Detection Of Human Enteric Viruses Research Articles

Detection of human enteric viruses in fresh produce of markets, farms and surface water used for irrigation in the Tehran, Iran

Considering the major role of vegetables in the transmission of gastrointestinal diseases, investigation of the presence of gastrointestinal viruses is particularly important for public health. Additionally, monitoring and investigating potential points of contamination at various stages of cultivation, harvesting, and distribution can be important in identifying the sources of transmission. This study was conducted with the aim of identifying norovirus, adenovirus, hepatitis A virus, hepatitis E virus, rotaviruses, and astroviruses in vegetable samples from the fields and fruit and vegetable centers of Tehran City, and to investigate their presence in irrigation water by RT-qPCR. This study was carried out in two phases: initial and supplementary. During phase I, a total of 3 farms and 5 fruit and vegetable centers and a total of 35 samples from farms, 102 samples from fruit and vegetable centers and 8 agricultural water samples were collected. Zero, 16 and 1 samples were positive for at least one of the viruses from each of the sources, respectively. During phase II, 88 samples from 23 farms, 226 samples from 50 fruit and vegetable centers and 16 irrigation water samples were collected, with 23, 57 and 4 samples were positive for at least one virus, respectively. Rotavirus was the most frequently identified virus among the samples, followed by NoV GII, NoV GI, AstV, and AdV. HAV and HEV were not detected in any of the tested samples. The results of this study suggest that there may be a wide presence of viruses in vegetables, farms, and fruit and vegetable centers in Tehran City, which could have significant consequences considering the fact that many of these foods are consumed raw. Additionally, the detection of some of these viruses in irrigation water suggests that this may be a potential route for viral contamination of produce.

A-293 Assessment of the Detection of Gastrointestinal Viruses Using Two Commercial Real-Time PCR Assays

Abstract Background Acute Gastroenteritis (AGE) are the second most common infectious diseases, only overtaken by Respiratory Tract Infections. Sign and symptoms of AGE illness include nausea, diarrhoea, vomits, fever and abdominal pain. In infants and children, gastroenteritis is one of the most important causes of morbidity and mortality all around the Globe. Since their identification in the 1970s, enteric viruses are considered a leading cause of gastroenteritis worldwide. Among these, rotaviruses (RV) are considered to be the main cause of AGE in young children, whereas noroviruses (NoV) affect people of all ages. In addition, sapoviruses (SaV), human astroviruses (hAstV), human adenoviruses (AdV) are also relevant. In the recent years, molecular methods, such as real-time PCR, have been widely used for detection of viral genome of enteric viruses due to their high sensitivity and specificity, becoming the gold standard. The aim of this study is the comparison of different commercial real-time PCR assays for the detection of human enteric viruses. Material and methods Three hundred and eighteen clinical stool samples were selected from different groups collected between 2016 and 2019, including specimens from symptomatic adults and symptomatic and asymptomatic children. Samples were stored at −20°C. The collection included a minimum of 25 samples positive for RV, 55 positives for AdV, 16 positives for AstV, 22 positives for SaV and 65 positives for NoV (31 for GI and 34 for GII). All specimens were extracted using the Maxwell®RSC Blood DNA Kit on the Maxwell RSC equipment (Promega). Samples were analysed using the following assays: RIDA®GENE Viral Stool Panel II, RIDA®GENE Sapovirus and RIDA®GENE Norovirus I & II (R-Biopharm), Vitassay qPCR kits for the detection of Rotavirus, Astrovirus, Norovirus GI, Norovirus GII, and Sapovirus. Results obtained with Vitassay were compared to R-Biopharm assays. Sensitivity and specificity were calculated using the software MetaDisc 1.4. Discrepant samples were tested in triplicate. Results After assessing the discrepant results, 29/114 samples were positive for RV in both RIDA®GENE and Vitassay kit. This latter reported 2 false negative (FN) and 4 false positive (FP) results when compared to RIDA®GENE. Sensitivity was 93.5% (78.6–99.2) and specificity was 95.2% (88.1–98.7). For AstV, 27/138 were positive in both kits. Vitassay qPCR reported 1 FN and 2 FP, obtaining sensitivity and specificity values of 96.4% (81.7–99.9) and 98.6% (94.9–99.8), respectively. Regarding SaV, Vitassay reported 100% (88.8%-100) of sensitivity and 97.8% (88.5–99.9) of specificity, as 1 FP was obtained. 30/89 samples tested for Norovirus GI were detected by Vitassay qPCR Norovirus GI and 25/89 RIDA®GENE Norovirus I & II (R-Biopharm). On the other hand, Vitassay qPCR Norovirus GII reported 31/81 positive samples for NoV I, whereas RIDA®GENE obtained 30 positive results. In both cases, sensitivity was of 100%, whereas specificity was over 92%. Conclusions Vitassay real-time PCR assays for the detection of different enteric viruses present high specificity and sensitivity, which is essential for a proper diagnosis and epidemiology assessment. In addition, Vitassay qPCR kit presents the advantage of being a ready-to-use lyophilised kit.

