Infectious Human Adenoviruses Research Articles

Human adenovirus in municipal solid waste leachate and quantitative risk assessment of gastrointestinal illness to waste collectors.

Leachate is a variable effluent from waste management systems generated during waste collection and on landfills. Twenty-two leachate samples from waste collection trucks and a landfill were collected from March to December 2019 in the municipality of Rio de Janeiro (Brazil) and were analyzed for Human Adenovirus (HAdV), bacterial indicators and physico-chemical parameters. For viral analysis, samples were concentrated by ultracentrifugation and processed for molecular analysis using QIAamp Fast DNA Stool mini kit® for DNA extraction followed by nested-PCR and qPCR/PMA-qPCR TaqMan® system. HAdV was detected by nested-PCR in 100% (9/9) and 83.33% (12/13) of the truck and landfill leachate samples, respectively. Viral concentrations ranged from 8.31×101 to 6.68×107 genomic copies per 100ml by qPCR and PMA-qPCR. HAdV species A, B, C, and F were characterized using nucleotide sequencing. HAdV were isolated in A549 culture cells in 100% (9/9) and 46.2% (6/13) from truck and landfill leachate samples, respectively. Regardless of the detection methods, HAdV concentration was predicted by the quantity of total suspended solids. A quantitative microbial risk assessment was performed to measure the probability of gastrointestinal (GI) illness attributable to inadvertent oral ingestion of truck leachate, revealing the higher probability of disease for the direct splashing into the oral cavity (58%) than for the gloved hand-to-mouth (33%). In a scenario where waste collectors do not wear gloves as protective personal equipment, the risk increases to 67%. This is the first study revealing infectious HAdV in solid waste leachate and indicates a potential health risk for waste collectors.

Applicability of infectivity assay for the quantification of infectious human adenovirus genotype 5 in UV-irradiated wastewater

AbstractThe use of infectivity assays in domestic wastewater samples is limited because of the concerns around cytotoxicity to host cells, thus, the UV inactivation efficiency of human adenoviruses (HAdVs) in wastewater remains unclear. In this study, a human adenovirus genotype five (HAdV-5) host cell line (A549 cells) was incubated with wastewater at varied dilutions from 1:1.5 to 1:4 (the ratio of wastewater to a mixture of wastewater and the cell culture medium) and the cytotoxicity was assessed by the cell morphology and viability. No change was observed in either cell viability or morphology in comparison with control samples, even at lowest dilution of 1:1.5, indicating the dilution allowed infectivity assays. The minimal degree of dilution to avoid cytotoxicity may differ with different water matrix. Consequently, the technique was applied to quantify spiked HAdV-5 after the UV disinfection of wastewater. A significant increase in UV disinfection efficiency was noted in wastewater and hydroxyl radicals (OH•) produced by the photosensitization of dissolved organic matter were suggested to be responsible for the enhancement. This study indicated that dilution can be a simple solution to avoid cytotoxicity, and UV inactivation may be enhanced in wastewater due to OH• radicals produced by UV radiation.

Decay of infectious adenovirus and coliphages in freshwater habitats is differentially affected by ambient sunlight and the presence of indigenous protozoa communities

