Newly recruited intraepithelial Ly6A+CCR9+CD4+ T cells protect against enteric viral infection.

The intestinal epithelium comprises the body's largest surface exposed to viruses. Additionally, the gut epithelium hosts a large population of intraepithelial T lymphocytes, or IELs, although their role in resistance against viral infections remains elusive. By fate-mapping T cells recruited to the murine intestine, we observed an accumulation of newly recruited CD4+ T cells after infection with murine norovirus CR6 and adenovirus type-2 (AdV), but not reovirus. CR6- and AdV-recruited intraepithelial CD4+ T cells co-expressed Ly6A and chemokine receptor CCR9, exhibited T helper 1 and cytotoxic profiles, and conferred protection against AdV in vivo and in an organoid model in an IFN-γ-dependent manner. Ablation of the T cell receptor (TCR) or the transcription factor ThPOK in CD4+ T cells prior to AdV infection prevented viral control, while TCR ablation during infection did not impact viral clearance. These results uncover a protective role for intraepithelial Ly6A+CCR9+CD4+ T cells against enteric adenovirus.

Human Sapovirus Replication in Human Intestinal Enteroids.

Human sapoviruses (HuSaVs), like human noroviruses (HuNoV), belong to the Caliciviridae family and cause acute gastroenteritis in humans. Since their discovery in 1976, numerous attempts to grow HuSaVs in vitro were unsuccessful until 2020, when these viruses were reported to replicate in a duodenal cancer cell-derived line. Physiological cellular models allowing viral replication are essential to investigate HuSaV biology and replication mechanisms such as genetic susceptibility, restriction factors, and immune responses to infection. In this study, we demonstrate replication of two HuSaV strains in human intestinal enteroids (HIEs) known to support the replication of HuNoV and other human enteric viruses. HuSaVs replicated in differentiated HIEs originating from jejunum, duodenum and ileum, but not from the colon, and bile acids were required. Between 2h and 3 to 6 days postinfection, viral RNA levels increased up from 0.5 to 1.8 log10-fold. Importantly, HuSaVs were able to replicate in HIEs independent of their secretor status and histo-blood group antigen expression. The HIE model supports HuSaV replication and allows a better understanding of host-pathogen mechanisms such as cellular tropism and mechanisms of viral replication. IMPORTANCE Human sapoviruses (HuSaVs) are a frequent but overlooked cause of acute gastroenteritis, especially in children. Little is known about this pathogen, whose successful in vitro cultivation was reported only recently, in a cancer cell-derived line. Here, we assessed the replication of HuSaV in human intestinal enteroids (HIEs), which are nontransformed cultures originally derived from human intestinal stem cells that can be grown in vitro and are known to allow the replication of other enteric viruses. Successful infection of HIEs with two strains belonging to different genotypes of the virus allowed discovery that the tropism of these HuSaVs is restricted to the small intestine, does not occur in the colon, and replication requires bile acid but is independent of the expression of histo-blood group antigens. Thus, HIEs represent a physiologically relevant model to further investigate HuSaV biology and a suitable platform for the future development of vaccines and antivirals.

Model correction of diagnostic coding-based RSV incidence for children 0-4 years in the US.

BACKGROUND: Although administrative claims data have a high degree of completeness, not all medically attended Respiratory Syncytial Virus-associated lower respiratory tract infections (RSV-LRTIs) are tested or coded for their causative agent. We sought to determine the attribution of RSV to LRTI in claims data via modeling of temporal changes in LRTI rates against surveillance data. METHODS: We estimated the weekly incidence of LRTI (inpatient, outpatient, and total) for children 0-4 years using 2011-2019 commercial insurance claims, stratified by HHS region, matched to the corresponding weekly NREVSS RSV and influenza positivity data for each region, and modelled against RSV, influenza positivity rates, and harmonic functions of time assuming negative binomial distribution. LRTI events attributable to RSV were estimated as predicted events from the full model minus predicted events with RSV positivity rate set to 0. RESULTS: Approximately 42% of predicted RSV cases were coded in claims data. Across all regions, the percentage of LRTI attributable to RSV were 15-43%, 10-31%, and 10-31% of inpatient, outpatient, and combined settings, respectively. However, when compared to coded inpatient RSV-LRTI, 9 of 10 regions had improbable corrected inpatient LRTI estimates (predicted RSV/coded RSV ratio < 1). Sensitivity analysis based on separate models for PCR and antigen-based positivity showed similar results. CONCLUSIONS: Underestimation based on coding in claims data may be addressed by NREVSS-based adjustment of claims-based RSV incidence. However, where setting-specific positivity rates is unavailable, we recommend modeling across settings to mirror NREVSS's positivity rates which are similarly aggregated, to avoid inaccurate adjustments.