A reduced graphene oxide-Fe3O4 composite functionalized with cetyltrimethylammonium bromide for efficient adsorption of SARS-CoV-2 spike pseudovirus and human enteric viruses

The latent dangers of waterborne viral transmission have become a major public health concern. In this study, reduced graphene oxide (rGO)-Fe3O4 nanoparticles were decorated with cetyltrimethylammonium bromide (CTAB) to adsorb severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike pseudovirus and three human enteric viruses (HuNoV, HRV, and HAdV). The successful combination of CTAB with rGO-Fe3O4 was confirmed by transmission electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, zeta potential, Brunner-Emmet-Teller, and vibrating sample magnetometer measurements. The adsorption of HuNoV and HAdV followed pseudo-first-order kinetics, while that of HRV conformed to the pseudo-second-order model. CTAB-functionalized rGO-Fe3O4 exhibited exceptionally high adsorption of HuNoV, HRV, HAdV and SARS-CoV-2 spike pseudovirus, with maximum adsorption capacities of 3.55 × 107, 7.01 × 107, 2.21 × 107 and 6.92 × 106 genome copies mg−1, respectively. Moreover, the composite could effectively adsorb the four types of virus particles from coastal, tap, and river water. In addition, concentrating the virions using CTAB functionalized rGO-Fe3O4 composites before qPCR analysis significantly improved the detection limit. The results indicate that viruses are captured on the surface of CTAB functionalized rGO-Fe3O4 composites through electrostatic interactions and the intrinsic adsorption ability of rGO. Overall, CTAB-functionalized rGO-Fe3O4 composites are promising materials for the adsorption and detection of human enteric viruses as well as SARS-CoV-2 from complex aqueous environments.

Detection of human enteric viruses in water and shellfish samples collected in Sicily (Italy)

Detection of human enteric viruses in water and shellfish samples collected in Sicily (Italy)

Detection of Human Enteric Viruses in French Polynesian Wastewaters, Environmental Waters and Giant Clams.

Lack of wastewater treatment efficiency causes receiving seawaters and bivalve molluscan shellfish to become contaminated, which can lead to public health issues. Six wastewater samples, five seawater samples and three batches of giant clams from Tahiti (French Polynesia) were investigated for the presence of enteric viruses, but also if present, for the diversity, infectivity and integrity of human adenoviruses (HAdV). Enteroviruses (EV), sapoviruses (SaV) and human polyomaviruses (HPyV) were detected in all wastewater samples. In decreasing frequency, noroviruses (NoV) GII and HAdV, rotaviruses (RoV), astroviruses (AsV), NoV GI and finally hepatitis E viruses (HEV) were also observed. Nine types of infectious HAdV were identified. HPyV and EV were found in 80% of seawater samples, NoV GII in 60%, HAdV and SaV in 40% and AsV and RoV in 20%. NoV GI and HEV were not detected in seawater. Intact and infectious HAdV-41 were detected in one of the two seawater samples that gave a positive qPCR result. Hepatitis A viruses were never detected in any water types. Analysis of transcriptomic data from giant clams revealed homologues of fucosyltransferases (FUT genes) involved in ligand biosynthesis that strongly bind to certain NoV strains, supporting the giant clams ability to bioaccumulate NoV. This was confirmed by the presence of NoV GII in one of the three batches of giant clams placed in a contaminated marine area. Overall, all sample types were positive for at least one type of virus, some of which were infectious and therefore likely to cause public health concerns.