BackgroundSanitary quality of recreational waters worldwide is assessed using fecal indicator bacteria (FIB), such as Escherichia coli and enterococci. However, fate and transport characteristics of FIB in aquatic habitats can differ from those of viral pathogens which have been identified as main etiologic agents of recreational waterborne illness. Coliphages (bacteriophages infecting E. coli) are an attractive alternative to FIB because of their many morphological and structural similarities to viral pathogens.MethodsIn this in situ field study, we used a submersible aquatic mesocosm to compare decay characteristics of somatic and F+ coliphages to those of infectious human adenovirus 2 in a freshwater lake. In addition, we also evaluated the effect of ambient sunlight (and associated UV irradiation) and indigenous protozoan communities on decay of somatic and F+ coliphage, as well as infectious adenovirus.ResultsOur results show that decay of coliphages and adenovirus was similar (p = 0.0794), indicating that both of these bacteriophage groups are adequate surrogates for decay of human adenoviruses. Overall, after 8 days the greatest log10 reductions were observed when viruses were exposed to a combination of biotic and abiotic factors (2.92 ± 0.39, 4.48 ± 0.38, 3.40 ± 0.19 for somatic coliphages, F+ coliphages and adenovirus, respectively). Both, indigenous protozoa and ambient sunlight, were important contributors to decay of all three viruses, although the magnitude of that effect differed over time and across viral targets.ConclusionsWhile all viruses studied decayed significantly faster (p < 0.0001) when exposed to ambient sunlight, somatic coliphages were particularly susceptible to sunlight irradiation suggesting a potentially different mechanism of UV damage compared to F+ coliphages and adenoviruses. Presence of indigenous protozoan communities was also a significant contributor (p value range: 0.0016 to < 0.0001) to decay of coliphages and adenovirus suggesting that this rarely studied biotic factor is an important driver of viral reductions in freshwater aquatic habitats.

Infectious human adenoviruses are shed in urine even after disappearance of urethral symptoms.

BackgroundUrethritis is a common sexually transmitted disease, and human adenoviruses (HAdVs) have been found to be associated with nonchlamydial nongonococcal urethritis. However, the level and viability of HAdV in the urine of patients with urethritis remain unclear.MethodsMale patients with urethritis and an asymptomatic group were screened using their First-void urine (FVU) for urethritis-related pathogens to identify those with HAdV DNA. FVU and gargle fluid were collected from all patients including from those in the asymptomatic group. A swab of eye discharge was also collected from patients with eye symptoms. The pharyngeal and/ or ocular fluid was also screened only in cases in which FVU was positive for HAdV DNA. HAdVs were isolated using A549 cell lines and typed by sequencing, and viral shedding during 2 years was quantified using real-time PCR. The prevalence of HAdV was assessed in the urethritis and asymptomatic groups, and viral load, isolated HAdV types, and urethral symptoms were compared between the groups.ResultsThe positive detection rate of HAdV DNA was significantly higher in the urethritis group than in the asymptomatic group. Of 398 patients with urethritis, HAdV was isolated in all 32 cases (23 cases in which only HAdV DNA was detected with a mean of 2 × 109 copies/mL in urine samples). Of 124 control cases, one had HAdV monoinfection. The most frequently detected HAdV type was 56, followed by types 37 and 64. Regarding the relationship between symptoms and isolated HAdVs, the virus was isolated for up to 12 days after urethritis symptoms disappeared.ConclusionsHAdVs were significantly detected and isolated from the FVU of patients with urethritis. Furthermore, high levels of infectious HAdVs are excreted in urine for a long period even after urethritis symptoms disappear.

Mineral Waste Containing High Levels of Iron from an Environmental Disaster (Bento Rodrigues, Mariana, Brazil) is Associated with Higher Titers of Enteric Viruses.

Although the effects of heavy metals on the behavior, including infectivity, of bacteria have been studied, little information is available about their effects on enteric viruses. We report an investigation of effects on the biosynthesis of human adenoviruses (HAdV) and hepatitis A (HAV) of waters contaminated with mineral waste following an environmental disaster in Mariana City, Minas Gerais State, Brazil. The study area was affected on November 5, 2015, by 60million m3 of mud (containing very high concentrations of iron salts) from a mining reservoir (Fundão), reaching the Gualaxo do Norte River (sites evaluated in this study), the "Rio Doce" River and finally the Atlantic Ocean. We found substantial counts of infectious HAdV and HAV (by qPCR) in all sampled sites from Gualaxo do Norte River, indicating poor basic sanitation in this area. The effects of iron on viral infection processes were evaluated using HAdV-2 and HAV-175, as DNA and RNA enteric virus models, respectively, propagated in the laboratory and exposed to this contaminated water. Experiments in field and laboratory scales found that the numbers of plaque forming units (PFU) of HAdV and HAV were significantly higher in contaminated water with high iron concentrations than in waters with low iron concentration (< 20µg/L of iron). These findings indicate that iron can potentiate enteric virus infectivity, posing a potential risk to human and animal health, particularly during pollution disasters such as that described here in Mariana, Brazil.

