Human Enterovirus Diversity by Next-Generation Sequencing Analysis in Urban Sewage Samples From Buenos Aires Metropolitan Area, Argentina: A Retrospective Study.

Human Enteroviruses (hEVs) are responsible for a wide variety of human diseases. During hEVs infection, virions are excreted in human feces and the fecal-oral route is the primary pathway for person-to-person transmission. Sewage surveillance could help in monitoring hEVs circulation and describing their diversity in a specific population. In this study, sewage samples collected in Buenos Aires Metropolitan Area (Argentina) were retrospectively studied through an amplicon-deep sequencing approach and phylogenetic analyses to characterize hEVs spread. We identified 17 different hEVs types belonging to A, B, and C species. To the best of our knowledge, this is the first report in Buenos Aires for 7 identified hEV-C types. Phylogenetic analyses suggest several introductions of coxsackievirus B4, echovirus 1, and echovirus 9 in the country, along with the national spread reached by some variants. Besides, well-supported monophyletic groups of Argentine, Uruguayan, and Brazilian strains unveiled regional circulation patterns for some variants. These results extend our knowledge about hEVs circulation in Buenos Aires and might exhort authorities to implement more active sewage surveillance in the region.

Proposal of a pathway for enteric virus groups detection as indicators of faecal contamination to enhance the evaluation of microbiological quality in freshwater in Argentina.

An environmental survey was conducted in order to assess the frequency of detection of picobirnavirus (PBV), human adenovirus (HAdV) and infective enterovirus (iEV) as indicators of faecal contamination in freshwater, and to determine their potential as reporters of the presence of other enteric viruses, such as group A rotavirus (RVA). The study was carried out over a three-year period (2013-2015) in the San Roque Dam, Córdoba, Argentina. The overall frequency detection was 62.9% for PBV, 64.2% for HAdV and 70.4% for iEV. No significant differences were observed in the rates of detection for any of these viruses through the years studied, and a seasonal pattern was not present. Whenever there was RVA detection in the samples analyzed, there was also detection of iEV and/or HAdV and/or PBV. At least one of the viral groups analyzed was demonstrated in the 100% of the samples with faecal coliforms values within the guideline limits. In this setting, especially in those samples which reveal faecal indicator bacteria within the guideline limit, we propose to carry out a pathway, involving PBV, HAdV and iEV detection in order to enhance the evaluation of microbiological quality in freshwater in Argentina. The proposed methodological strategy could report faecal contamination in water, mainly of human origin, and the condition of the matrix to maintain viral viability. In addition, the viral groups selected could report the presence of RV.

Evaluation of Bacterial Contamination as an Indicator of Viral Contamination in a Sedimentary Aquifer in Uruguay.

In Uruguay, groundwater is frequently used for agricultural activities, as well as for human consumption in urban and rural areas. As in many countries worldwide, drinking water microbiological quality is evaluated only according to bacteriological standards and virological analyses are not mentioned in the legislation. In this work, the incidence of human viral (Rotavirus A, Norovirus GII, and human Adenovirus) and bacterial (total and thermotolerant coliform and Pseudomonas aeruginosa) contamination in groundwater in the Salto district, Uruguay, as well as the possible correlation between these groups of microorganisms, was studied. From a total of 134 groundwater samples, 42 (32.1%) were positive for Rotavirus, only 1 (0.7%) for both Rotavirus and Adenovirus, and 96 (72.6%) samples were positive for bacterial indicators. Results also show that Rotavirus presence was not associated with changes in chemical composition of the aquifer water. Bacteriological indicators were not adequate to predict the presence of viruses in individual groundwater samples (well scale), but a deeper spatial-temporal analysis showed that they are promising candidates to assess the viral contamination degree at aquifer scale, since from the number of wells with bacterial contamination the number of wells with viral contamination could be estimated.

Human enteric viruses in a wastewater treatment plant: evaluation of activated sludge combined with UV disinfection process reveals different removal performances for viruses with different features.