A soft-sensor approach for predicting an indicator virus removal efficiency of a pilot-scale anaerobic membrane bioreactor (AnMBR).

The anaerobic membrane bioreactor (AnMBR) is a promising technology for not only water reclamation but also virus removal; however, the virus removal efficiency of AnMBR has not been fully investigated. Additionally, the removal efficiency estimation requires datasets of virus concentration in influent and effluent, but its monitoring is not easy to perform for practical operation because the virus quantification process is generally time-consuming and requires specialized equipment and trained personnel. Therefore, in this study, we aimed to identify the key, monitorable variables in AnMBR and establish the data-driven models using the selected variables to predict virus removal efficiency. We monitored operational and environmental conditions of AnMBR in Sendai, Japan and measured virus concentration once a week for six months. Spearman's rank correlation analysis revealed that the pH values of influent and mixed liquor suspended solids (MLSS) were strongly correlated with the log reduction value of pepper mild mottle virus, indicating that electrostatic interactions played a dominant role in AnMBR virus removal. Among the candidate models, the random forest model using selected variables including influent and MLSS pH outperformed the others. This study has demonstrated the potential of AnMBR as a viable option for municipal wastewater reclamation with high microbial safety.

Late-onset enteric virus infection associated with hepatitis (EVAH) in transplanted SCID patients.

BACKGROUND: Allogenic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) are potentially curative treatments for severe combined immunodeficiency (SCID). Late-onset posttreatment manifestations (such as persistent hepatitis) are not uncommon. OBJECTIVE: We sought to characterize the prevalence and pathophysiology of persistent hepatitis in transplanted SCID patients (SCIDH+) and to evaluate risk factors and treatments. METHODS: We used various techniques (including pathology assessments, metagenomics, single-cell transcriptomics, and cytometry by time of flight) to perform an in-depth study of different tissues from patients in the SCIDH+ group and corresponding asymptomatic similarly transplanted SCID patients without hepatitis (SCIDH-). RESULTS: Eleven patients developed persistent hepatitis (median of 6 years after HSCT or GT). This condition was associated with the chronic detection of enteric viruses (human Aichi virus, norovirus, and sapovirus) in liver and/or stools, which were not found in stools from the SCIDH- group (n = 12). Multiomics analysis identified an expansion of effector memory CD8+ T cells with high type I and II interferon signatures. Hepatitis was associated with absence of myeloablation during conditioning, split chimerism, and defective B-cell function, representing 25% of the 44 patients with SCID having these characteristics. Partially myeloablative retransplantation or GT of patients with this condition (which we have named as "enteric virus infection associated with hepatitis") led to the reconstitution of T- and B-cell immunity and remission of hepatitis in 5 patients, concomitantly with viral clearance. CONCLUSIONS: Enteric virus infection associated with hepatitis is related to chronic enteric viral infection and immune dysregulation and is an important risk for transplanted SCID patients with defective B-cell function.

Occurrence of human pathogenic viruses in drinking water and in its sources: A review.

Since many waterborne diseases are caused by human pathogenic viruses, virus monitoring of drinking water (DW) and DW sources is crucial for public health. Therefore, the aim of this review was to describe the occurrence of human pathogenic viruses in DW and DW sources; the occurrence of two viruses proposed as novel indicators of human faecal contamination (Pepper mild mottle virus and Tobacco mosaic virus) was also reported. This research was focused on articles that assessed viral occurrence using molecular methods in the surface water used for DW production (SW-D), groundwater used for DW production (GW-D), DW and bottled-DW (BW). A total of 1544 studies published in the last 10 years were analysed, and 79 were ultimately included. In considering the detection methods, filtration is the most common concentration technique, while quantitative polymerase chain reaction is the most common quantification technique. Regarding virus occurrence in SW-D, GW-D, and DW, high percentages of positive samples were reported for adenovirus, polyomavirus and Pepper mild mottle virus. Viral genomes were frequently detected in SW-D and rarely in GW-D, suggesting that GW-D may be a safe DW source. Viral genomes were also detected in DW, posing a possible threat to human health. The lowest percentages of positive samples were found in Europe, while the highest were found in Asia and South America. Only three articles assessed viral occurrence in BW. This review highlights the lack of method standardization and the need for legislation updates.