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.

Quantification of human adenovirus and norovirus in river water in the north-east of France.

Human adenoviruses (HAdVs) are a major cause of infection and have been proposed as viral indicators of water quality. Human noroviruses (NoV) are the main cause of viral acute gastroenteritis. Quantitative data on the environmental prevalence of both viruses are needed. The genomes of HAdVs enteric adenovirus type 41 (HAdV41) and noroviruses of genogroups I and II (NoV GGI and GGII) were quantified over a 6-month period in a river located in north-eastern France. The samples were collected downstream from the discharge of a wastewater treatment plant. The viruses were concentrated using a glass wool method and the viral genomes were quantified using digital droplet PCR (ddPCR). All river water samples (15/15) were positive for the genomes of HAdVs, HAdV41, NoV GGI and NoV GGII. Concentrations of HAdVs, HAdV41 and NoV GII genomes were similar and HAdV41 represented ~ 80% of HAdVs. Infectious HAdVs were quantified in these samples using an integrated cell culture-quantitative PCR method (ICC-qPCR); they were detected in 93% (14/15) and quantified in 53% (8/15) of the samples. Thus, infectious HAdVs represented 0.3 to 12.2% of total HAdV particles detected by ddPCR. Infectious HAdV41 particles were found in 73% (11/15) of the samples. This common presence of pathogenic enteric viruses underlines the impact of wastewater discharge on quality of surface waters and may constitute a threat for human health. The relative abundance of genome of HAdV41 underlines the need for studies focusing on the specific detection of its infectious forms along water cycle.

Viral disinfection of organic fresh produce comparing Polyphenon 60 from green tea with chlorine

Contamination of fresh produce with enteric viruses is a matter of concern because viruses may persist in food surfaces under normal storage conditions for long periods. Chlorinated compounds are the most common substances used to disinfect fresh produce but they present some disadvantages such as the formation of by-products. In this study, green tea extract (GTE), a natural compound from Camellia sinensis, was tested to disinfect fresh produce contaminated with 8 log10 of human adenoviruses (HAdV) and 6 log10 of infectious murine noroviruses (MNV), a surrogate of human norovirus. Chorine was also employed to compare the action of GTE with a common disinfectant. Results demonstrated that, although GTE was not as efficient as chlorine, it demonstrated good effectiveness to decrease the amount HAdV in fresh produce. Both disinfectants were more effective than just using water to reduce the load of infectious HAdV. It was also observed that both chlorine and GTE were more effective to remove HAdV than MNV, probably due to different interactions of the viruses with the plant tissues.

Detection of human adenoviruses in organic fresh produce using molecular and cell culture-based methods