Detection of human enteric viruses from shellfish, vegetable and water samples collected in Sicily

Background: Numerous viruses of human or animal origin can spread in the environment and infect people. Foodborne and waterborne infections cause outbreaks effecting a large number of people and are a common cause of gastroenteritis. Among foods, an important role is played by raw and undercooked shellfishes and vegetables. The aim of this study was to evaluate the presence of viral contamination in some food and water samples collected in South of Italy (Sicily). Methods & Materials: The survey was conducted over a period of five years (2013–2017). A total of 49 vegetable and 24 water samples were collected. Eighty-seven fresh and frozen shellfishes were sampled from harvesting areas, restaurants, and markets during regional official control or checked at Veterinary Border Inspection Posts. Hepatitis A (HAV), Norwalk virus (NoVs) GI and GII genogroups, Adenovirus, Rotavirus, Enterovirus and Bocavirus were detected by nucleic acid amplification (end-point and Real Time PCR/RT-PCR) in shellfish, vegetable and water samples. Results: The shellfish samples showed high positivity for HAV (11.5%), NoV (8.04% GI, 6.89% GII) and Enterovirus (4.6%). Enteroviruses were detected positive particularly in the vegetable samples (22.4%) and NoV GII in water samples (16.6%). No HAV and Bocavirus genoma was detected in water and vegetable samples. Conclusion: Data on viral contamination of shellfish, vegetables and water provided information on the presence of environment contamination, chiefly in shellfish production areas, and were useful to generate a picture of the circulation of viral pathogens in local territory. Molecular surveillance of water and food samples showed that the exposure to environmental viruses could be a real and probably underestimated public health hazards.

Molecular detection of human enteric viruses circulating among children with acute gastroenteritis in Valencia, Venezuela, before rotavirus vaccine implementation

BackgroundThe role of rotavirus as main etiologic agent of diarrhea has been well documented worldwide, including in Venezuela. However, information about the prevalence of gastrointestinal viruses such as calicivirus, adenovirus and astrovirus is limited and the contribution of other agents as Aichi virus and klassevirus is largely unknown. To explore the etiological spectrum of diarrhea associated with agents other than rotaviruses, 227 stool samples from children under 5 years old with acute gastroenteritis, collected in Valencia (Venezuela) from 2001 to 2005, and previously tested as rotavirus-negative, were analyzed for caliciviruses, adenoviruses, astroviruses, Aichi viruses, klasseviruses, picobirnaviruses and enteroviruses by specific RT-PCRs.ResultsAt least one viral agent was detected in 134 (59%) of the samples analyzed, mainly from children under 24 months of age and most of them belonging to the lowest socioeconomic status. Overall, enterovirus was identified as the most common viral agent (37.9%), followed by calicivirus (23.3%), adenovirus (11.5%), astrovirus (3.5%), klassevirus (1.3%) and Aichi virus (0.4%), while no picobirnavirus was detected. Klasseviruses were found during 2004 and 2005 and Aichi viruses only in 2005, indicating their circulation in Venezuela; meanwhile, the rest of the viruses were detected during the whole study period. Coinfections with two or more viruses were found in 39 (29.1%) of the infected children, most under 24 months of age. Adenovirus was involved as the coinfecting agent in at least 46.9% of the cases, but no differences concerning socio-demographic variables were observed between the coinfected and the single infected children.ConclusionsThe results show that various enteric viruses, including enteroviruses, caliciviruses and adenoviruses, accounted for a significant proportion of infantile diarrhea cases in Venezuela before rotavirus vaccine implementation. In addition, emerging viruses as Aichi virus and klassevirus were found, indicating the need to continue monitoring their spreading into the communities. Efforts are needed to develop more accurate methods to identify the major causes of diarrhea and to provide tools for more effective preventive measures.

A one-step centrifugal ultrafiltration method to concentrate enteric viruses from wastewater

A one-step centrifugal ultrafiltration method was developed to enhance rapid detection of human enteric viruses and co-occurring viruses in wastewater. Samples were collected pre- and post-UV treatment at two full-scale tertiary municipal wastewater treatment plants in Calgary, Canada. Viruses were concentrated from 100mL wastewater samples through direct centrifugation using the Centricon Plus-70 ultrafilter. Seven viruses, including norovirus, rotavirus, sapovirus, astrovirus, enterovirus, adenovirus and JC virus, were tested using real-time quantitative PCR (rt-qPCR) and cell culture. All of the viruses were detected in pre- and post-UV samples by rt-qPCR, with rotavirus the most numerous (6.6 log10 GE copies/L). Infectious viruses, by cell culture, were found in all tested pre-UV samples but only in one post-UV sample. The results were comparable and consistent to that obtained using virus adsorption-elution method, indicating that the centrifugal ultrafiltration method is adequate to retain the viruses and maintain their infectivity during processing. As a simple, rapid and cost-effective method to screen wastewater viruses, this one-step centrifugal ultrafiltration method may serve as an effective approach to assess virus removal and gain knowledge of human virus activity during wastewater treatment.

Detection of Human Enteric Viruses in Freshwater from European Countries.