This study assess the quality of wastewater through the detection and quantification of important viruses causing gastroenteritis at different stages of the wastewater treatment process in an activated-sludge wastewater treatment plant with ultraviolet disinfection. Ten sampling events were carried out in a campaign along a period of 18 months collecting wastewater samples from the influent, after the activated-sludge treatment, and after the final disinfection with UV radiation. Samples were concentrated through ultracentrifugation and analysed using retro-transcription, PCR and real time quantitative PCR protocols, for detection and quantification of Group A Rotavirus (RVA), Human Astrovirus (HAstV), Norovirus Genogroup II (NoV GII) and Human Adenovirus (HAdV). HAdV (100%), NoV GII (90%), RVA (70%) and HAstV (60%) were detected in influent samples with concentration from 1·4 (NoV GII) to 8·0 (RVA) log10  gc l-1 . Activated-sludge treatment reached well quality effluents with low organic material concentration, although nonstatistical significant differences were registered among influent and postactivated sludge treatment samples, regarding the presence and concentration for most viruses. All post-UV samples were negative for NoV GII and HAstV, although RVA and HAdV were detected in 38% and 63% of those samples respectively, with concentration ranging from 2·2 to 5·5 and 3·1 to 3·4 log10  gc l-1 . SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that an activated-sludge wastewater treatment plant with UV disinfection reduces to levels below the detection limit those single-stranded RNA viruses as noroviruses and astroviruses and reach significant lower levels of rotaviruses and adenoviruses after the complete treatment process.

Wastewater contamination in Antarctic melt-water streams evidenced by virological and organic molecular markers.

Human activities in the Antarctica including tourism and scientific research have been raised substantially in the last century with the concomitant impact on the Antarctic ecosystems through the release of wastewater mainly from different scientific stations activities. The aim of this study was to assess the wastewater contamination of surface waters and sediments of three melt-water streams (11 sites) by leaking septic tanks located in the vicinity of the Uruguayan Scientific Station in the Fildes Peninsula, King George Island, Antarctica, during summer 2015. For this purpose, we combined the analysis of fecal steroids in sediments by using gas chromatography and six enteric viruses in surface waters by quantitative and qualitative PCR. Coprostanol concentrations (from 0.03 to 3.31µgg-1) and fecal steroids diagnostic ratios indicated that stations C7 and C8 located in the kitchen stream presented sewage contamination. Rotavirus was the only enteric virus detected in five sites with concentration ranging from 1.2×105gcL-1 to 5.1×105gcL-1 being three of them located downstream from the leaking AINA and Kitchen septic tanks. This study shows for the first time the presence of both virological and molecular biomarkers of wastewater pollution in surface waters and sediments of three melt-water streams in the vicinity of a scientific station in the Antarctica. These results highlight the importance of the complementation of these biomarkers in two different matrices (surface waters and sediments) to assess wastewater pollution in an Antarctic environment related to anthropogenic activities in the area.

Norovirus molecular detection in Uruguayan sewage samples reveals a high genetic diversity and GII.4 variant replacement along time.

AIMS: To determine the prevalence and molecular epidemiology of norovirus (NoV) genogroup I (GI) and GII in Uruguay. METHODS AND RESULTS: One hundred and sixteen sewage samples were collected in six cities (Bella Unión, Salto, Paysandú, Fray Bentos, Melo and Treinta y Tres) from March 2011 to April 2013, viruses were concentrated by ultracentrifugation and NoV studies were performed by semi-nested RT-PCR (partial capsid region). NoV were detected in samples from all the cities and detected in 72% (84/116) of the samples with nine of them belonging to GI, 48 to GII and 27 to both genogroups. Remarkably, a high genetic diversity was identified: GII.2 (n = 13), GII.4 (n = 13), GI.1 (n = 5), GI.4 (n = 5), GI.8 (n = 4), GII.13 (n = 4), GII.1 (n = 3), GII.6 (n = 3), GI.3 (n = 1), GI.5 (n = 1), GI.6 (n = 1), GII.3 (n = 1), GII.17 (n = 1). Interestingly, a complete replacement of GII.4 New Orleans 2009 by GII.4 Sydney 2012 variants during 2012 was evidenced. CONCLUSION: This study reveals a high circulation of different NoV GI and GII genotypes in sewage evidencing a replacement of GII.4 variants. SIGNIFICANCE AND IMPACT OF STUDY: This approach can be used as an indicator of the presence of a new GII.4 variant which can originate an increase in acute gastroenteritis outbreaks worldwide.

Detection and Molecular Characterization of Aichivirus 1 in Wastewater Samples from Uruguay.

