.4, GII.4, murine norovirus (MNV-1), a culturable surrogate of hNoV, and human adenovirus (hAdV-2), a human pathogen proposed as an indicator for human faecal pollution, between gloved fingertips and raspberries, strawberries, and lettuce, by quantitative RT-PCR and cell culture if applicable. Virus transfer proportions were corrected for virus-matrix specific recoveries, and variability and uncertainty of the parameters were estimated. Virus transfer from gloves to soft berries was generally lower as compared to lettuce, with mean transfer proportions ranging between 0.1 to 2.3 % and 9 to 10 % for infectious MNV-1 and hAdV-2, respectively. Transfer from produce to glove was mostly greater than transfer from glove to produce, adding to the likelihood of virus transfer due to cross contamination from contaminated produce via food handlers. HNoV GI.4 and hNoV GII.4 showed no significant difference between their mean transfer proportions.

Using the estimated transfer proportions, we studied the impact of low and high transfer proportions on the public health risk, based on a scenario in which a food handler picked raspberries with contaminated fingertips. Given the made assumptions, we could show that for a pathogen as infectious as hNoV, low transfer proportions may pose a greater public health risk than high transfer proportions, due to a greater viral spread. We demonstrated the potential of food handlers in spreading hNoV in food chains, showing that prevention of virus contamination on food handlers’ hands is crucial for food safety. Nevertheless, complete prevention of virus contamination on fresh produce cannot be achieved in reality, and reliable and effective intervention measures are consequently required. We estimated that, especially for low transfer proportions, a robust one log10-unit reduction of infectious hNoV on contaminated produce, and on food handlers’ hands, could lower the public health risk substantially. Using the obtained data in quantitative risk assessment will aid in elucidating the contribution of food handlers in hNoV transmission.

There is a growing concern of the “safety status” of South African agricultural produce, especially those that are consumed raw. If these products are contaminated they will impact not only the health of the final consumer but also that of people living next to rivers and the producers. This will immediately impact both the national and international “trading status” and cause a suspension of exports. Furthermore, there were, and still are regular articles in the local press reporting on the shocking “environmental status” of local rivers. The source of contamination of the agricultural products was identified as irrigation water that had been contaminated before irrigation took place. The health risks associated with the use of contaminated irrigation water on agricultural products thus became an increasing concern. The main objective of solicited research project was to do a quantitative investigation into the link between irrigation water quality and food safety. Based on the results from this research project, the microbial pollution levels of rivers and fresh produce monitored at selected sites in different provinces of South Africa over a period of 3-4 years were of an unacceptable microbiological standard and did not meet either the international or national faecal guidelines for safe irrigation or human consumption.

Other potential waterborne bacterial, virus and protozoan pathogens were frequently recovered from both the water and the produce. It was concluded that there is a high risk of exposure to pathogens when water from these rivers is used to irrigate produce that is consumed raw or without any further processing steps. In the research it was shown using phenotypic and genotypic identifications that direct water to produce linkages could be made. It was concluded that species from the surface of produce were present as a result of transfer from the contaminated irrigation water. There can now be no doubt that specific carry-over does take place. The potential of pathogenic organisms being transferred from irrigation water to the surface of fresh produce plus their ability to survive in these unfavourable conditions presents the scenario where consumers unknowingly face a high risk of being infected with harmful organisms when consuming fresh produce. Various recommendations for further research are made ranging from distribution profiles of pathogenic bacteria, seasonal variations and monitoring in irrigation water, development of effective quality assurance measures for detection of enteric viruses to investigation of effective on-farm treatment options of contaminated irrigation water.

Background and motivation
The presence of dense settlements on the river banks in the Western Cape give rise to water pollution of nearby rivers and severely affect the water quality downstream. Most of the water pollution can be attributed to inadequate sanitation in these settlements, severe overcrowding, as well as failing sewerage systems. The resultant burden of disease, loss of productivity and health costs should make the reduction of pollution and improvement of sanitation in Kayamandi a priority for the local authorities. The study site chosen was the Plankenbrug River running through Stellenbosch and the dense settlement of Kayamandi on its banks.

The promotion of a high level of food safety and quality is of major importance world-wide. Aspects of food quality such as genetically modified organisms (GMOs), food allergens and food authentication have become increasingly important while food-borne diseases caused by bacteria, viruses and parasites continue to be a significant problem. The application of real-time PCR is one of the most promising advances in food safety and quality providing rapid, reliable and quantitative results. In recent years real-time PCR has become a valuable alternative to traditional detection methods in the agricultural and food industries. The advantages of quantitative real-time PCR include speed, an excellent detection limit, selectivity, specificity, sensitivity and the potential for automation.

Written by experts in the field, this book is an indispensable manual for scientists in the food industry. The first section, Real-Time PCR Basics, provides an introduction to real-time PCR, discusses the use of PCR diagnostics in food science, describes the principles and methods of sample preparation, and covers the verification and control of PCR procedures. The eleven chapters in the second section, Food Microbiology, cover the use of real-time PCR to detect various pathogens including Salmonella, Listeria, E. coli, Campylobacter, Yersinia, Staphylococcus, Clostridium, viruses and parasites. Also included is a chapter on the standardisation of real-time PCR methods in food microbiology. The final section, Food Quality, covers the use of real-time PCR for the analysis of GMOs, food allergens and for the identification of animal or plant species.

An invaluable book for anyone involved in food science or the detection of foodborne pathogens and a recommended volume for all microbiology laboratories.

- explores methods of detection, surveilance and risk assessment of viruses in food and water
- considers virus transmission routes and control of food and water contamination
- highlights advances in the understanding of specific pathogens, including norovirus, hepatitis A and rotaviruses and the advances in vaccine development

Viruses can be highly infectious and are capable of causing widespread disease outbreaks. The significance of viral pathogens in food and waterborne illness is increasingly being recognised and viruses transferred by these routes are important areas of research. Viruses in food and water reviews the risks, surveillance and control of food and waterborne viral disease.

Part one provides an introduction to food and environmental virology. Part two goes on to explore methods of detection, surveillance and risk assessment of viruses in food and water; it includes chapters on molecular detection of viruses in foods and food processing environments, quality control in the analytical laboratory, and quantitative risk assessment for food and waterborne viruses. Part three focuses on virus transmission routes and control of food and water contamination. It contains chapters on fresh produce, shellfish and viral presence, and control methods in waste water and sewage. Finally, part four highlights particular pathogens including norovirus, hepatitis A and emerging zoonotic viruses.

Viruses in food and water is a standard reference book for microbiologists in academia, analytical labs and the food and water treatment industries, as well as environmental health professionals and researchers working on foodborne viruses.