The consumption of organic fresh produce has increased in recent years due to consumer demand for healthy foods without chemical additives. However, the number of foodborne outbreaks associated with fresh produce has also increased. Contamination of food with enteric viruses is a major concern because the viruses have a low infectious dose and high persistence in the environment. Human adenovirus (HAdV) has been proposed as a good marker of faecal contamination. Therefore, the aim of this study was to evaluate the efficiency of the plaque assay (PA), real time PCR (qPCR) and integrated cell culture-RT-qPCR (ICC-RT-qPCR) for the recovery of HAdV from artificially and naturally contaminated fresh produce. Organic lettuce, strawberries and green onions were selected because these fresh products are frequently associated with foodborne outbreaks. The virus extraction efficiencies from artificially contaminated samples varied from 2.8% to 32.8% depending on the food matrix and the quantification method used. Although the HAdV recoveries determined by qPCR were higher than those determined by PA and ICC-RT-qPCR, PA was defined as the most reproducible method. The qPCR assays were more sensitive than the PA and ICC-RT-qPCR assays; however, this technique alone did not provide information about the viability of the pathogen. ICC-RT-qPCR was more sensitive than PA for detecting infectious particles in fresh produce samples. HAdV genome copies were detected in 93.3% of the analysed naturally contaminated samples, attesting to the common faecal contamination of the fresh produce tested. However, only 33.3% of the total samples were positive for infectious HAdV particles based on ICC-RT-qPCR. In conclusion, this study reported that HAdV can be an efficient viral marker for fresh produce contamination. Good detection of infectious HAdV was obtained with the ICC-RT-qPCR and PA assays. Thus, we suggest that the ICC-RT-qPCR and PA assays should be considered when quantitative microbial risk assessment (QMRA) studies are required and to establish reliable food safety guidelines.

Human and animal enteric virus in groundwater from deep wells, and recreational and network water.

This study was designed to assess the presence of human adenovirus (HAdV), rotavirus-A (RVA), hepatitis A virus (HAV), and porcine circovirus-2 (PCV2) in groundwater from deep wells, and recreational and network waters. The water samples were collected and concentrated and the virus genomes were assessed and quantified by quantitative PCR (qPCR). Infectious HAdV was evaluated in groundwater and network water samples by integrated cell culture using transcribed messenger RNA (mRNA) (ICC-RT-qPCR). In recreational water samples, HAdV was detected in 100 % (6/6), HAV in 66.6 % (4/6), and RVA in 66.6 % (4/6). In network water, HAdV was detected in 100 % (6/6) of the samples (these 83 % contained infectious HAdV), although HAV and RVA were not detected and PCV2 was not evaluated. In groundwater from deep wells, during rainy period, HAdV and RVA were detected in 80 % (4/5) of the samples, and HAV and PCV2 were not detected; however, during dry period, HAdV and RVA were detected in 60 % (3/5), HAV in only one sample, and PCV2 in 60 % (4/5). In groundwater, all samples contained infectious HAdV. PCV2 presence in groundwater is indicative of contamination caused by swine manure in Concórdia, Santa Catarina, Brazil. The disinfection of human and animal wastes is urgent, since they can contaminate surface and groundwater, being a potential threat for public and animal health.

Enteric viruses in surface water and sediment samples from the catchment area of Peri Lagoon, Santa Catarina State, Brazil.

This paper aims to quantify human adenovirus (HAdV), rotavirus species A (RVA), and hepatitis A virus (HAV) in surface water and sediments and to determine the viability of HAdV in these samples. Water and sediment samples were collected, and HAdV, RVA, and HAV were quantified by real-time polymerase chain reaction (PCR); HAdV was also evaluated for infectivity by a plaque assay (PA). For the water samples, HAdV was detected in 70.8% of the summer collections, with 82.4% containing infectious HAdV; the HAdV incidence in winter was 62.5%. For the sediment samples, the incidence of HAdV was 37.5% in the summer collections, with 66.7% containing infectious HAdV; the HAdV incidence in winter was 37.5%. RVA was detected in 20.8 and 45.8% of surface water samples collected in summer and winter, respectively, and 8.3 and 12.5% of sediment samples collected in summer and winter, respectively. HAV was detected only in surface waters, with 54.8 and 12.5% positivity in summer and winter samples, respectively. This study demonstrated that enteric viruses are present in water and sediments and that the presence of infectious viruses should be investigated whenever possible for quantitative microbial risk assessment studies. Combined analyses of water and sediments are important for reliable public health risk analysis of recreational and lagoon waters.