The transmission of water-borne pathogens typically occurs by a faecal-oral route, through inhalation of aerosols, or by direct or indirect contact with contaminated water. Previous molecular-based studies have identified viral particles of zoonotic and human nature in surface waters. Contaminated water can lead to human health issues, and the development of rapid methods for the detection of pathogenic microorganisms is a valuable tool for the prevention of their spread. The aims of this work were to determine the presence and identity of representative human pathogenic enteric viruses in water samples from six European countries by quantitative polymerase chain reaction (q-PCR) and to develop two quantitative PCR methods for Adenovirus 41 and Mammalian Orthoreoviruses. A 2-year survey showed that Norovirus, Mammalian Orthoreovirus and Adenoviruses were the most frequently identified enteric viruses in the sampled surface waters. Although it was not possible to establish viability and infectivity of the viruses considered, the detectable presence of pathogenic viruses may represent a potential risk for human health. The methodology developed may aid in rapid detection of these pathogens for monitoring quality of surface waters.

A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System.

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health.

A 1-Year Study on the Detection of Human Enteric Viruses in New Caledonia.

Human enteric viruses occur in high concentrations in wastewater and can contaminate receiving environmental waters. Due to the lack of data on the prevalence of enteric viruses in New Caledonia, the presence and the concentrations of enteric viruses in wastewater and seawater were determined. Untreated wastewater and seawater samples were collected monthly for 1 year from a wastewater treatment plant (WWTP) and from the WWTP's outlet, located directly on a popular recreational beach. Samples were tested for norovirus genogroups I and II (NoV GI and GII), astroviruses (AsV), sapoviruses (SaV), enteroviruses (EV), hepatitis A viruses (HAV), rotaviruses (RoV), human adenoviruses (HAdV) and human polyomaviruses (HPyV). To support these data, faecal samples from cases of gastroenteritis were tested for the first time for NoV and detected in the population. NoV GI, NoV GII, EV, SaV, HAdV and HPyV were detected in all wastewaters, RoV in 75% and AsV in 67%. HAV were not detected in wastewater. Overall, 92% of seawater samples were positive for at least one virus. HPyV were detected most frequently in 92% of samples and at concentrations up to 7.7 × 10(3) genome copies/L. NoV GI, NoV GII, EV, SaV, RoV and HAdV were found in 33, 66, 41, 33, 16 and 66% of seawater samples, respectively. AsV were not detected in seawater. This study reports for the first time the presence of NoV and other enteric viruses in New Caledonia and highlights the year-round presence of enteric viruses in the seawater of a popular beach.

Detection of human enteric viruses in Umgeni River, Durban, South Africa.

The prevalence of adenovirus (AdV), rotaviruses (RV) and enteroviruses (EV) in Umgeni River waters of Durban, South Africa was assessed qualitatively and quantitatively during April 2011 to January 2012 using polymerase chain reaction (PCR)/reverse transcription-polymerase chain reaction (RT-PCR), nested PCR and quantitative PCR (qPCR), as well as nested integrated cell culture PCR (nested ICC-PCR). The phylogenetic analysis of the adenovirus and enterovirus amplicons was also performed. The nested PCR results effectively detected the presence of AdV and EV in all water samples. The results of qPCR demonstrated that higher populations of EV and of AdV were widely found in the Umgeni River. Rotavirus could only be detected in the upper Umgeni River, mainly during drier seasons. Nested ICC-PCR further confirmed the presence of infectious AdV and EV particles in 100% of water samples using various cell lines. The present study identifies potential viral hazards of Umgeni River water for domestic water supply and recreational activities.

Development of a Luminex assay for the simultaneous detection of human enteric viruses in sewage and river water

Real time PCR (qPCR) is increasingly being used for viral detection in aquatic environments because it enables high specificity and sensitivity of detection. However, the limited number of fluorescent reporter dyes restricts its multiplex application. In this study, a multiplex Luminex assay was established for the simultaneous detection of human adenovirus (HAdV), human polyomavirus (HPyV), enterovirus (EV), rotavirus (RoV), norovirus GI (NoVGI) and norovirus GII (NoVGII). Different river water and wastewater samples were tested for the viruses using both qPCR and the multiplex Luminex xMAP assay. HAdV and HPyV were the most abundant in all environmental samples. HAdV was detected in all river water and wastewater samples, and HPyV was detected in 79% of river water and 95.8% of wastewater samples. The multiplex xMAP assay revealed high specificity and no cross-reactivity. Using the multiplex Luminex assay, the viral detection rates in river water samples were lower than the rates obtained by qPCR for all viruses. Conversely, in wastewater samples, the viral detection rates were the same for both methods. In addition, the analytical sensitivity of the monoplex Luminex assay was comparable to or lower than qPCR. Results suggest that the multiplex Luminex assay could be a reliable method for the simultaneous detection of viral pathogens in wastewater.