RSV testing practice and positivity by patient demographics in the United States: integrated analyses of MarketScan and NREVSS databases.

BACKGROUND: RSV-incidence estimates obtained from routinely-collected healthcare data (e.g., MarketScan) are commonly adjusted for under-reporting using test positivity reported in national Surveillance Systems (NREVSS). However, NREVSS lacks detail on patient-level characteristics and the validity of applying a single positivity estimate across diverse patient groups is uncertain. We aimed to describe testing practices and test positivity across subgroups of private health insurance enrollees in the US and illustrate the possible magnitude of misclassification when using NREVSS to correct for RSV under ascertainment. METHODS: Using billing records, we determined distributions of RSV-test claims and test positivity among a national sample of private insurance enrollees. Tests were considered positive if they coincided with an RSV-diagnosis. We illustrated the influence of positivity variation across sub-populations when accounting for untested acute respiratory infections. RESULTS: Most tests were for children (age 0-4: 65.8%) and outpatient encounters (78.3%). Test positivity varied across age (0-4: 19.8%, 5-17: 1.8%, adults: 0.7%), regions (7.6-16.1%), settings (inpatient 4.7%, outpatient 14.2%), and test indication (5.0-35.9%). When compared to age, setting or indication-specific positivity, bias due to using NREVSS positivity to correct for untested ARIs ranged from - 76% to 3556%. CONCLUSIONS: RSV-test positivity depends on the characteristics of patients for whom those tests were ordered. NREVSS-based correction for RSV-under-ascertainment underestimates the true incidence among children and overestimate rates among adults. Demographic-specific detail on testing practice and positivity can improve the accuracy of RSV-incidence estimates.

Epidemiology of major entero-pathogenic viruses and genetic characterization of Group A rotaviruses among children (≤5 years) with acute gastroenteritis in eastern India, 2018-2020.

AIMS: This study was carried out from January 2018 to March 2020 in Kolkata, eastern India to determine the prevalence rates and epidemiological patterns associated with the major viral agents of gastroenteritis among children ≤5 years of age. Molecular characterization of GARV, the predominant agent of viral gastroenteritis, was done to understand their genotype diversity. METHODS AND RESULTS: 1284 of 3157 stool samples (~40%) from children (≤5 years) with acute gastroenteritis tested positive for one or more enteric viruses with positivity rates 25.11%, 8.74%, 6.62% and 6.11% for GARV, HAdV-F, AstV and NoV respectively. Co-infection was observed in 5.31% of cases. Associated clinical/meteorological variables like age, sex, symptoms, temperature and precipitation were assessed to find any correlation between these and enteric virus infection rates. >70% of viral gastroenteritis cases were observed in 6-24 months' age group. GARV and AstV infection occurred mostly during cooler months while HAdV-F infection mostly occurred during warmer periods. No definite seasonality was observed for NoV infections. Clinical severity associated with GARV infection was higher compared to other enteric viruses. Genotyping of rotavirus positive samples revealed G3P[8] was the predominantly circulating GARV genotype throughout the study period. CONCLUSIONS: GARV remained the predominant viral agent of acute gastroenteritis among children though its prevalence rates in this region declined significantly compared to the previous years (2010-2016). The prevalence of other enteric viruses was below 10%. SIGNIFICANCE AND IMPACT OF STUDY: This study provides valuable insights regarding the current burden of viral gastroenteritis in Eastern India. The 2-year study in children will provide the baseline data for future surveillance studies in evaluating the impact of the introduced GARV vaccine on the overall prevalence of viral gastroenteritis.

Detection of viruses from feces of wild endangered Macaca maura: a potential threat to moor macaque survival and for zoonotic infection.