Behaviour and recovery of human adenovirus from tropical sediment under simulated conditions

This study assessed the contributions of pH and organic matter (OM) on the recovery of infectious human adenovirus 5 (HAdV-5) and genome copies (GCs) in waters that were artificially contaminated with tropical soil. The use of a mathematical equation was proposed based on the flocculation index of clay to assess the recovery of total GCs in these controlled assays. The results suggest that solids in the water reduced the viral genome copy loads per millilitre (GC·mL−1) and viral infectivity. OM did not influence the GC·mL−1 recovery rate (p>0.05) but led to a 99% (2 log10) reduction in plaque-forming unit counts per millilitre (PFU/mL), which indicates that infectivity and gene integrity were non-related parameters. Our findings also suggest that acidic pH levels hinder viral inactivation and that clay is the main factor responsible for the interactions of HAdV-5 with soil. These findings may be useful for future eco-epidemiological investigations and studies of viral inactivation or even as parameters for future research into water quality analysis and water treatment.

Human Adenovirus Diversity in Water Samples Using a Next-Generation Amplicon Sequencing Approach.

This study aims to establish a straightforward and original workflow for high-throughput typing of human adenoviruses (HAdVs) in environmental samples. Occurrence of HAdVs in water is well documented worldwide, but data on diversity of HAdV types circulating in water are scarcely available. Here, the characterisation of viral particles was performed by determination of amplicon sequences using a next-generation sequencing (NGS) approach. Adenoviral DNA was either directly isolated from wastewater or river water concentrates or after a cell culture passage. Genome amplification targeted a hyper variable region of the hexon gene, allowing the discrimination of the 54 human adenoviral types described until now. After read generation on the benchtop MiSeq platform (Illumina), data were analysed using the Mothur software for identification of all HAdV species and types simultaneously present in a unique sample. NGS results showed a relatively wide HAdV diversity of up to six types in one sample, whereas Sanger sequencing always only retrieved the dominant one. Detected types included HAdV-1, HAdV-2, HAdV-3, HAdV-6, HAdV-12, HAdV-31, HAdV-40 and HAdV-41, HAdV-41 being the most abundant in tested samples. In addition, the influence of the cell line (A549 vs 293A cells) on the infectious HAdV typing results was clearly determined. The 293A appeared to be the most suitable cell line allowing the detection of a larger diversity of infectious HAdVs and reflecting a more realistic initial species distribution than using the A549 cells. These findings demonstrated the feasibility of amplicon sequencing NGS approach to identify viruses in complex environmental water samples.

Assessment of water quality in a border region between the Atlantic forest and an urbanised area in Rio de Janeiro, Brazil.

The preservation of water resources is one of the goals of the designation of parks that act as natural reservoirs. In order to assess the impact of the presence of humans in an environmental preservation area bordering urban areas, the presence of four pathogenic enteric viruses [group A rotavirus (RV-A), norovirus (NoV), human adenoviruses (HAdV), and hepatitis A virus (HAV)], as well as the physico-chemical parameters, and Escherichia coli levels were assessed in riverine water samples. From June 2008 to May 2009, monthly monitoring was performed along the Engenho Novo River. RV-A, NoV, and HAdV were observed in 29% (31/108) of the water samples, with concentrations of up to 10(3) genome copies/liter. The natural occurrence of infectious HAdV was demonstrated by Integrated Cell Culture-PCR (ICC-PCR). This study confirms the suitability of using the detection of fecal-oral transmitted viruses as a marker of human fecal contamination in water matrices and indicates the spread of pathogenic viruses occurring in an alleged area of environmental protection.