Enhanced microarray-based detection of human enteric viruses using cDNA targets

Enhanced microarray-based detection of human enteric viruses using cDNA targets

Detection of human enteric viruses in shellfish, vegetables, waters and environmental samples: a preliminary study

Detection of human enteric viruses in shellfish, vegetables, waters and environmental samples: a preliminary study

Detection of human enteric viruses in Japanese clams.

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.

Detection of human enteric viruses in shellfish collected in Tunisia

The aim of this study was to detect the main pathogenic human RNA enteric viruses able to persist in the environment such as astrovirus, enterovirus, norovirus and hepatitis A virus (HAV) in shellfish collected from two locations in northern Tunisia. Viruses were eluted from digestive tissues and concentrated by polyethylene glycol precipitation before nucleic acid extraction and purification. After checking for inhibitors, all viruses were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by hybridization. Overall, 83% of the samples were found positive for at least one virus. Astrovirus was detected in 61% of the samples, norovirus in 35% and HAV in 26%. Surprisingly, only one sample was found positive for enterovirus. The mean number of positive samples found in this study is in accordance with the data found in the literature, indicating that no real difference exists in this respect among countries studied. A notable exception is HAV, which reflects the epidemiological status of the population. This study highlights the interest to analyse shellfish samples from different production areas. These data will be helpful to understand virus circulation and to improve shellfish safety. The results, which confirm contamination, necessitate the development of appropriate studies and monitoring in all shellfish-producing countries.

Nested multiplex PCR assay for detection of human enteric viruses in shellfish and sewage

Environmental samples and contaminated shellfish present frequently low concentrations of more than one viral species. For this reason, a nested multiplex RT-PCR was developed for the detection of adenoviruses, enteroviruses and hepatitis A viruses in different environmental samples such as urban sewage and shellfish. This assay will save time and cost for detection of these enteric viruses with a smaller sample volume, which otherwise can be a limiting factor in routine analysis. The limit of detection was approximately 1 copy for adenovirus and 10 copies for enterovirus and hepatitis A virus per PCR reaction using titrated cell-cultured viruses as template material. In shellfish and environmental samples, this multiplex PCR was optimized to detect all three viruses simultaneously when the concentration of each virus was equal or lower than 1000 copies per PCR reaction. This is the level found predominantly in the environment and in shellfish when the numbers of fecal bacterial and phage indicators are low. The detection of human adenoviruses by PCR has been suggested as a molecular index of fecal contamination of human origin in the environment and food and the multiplex assay developed may be a tool for evaluating the presence of viral contamination in shellfish and water and to expand microbiological control to include viral markers.

Round-Robin Comparison of Methods for the Detection of Human Enteric Viruses in Lettuce

Five methods that detect human enteric virus contamination in lettuce were compared. To mimic multiple contaminations as observed after sewage contamination, artificial contamination was with human calicivirus and poliovirus and animal calici-virus strains at different concentrations. Nucleic acid extractions were done at the same time in the same laboratory to reduce assay-to-assay variability. Results showed that the two critical steps are the washing step and removal of inhibitors. The more reliable methods (sensitivity, simplicity, low cost) included an elution/concentration step and a commercial kit. Such development of sensitive methods for viral detection in foods other than shellfish is important to improve food safety.

Development of homologous viral internal controls for use in RT-PCR assays of waterborne enteric viruses

Enteric viruses often contaminate water sources causing frequent outbreaks of gastroenteritis. Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental and drinking water samples. RT-PCR provides a means to rapidly detect low levels of these viruses, but it is sensitive to inhibitors that are present in water samples. Inhibitors of RT-PCR are concentrated along with viruses during sample processing. While procedures have been developed to remove inhibitors, none of them completely remove all inhibitors from all types of water matrices. This problem requires that adequate controls be used to distinguish true from potentially false-negative results. To address this problem, we have developed homologous viral internal controls for hepatitis A virus (HAV), poliovirus, Norwalk virus and rotavirus. These internal controls can be used in RT-PCR assays for the detection of the above viruses by competitive amplification, thereby allowing the detection of false negatives in processed water samples. The internal controls developed in this study were successfully tested with virus-seeded environmental water sample concentrates.