BACKGROUND: To date, there is a scarcity of information and literature on Macaca maura health status relative to viral diseases. The objectives of the present study were to investigate on the potential spread of enteric and non-enteric viruses shed in the environment through a wild macaque feces and to understand the possible interrelation in the spread of zoonotic viruses in a poorly studied geographical area, the Sulawesi Island. This study will also contribute providing useful information on potential threats to the health of this endangered species. METHODS: The sampling was conducted between 2014 and 2016 in the Bantimurung Bulusaraung National Park, in the south of the Sulawesi Island and non-invasive sampling methods were used to collect fresh stools of the M. maura, one of the seven macaque species endemic to the island of Sulawesi, Indonesia. The population under study consisted in two wild, neighboring social macaque groups with partially overlapping home ranges; twenty-four samples were collected and examined using negative staining electron microscopy and a panel of PCR protocols for the detection of ten RNA and two DNA viruses. RESULTS: Viral particles resembling parvovirus (5 samples), picornavirus (13 samples) and calicivirus (13 samples) were detected by electron microscopy whereas the PCR panel was negative for the 12 viruses investigated, except for one sample positive for a mosquito flavivirus. The results did not correlate with animal sex; furthermore, because all of the animals were clinically healthy, it was not possible to correlate feces consistency with viral presence. CONCLUSIONS: As information on viral infections in wild moor macaques remains limited, further studies are yet required to identify the fecal-oral and blood transmitted potentially zoonotic viruses, which may infect the moor macaque and other macaque species endemic to the South Sulawesi Island.

Prevalence, distribution and environmental effects on faecal indicator bacteria and pathogens of concern in commercial shellfish production areas in a subtropical region of a developing country (Santa Catarina, Brazil).

This paper reviews recent literature on the abundance and distribution of faecal indicator bacteria and pathogens in shellfish production areas in the state of Santa Catarina, on the subtropical coast of Brazil. This state supplies > 95% of the national production of shellfish. Microbiological monitoring data were mapped using GIS and the results compared with those from other countries. Coastal human population is the main predictive parameter for faecal bacteria in the production areas. Temporal variations of the bacteria can also be predicted by solar radiation and rainfall. The prevalence of pathogens such as hepatitis A virus, human norovirus, Salmonella spp. and Vibrio spp. does not differ substantially from that in developed countries. The information reported here can be used to inform development of microbiological risk profiles for shellfish production areas.

Egress of non-enveloped enteric RNA viruses.

A long-standing paradigm in virology was that non-enveloped viruses induce cell lysis to release progeny virions. However, emerging evidence indicates that some non-enveloped viruses exit cells without inducing cell lysis, while others engage both lytic and non-lytic egress mechanisms. Enteric viruses are transmitted via the faecal-oral route and are important causes of a wide range of human infections, both gastrointestinal and extra-intestinal. Virus cellular egress, when fully understood, may be a relevant target for antiviral therapies, which could minimize the public health impact of these infections. In this review, we outline lytic and non-lytic cell egress mechanisms of non-enveloped enteric RNA viruses belonging to five families: Picornaviridae, Reoviridae, Caliciviridae, Astroviridae and Hepeviridae. We discuss factors that contribute to egress mechanisms and the relevance of these mechanisms to virion stability, infectivity and transmission. Since most data were obtained in traditional two-dimensional cell cultures, we will further attempt to place them into the context of polarized cultures and in vivo pathogenesis. Throughout the review, we highlight numerous knowledge gaps to stimulate future research into the egress mechanisms of these highly prevalent but largely understudied viruses.

Use of a Glycan Library Reveals a New Model for Enteric Virus Oligosaccharide Binding and Virion Stabilization.