Adenovirus and Norovirus Contaminants in Commercially Distributed Shellfish

Shellfish complying with European Regulations based on quantification of fecal bacterial indicators (FIB) are introduced into markets; however, information on viruses, more stable than FIB, is not available in the literature. To assess the presence of noroviruses (NoVs) GI and GII and human adenoviruses (HAdV) in domestic and imported mussels and clams (n=151) their presence was analyzed during winter seasons (2004-2008) in north-west Spanish markets through a routine surveillance system. All samples tested negative for NoV GI and 13% were positive for NoV GII. The role of HAdV as viral indicator was evaluated in 20 negative and 10 positive NoV GII samples showing an estimated sensitivity and specificity of HAdV to predict the presence of NoV GII of 100 and 74% (cut-off 0.5). The levels of HAdV and NoVs and the efficiency of decontamination in shellfish depuration plants (SDP) were evaluated analyzing pre- and post-depurated mussels collected in May-June 2010 from three different SDP. There were no statistically significant differences in the prevalence and quantification of HAdV between pre- and post-depurated shellfish and between seawater entering and leaving the depuration systems. Moreover, infectious HAdV were detected in depurated mussels. These results confirm previous studies showing that current controls and depuration treatments limiting the number of FIB do not guarantee the absence of viruses in shellfish.

Evaluation and molecular characterization of human adenovirus in drinking water supplies: viral integrity and viability assays

BackgroundHuman adenoviruses (HAdVs) are the second-leading cause of childhood gastroenteritis worldwide. This virus is commonly found in environmental waters and is very resistant to water disinfection and environmental stressors, especially UV light inactivation. Molecular techniques, such as PCR-based methods (Polymerase Chain Reaction), are commonly used to detect and identify viral contamination in water, although PCR alone does not allow the discrimination between infectious and non-infectious viral particles. A combination of cell culture and PCR has allowed detection of infectious viruses that grow slowly or fail to produce cytopathic effects (CPE) in cell culture. This study aimed to assess the integrity and viability of human adenovirus (HAdV) in environmental water and evaluate circulating strains by molecular characterization in three sites of the water supply in Florianópolis, Santa Catarina Island, Brazil: Peri Lagoon water, spring source water, and water from the public water supply system.MethodsWater samples were collected, concentrated and HAdV quantified by real-time PCR. Viral integrity was evaluated by enzymatic assay (DNase I) and infectivity by plaque assay (PA) and integrated cell culture using transcribed mRNA (ICC-RT-qPCR). Samples containing particles of infectious HAdV were selected for sequencing and molecular characterization.ResultsThe analyzed sites contained 83, 66 and 58% undamaged HAdV particles (defined as those in which the genetic material is protected by the viral capsid) at Peri Lagoon, spring source water and public supply system water, respectively. Of these, 66% of the particles (by PA) and 75% (by ICC-RT-qPCR) HAdV were shown to be infectious, due to being undamaged in Peri Lagoon, 33% (by PA) and 58% (by ICC-RT-qPCR) in spring source water and 8% (by PA) and 25% (by ICC-RT-qPCR) in the public water supply system. ICC-RT-qPCR, a very sensitive and rapid technique, was able to detect as low as 1 × 102 HAdV genome copies per milliliter of infectious viral particles in the environmental water samples. The molecular characterization studies indicated that HAdV-2 was the prevalent serotype.ConclusionsThese results indicate a lack of proper public health measures. We suggest that HAdV can be efficiently used as a marker of environmental and drinking water contamination and ICC-RT-qPCR demonstrated greater sensitivity and speed of detection of infectious viral particles compared to PA.

Effect of temperature and sunlight on the stability of human adenoviruses and MS2 as fecal contaminants on fresh produce surfaces

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.

Two-day detection of infectious enteric and non-enteric adenoviruses by improved ICC-qPCR