Enteric viruses infect the gastrointestinal tract, and bacteria can promote replication and transmission of several enteric viruses. Viruses can be inactivated by exposure to heat or bleach, but poliovirus, coxsackievirus B3, and reovirus can be stabilized by bacteria or bacterial polysaccharides, limiting inactivation and aiding transmission. We previously demonstrated that certain N-acetylglucosamine (GlcNAc)-containing polysaccharides can stabilize poliovirus. However, the detailed virus-glycan binding specificity and glycan chain length requirements, and thus the mechanism of virion stabilization, have been unclear. A previous limitation was our lack of defined-length glycans to probe mechanisms and consequences of virus-glycan interactions. Here, we generated a panel of polysaccharides and oligosaccharides to determine the properties required for binding and stabilization of poliovirus. Poliovirus virions are nonenveloped icosahedral 30-nm particles with 60 copies of each of four capsid proteins, VP1 to VP4. VP1 surrounds the 5-fold axis, and our past work indicates that this region likely contains the glycan binding site. We found that relatively short GlcNAc oligosaccharides, such as a six-unit GlcNAc oligomer, can bind poliovirus but fail to enhance virion stability. Virion stabilization required binding of long GlcNAc polymers of greater than 20 units. Our data suggest a model where GlcNAc polymers of greater than 20 units bind and bridge adjacent 5-fold axes, thus aiding capsid rigidity and stability. This study provides a deeper understanding of enteric virus-bacterial glycan interactions, which are important for virion environmental stability and transmission.IMPORTANCE Enteric viruses are transmitted through the fecal-oral route, but how enteric viruses survive in the environment is unclear. Previously, we found that bacterial polysaccharides enhance poliovirus stability against heat or bleach inactivation, but the specific molecular requirements have been unknown. Here, we showed that certain short-chain oligosaccharides can bind to poliovirus but do not increase virion stability. Long-chain polysaccharides bind and may bridge adjacent sites on the viral surface, thus increasing capsid rigidity and stability. This work defines the unique interactions of poliovirus and glycans, which provides insight into virion environmental stability and transmission.

SARS-CoV-2 in environmental perspective: Occurrence, persistence, surveillance, inactivation and challenges.

The unprecedented global spread of the severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 is depicting the distressing pandemic consequence on human health, economy as well as ecosystem services. So far novel coronavirus (CoV) outbreaks were associated with SARS-CoV-2 (2019), middle east respiratory syndrome coronavirus (MERS-CoV, 2012), and SARS-CoV-1 (2003) events. CoV relates to the enveloped family of Betacoronavirus (ßCoV) with positive-sense single-stranded RNA (+ssRNA). Knowing well the persistence, transmission, and spread of SARS-CoV-2 through proximity, the faecal-oral route is now emerging as a major environmental concern to community transmission. The replication and persistence of CoV in the gastrointestinal (GI) tract and shedding through stools is indicating a potential transmission route to the environment settings. Despite of the evidence, based on fewer reports on SARS-CoV-2 occurrence and persistence in wastewater/sewage/water, the transmission of the infective virus to the community is yet to be established. In this realm, this communication attempted to review the possible influx route of the enteric enveloped viral transmission in the environmental settings with reference to its occurrence, persistence, detection, and inactivation based on the published literature so far. The possibilities of airborne transmission through enteric virus-laden aerosols, environmental factors that may influence the viral transmission, and disinfection methods (conventional and emerging) as well as the inactivation mechanism with reference to the enveloped virus were reviewed. The need for wastewater epidemiology (WBE) studies for surveillance as well as for early warning signal was elaborated. This communication will provide a basis to understand the SARS-CoV-2 as well as other viruses in the context of the environmental engineering perspective to design effective strategies to counter the enteric virus transmission and also serves as a working paper for researchers, policy makers and regulators.

A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection.

The intestinal epithelium can limit enteric pathogens by producing antiviral cytokines, such as IFNs. Type I IFN (IFN-α/ß) and type III IFN (IFN-λ) function at the epithelial level, and their respective efficacies depend on the specific pathogen and site of infection. However, the roles of type I and type III IFN in restricting human enteric viruses are poorly characterized as a result of the difficulties in cultivating these viruses in vitro and directly obtaining control and infected small intestinal human tissue. We infected nontransformed human intestinal enteroid cultures from multiple individuals with human rotavirus (HRV) and assessed the host epithelial response by using RNA-sequencing and functional assays. The dominant transcriptional pathway induced by HRV infection is a type III IFN-regulated response. Early after HRV infection, low levels of type III IFN protein activate IFN-stimulated genes. However, this endogenous response does not restrict HRV replication because replication-competent HRV antagonizes the type III IFN response at pre- and posttranscriptional levels. In contrast, exogenous IFN treatment restricts HRV replication, with type I IFN being more potent than type III IFN, suggesting that extraepithelial sources of type I IFN may be the critical IFN for limiting enteric virus replication in the human intestine.

Transfer of Enteric Viruses Adenovirus and Coxsackievirus and Bacteriophage MS2 from Liquid to Human Skin.

Indirect exposure to waterborne viruses increases the risk of infection, especially among children with frequent hand-to-mouth contacts. Here, we quantified the transfer of one bacteriophage (MS2) and two enteric viruses (adenovirus and coxsackievirus) from liquid to skin. MS2, a commonly used enteric virus surrogate, was used to compare virus transfer rates in a volunteer trial to those obtained using human cadaver skin and synthetic skin. MS2 transfer to volunteer skin was similar to transfer to cadaver skin but significantly different from transfer to synthetic skin. The transfer of MS2, adenovirus, and coxsackievirus to cadaver skin was modeled using measurements for viruses attaching to the skin (adsorbed) and viruses in liquid residual on skin (unadsorbed). We find virus transfer per surface area is a function of the concentration of virus in the liquid and the film thickness of liquid retained on the skin and is estimable using a linear model. Notably, the amount of MS2 adsorbed on the skin was on average 5 times higher than the amount of adenovirus and 4 times higher than the amount of coxsackievirus. Quantification of pathogenic virus retention to skin would thus be overestimated using MS2 adsorption data. This study provides models of virus transfer useful for risk assessments of water-related activities, demonstrates significant differences in the transfer of pathogenic virus and MS2, and suggests cadaver skin as an alternative testing system for studying interactions between viruses and skin.IMPORTANCE Enteric viruses (viruses that infect the gastrointestinal tract) are responsible for most water-transmitted diseases. They are shed in high concentrations in the feces of infected individuals, persist for an extended period of time in water, and are highly infective. Exposure to contaminated water directly (through ingestion) or indirectly (for example, through hand-water contacts followed by hand-to-mouth contacts) increases the risk of virus transmission. The work described herein provides a quantitative model for estimating human-pathogenic virus retention on skin following contact with contaminated water. The work will be important in refining the contribution of indirect transmission of virus to risks associated with water-related activities.

Monitoring of Poyang lake water for sewage contamination using human enteric viruses as an indicator.

BACKGROUND: Recreational water contaminated with fecal pollution poses a great public health concern, as fecal waste may cause serious waterborne illnesses. Current recreational water standards using fecal indicator bacteria (FIB) have their limitations for human protection especially in developing countries such as China. METHODS: To explore the potential use of enteric viruses as a potential indicator of fecal contamination, four viruses: norovirus geno-groups I and II, enteroviruses, and adenoviruses were tested in this study using molecular detection methods and sensitive RT-PC developed in the University of Hawaii. Water samples were also tested for FIB in order to determine their association with enteric virus detection. RESULTS: All sample sites tested positive for four enteric viruses. Human enterovirus (58%) and adenovirus (67%) were more frequently detected from these six sites, followed by norovirus I (50%) and norovirus II (38%). Six sampling sites all met the level-I water quality of GB3838-2002 criteria in microbiological level, but they all tested positive for enteric viruses. CONCLUSION: These findings indicate the current sewage contamination of Poyang Lake and also support the essential need of additional indicator such as human enteric viruses for enhanced monitoring of water quality since the presence of enteric viruses does not always correlate with fecal bacterial indicator detection.

Human virus and microbial indicator occurrence in public-supply groundwater systems: meta-analysis of 12 international studies.

Groundwater quality is often evaluated using microbial indicators. This study examines data from 12 international groundwater studies (conducted 1992-2013) of 718 public drinking-water systems located in a range of hydrogeological settings. Focus was on testing the value of indicator organisms for identifying virus-contaminated wells. One or more indicators and viruses were present in 37 and 15% of 2,273 samples and 44 and 27% of 746 wells, respectively. Escherichia coli (E. coli) and somatic coliphage are 7-9 times more likely to be associated with culturable virus-positive samples when the indicator is present versus when it is absent, while F-specific and somatic coliphages are 8-9 times more likely to be associated with culturable virus-positive wells. However, single indicators are only marginally associated with viruses detected by molecular methods, and all microbial indicators have low sensitivity and positive predictive values for virus occurrence, whether by culturable or molecular assays, i.e., indicators are often absent when viruses are present and the indicators have a high false-positive rate. Wells were divided into three susceptibility subsets based on presence of (1) total coliform bacteria or (2) multiple indicators, or (3) location of wells in karst, fractured bedrock, or gravel/cobble settings. Better associations of some indicators with viruses were observed for (1) and (3). Findings indicate the best indicators are E. coli or somatic coliphage, although both indicators may underestimate virus occurrence. Repeat sampling for indicators improves evaluation of the potential for viral contamination in a well.