In order to provide a more suitable response to public health concerns, we improved the detection of infectious human adenoviruses in water by optimising the commonly used integrated cell culture-PCR method. Risk evaluation studies seek for rapid detection of infectious adenoviruses, including the enteric types 40 and 41 that are considered as the second most common agents of gastroenteritis in children next to rotaviruses. The here-employed 293A cell line used for infectious status assessment showed its ability to multiply adenoviruses including type 41. Two modifications were moreover applied to the workflow for viral detection. The first occurred at the nucleic acid extraction step performed directly on all infected cells, while the second was the application of real-time quantitative PCR as detection tool. All adaptations led to a 3-day reduction of the response delay and an improved sensitivity especially for the enteric adenoviral types. The infectious status of laboratory strain types 2 and 41 was demonstrated by a more than 2-log10 increase in genome quantity. These conclusions were confirmed and reinforced by the analysis of water samples applying the improved assay. Naturally occurring infectious adenoviruses were detected in wastewater and river water, within 2 days. Types belonging to the species human adenoviruses C and type 31 were observed, but the most frequently identified type was 41 (71 % of identified sequences, n = 34). This highlights the usefulness of our method for a wide range of types, and especially for the most prevalent and public health-relevant enteric adenoviruses.

An improved infectivity assay combining cell culture with real-time PCR for rapid quantification of human adenoviruses 41 and semi-quantification of human adenovirus in sewage

A protocol for the rapid detection and semi-quantification of human enteric adenovirus based on the quantification of viral mRNA during cell culture infectivity assay was developed. Infectivity assays for adenovirus incorporated cell culture and reverse transcription real-time PCR, where viral mRNA detection was used to monitor the progress of adenovirus infection (CC/mRNA qPCR). The cell line used was G293. This specific infectivity assay was calibrated against different initial concentrations of human adenovirus 41. In addition, the expression of the host's housekeeping (HK) gene, GAPDH, served as internal control for the mRNA assays for quality assurance of the mRNA extraction and reverse transcription steps. The concentrations of infectious human adenoviruses in different sewage samples were estimated semi-quantitatively using the CC/mRNA qPCR assay and calibration obtained for adenovirus 41. A linear relationship between concentrations of viral mRNA (hexon gene) and infectious units was observed between 107 to 101 infectious units per assay (R2 = 0.97) in samples analyzed 3–5 days post infection. The expressions of host cell GAPDH gene were not significantly affected by infections with different concentrations of human adenovirus 41, and between virus positive and negative cell cultures (p > 0.1). The estimated concentrations of human adenoviruses in sewage samples ranged between 102 to 103 mRNA-IU/L. Most of the viruses detected in sewage samples were from human adenovirus species F. The CC/mRNA qPCR assay can be used for quantifying infectious human adenovirus 41, estimating the levels of human adenoviruses in sewage samples, and applied to other sample settings. The CC/mRNA qPCR protocol described here represents an improvement in the detection of human enteric adenoviruses by reducing incubation time (5 days); whereas the conventional cell culture method requires longer incubation periods (10–20 days). More importantly, this protocol can be used to more rapidly and semi-quantitatively estimate the levels of infectious human adenoviruses in environmental samples.

Detection and molecular identification of human adenoviruses and enteroviruses in wastewater from Morocco

Reclaimed wastewater is a considerable water resource in Morocco. Its agricultural reuse requires an assessment of viral contamination. The aim of this study was to detect both infectious and noninfectious human adenoviruses (HAdV) and enteroviruses (EV) in raw wastewater and treat effluent from wastewater treatment plants (WWTPs) and domestic sewage in Morocco. A total of 22 samples were analysed. A polyethylene glycol (PEG) precipitation method was used, followed by integrated cell culture-PCR (ICC-PCR) using two cell lines: human rhabdomyosarcoma tumour tissue and laryngeal carcinoma cells (RD and Hep2 cells). Furthermore, viral genome amplification was confirmed by sequencing. HAdV were detected in 10 (45·5%) of the 22 samples involving two species: HAdV-B and HAdV-D. EV was detected in 5 (23%) samples belonging to Coxsackievirus B5 and poliovirus vaccine strain (Sabin 2). Human adenoviruses and EV were detected in the analysed samples from two WWTPs and HAdV in domestic sewage. This work is the first study in Morocco using cell culture, PCR and sequencing of enteric viruses from wastewater. The presence of infectious HAdV and EV in treated effluent emphasizes the need of wastewater treatment surveillance.