Isolation of chicken astrovirus from specific pathogen-free chicken embryonated eggs.

Astroviruses have been associated with enteric disorders in many animal species, including chickens. Here, we describe the isolation, propagation, and pathological characteristics of chicken astrovirus (CAstV) in specific pathogen free (SPF) chicken embryonated eggs (CEE) from chickens with diarrhea and runting-stunting syndrome. The CEE were inoculated via the yolk sac route. Viral confirmation was carried out using PCR techniques and transmission electron microscopy negative staining with ammonium molybdate. The intestinal contents were screened for CAstV, and differential diagnostic testing was performed for avian nephritis virus, avian rotavirus, avian reovirus, chicken parvovirus, infectious bronchitis virus, and fowl adenovirus Group I to detect co-infection with other infectious agents. Seven- or 14-day-old CEEs presented with hemorrhages, edema, a gelatinous aspect, deformities, and dwarfism. The supporting membranes did not show any alterations. Here, we have described the isolation of CAstV and its pathological characteristics in SPF CEE.

Applicability of Bio-wipes for the collection of human faecal specimens for detection and characterisation of enteric viruses.

OBJECTIVE: To determine whether gastroenteritis viruses and other enteric viruses could be detected in faecal specimens collected with Bio-wipes. METHODS: Faecal specimens, self-collected with Bio-wipes, from 190 individuals (94 diarrhoeal, 93 non-diarrhoeal, 3 unknown) were screened for eight human enteric viruses (enterovirus, hepatitis A virus, adenovirus, astrovirus, norovirus GI and GII, sapovirus and rotavirus) by real-time (reverse transcription)-polymerase chain reaction. Rotaviruses and noroviruses from positive specimens were genotyped. RESULTS: At least one enteric virus could be detected in 82.6% (157/190) of faecal specimens. Mixed infections of up to four different viruses could be detected in both diarrhoeal and non-diarrhoeal specimens. Enteroviruses were detected most frequently (63.7%), followed by adenoviruses (48.4%) and noroviruses (32.2%). Genotyping was successful for 78.6% of rotaviruses and 44.8% of noroviruses. CONCLUSIONS: Bio-wipes provide a user friendly, easier method for stool collection that facilitates enteric virus detection and genetic characterisation.

Human intestinal enteroids platform to assess the infectivity of gastroenteritis viruses in wastewater.

Fecal-orally transmitted gastroenteritis viruses, particularly human noroviruses (HuNoVs), are a public health concern. Viral transmission risk through contaminated water results underexplored as they have remained largely unculturable until recently and the robust measuring of gastroenteritis viruses infectivity in a single cell line is challenging. This study primarily aimed to test the feasibility of the human intestinal enteroids (HIE) model to demonstrate the infectivity of multiple gastroenteritis viruses in wastewater. Initially, key factors affecting viral replication in HIE model were assessed, and results demonstrated that the reagent-assisted disruption of 3D HIE represents an efficient alternative to syringe pass-through, and the filtering of HuNoV stool suspensions could be avoided. Moreover, comparable replication yields of clinical strains of HuNoV genogroup I (GI), HuNoV GII, rotavirus (RV), astrovirus (HAstV), and adenoviruses (HAdV) were obtained in single and multiple co-infections. Then, the optimized HIE model was used to demonstrate the infectivity of multiple naturally occurring gastroenteritis viruses from wastewater. Thus, a total of 28 wastewater samples were subjected to (RT)-qPCR for each virus, with subsequent testing on HIE. Among these, 16 samples (57 %) showed replication of HuNoVs (n = 3), RV (n = 5), HAstV (n = 8), and/or HAdV (n = 5). Three samples showed HuNoV replication, and sequences assigned to HuNoV GI.3[P13] and HuNoV GII.4[P16] genotypes. Concurrent replication of multiple gastroenteritis viruses occurred in 4 wastewater samples. By comparing wastewater concentrate and HIE supernatant sequences, diverse HAstV and HAdV genotypes were identified in 4 samples. In summary, we successfully employed HIE to demonstrate the presence of multiple infectious human gastroenteritis viruses, including HuNoV, in naturally contaminated wastewater samples.