Aichivirus 1 (AiV-1) is an enteric virus with 30 nm in diameter, belonging to the genus Kobuvirus in the Picornaviridae family being a causative agent of gastroenteritis in humans. The transmission is via the fecal-oral route, through person to person contact, recreation in contaminated waters, or through the consumption of contaminated food or water. The aim of this study was to determine the frequency and the molecular characterization of AiV-1 in wastewater from Uruguay. Biweekly collections from March 2011 to February 2012 were performed in the cities of Bella Unión, Salto, Paysandú, and Fray Bentos, northwestern region of Uruguay. A total of 96 samples were collected; viruses were concentrated by ultracentrifugation, and AiV-1 was detected by using a nested PCR with primers directed to a conserved region (3CD junction) of the viral genome. A high frequency of AiV-1 (n = 54) was observed at all the cities analyzed mainly in the colder months of the year. AiV-1 was not evidenced as an appropriate viral fecal indicator since when compared with other previously detected enteric viruses, no correlation was observed. All 13 characterized AiV-1 belonged to the genotype B after the phylogenetic analysis performed with the sequences obtained from the first round PCR amplicon. This study demonstrates that AiV-1 is a frequently detected enteric viruses present in wastewater and excreted by infected persons in the northwestern region of Uruguay.

Sewage surveillance reveals the presence of canine GVII norovirus and canine astrovirus in Uruguay.

Canine norovirus (NoV) and astrovirus (AstV) were studied in 20 domestic sewage samples collected in two cities in Uruguay. Four samples were characterized as canine AstV after phylogenetic analysis clustering with strains detected in Italy and Brazil in 2008 and 2012, respectively. One sample was characterized as canine NoV and clustered with a strain detected in Hong Kong and recently classified as GVII. This study shows the occurrence of a canine NoV GVII strain for the first time in the American continent and also warns about possible zoonotic infection, since canine strains were detected in domestic sewage.

Environmental assessment reveals the presence of MLB-1 human astrovirus in Uruguay.

AIMS: The aim of this study was to investigate the presence of the recently identified human astrovirus (HAstV) and to increase the knowledge of the molecular epidemiology of classical HAstV detected in Uruguay. METHODS AND RESULTS: Recently identified and classical HAstV genotypes were investigated by RT-PCR targeting the ORF1b and ORF2 genome regions in 20 samples obtained between September 2011 and April 2013 in two cities of the eastern region of Uruguay. Four of 20 samples (20%) were identified as MLB-1 genotype and it was found a new MLB-1 classification through the segregation of the worldwide reported MLB-1 strains in two genetic lineages proposed and named: MLB-1a and MLB-1b. Fourteen (70%) samples were positive for classical HAstV and 12 of them were successfully sequenced and genotyped as: HAstV-1 (n = 10), HAstV-2 and HAstV-5 (one sample each). CONCLUSION: These results constitute the first report in the Latin American region concerning the molecular detection and characterization of MLB-1 HAstV strains in environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the benefits of an environmental surveillance to study emerging enteric viruses circulating in human societies.

Molecular epidemiology of group A rotavirus among children admitted to hospital in Salto, Uruguay, 2011-2012: first detection of the emerging genotype G12.

Group A rotavirus (RVA) is the most important etiologic agent of infant acute gastroenteritis (AGE) worldwide. Detection and molecular characterization of RVA in Salto department, Northwestern region of Uruguay, was conducted on 175 clinical samples, being 153 stool and 22 vomit samples, collected from hospitalized children with AGE, between 0-15 years old, from two hospitals of Salto city during 2011 and 2012. RVA was detected and genotyped by seminested multiplex RT-PCR in order to determine G- and P-genotypes. Positive samples were sequenced and phylogenetic analyses were carried out in order to determine lineages and sub-lineages. RVA were detected in 64 (37%) of the samples and the G and P genotypes observed were: 6% G1P[8], 23% G2P[4]/G2P[X]/GXP[4], 23% G3P[8]/G3P[X], 14% G12P[8]/G12P[X], 16% GXP[8], 1,5% G12P[9], 3% G2P[4]/[8], and 16% non-typeable. VP7 and VP4 genotypes related to DS-1 like gene constellation were prevalent during 2011 and those VP7 and VP4 genotypes related to Wa-like constellation were prevalent during 2012 (mainly represented by G3P[8]). Interestingly, RVA was detected in vomit samples in a high prevalence (41%). RVA was observed mainly in the age group between 1 and 5 years old (75% of the cases), and seasonality with a high detection rate in winter season was observed for the two consecutive years of surveillance. To our knowledge, this study represents the first detection and molecular characterization of RVA in Salto department, Northwestern region of Uruguay; and the first identification of the emerging genotype G12 in the country.

Environmental Assessment of Classical Human Astrovirus in Uruguay.

The aim of this study was to determine the molecular epidemiology of classical human astrovirus (HAstV) strains in sewage samples from four Uruguayan cities: Bella Unión, Salto, Paysandú, and Fray Bentos, located in the Northwestern region of the country. Overall, 96 sewage samples were collected biweekly between March 2011 and February 2012 and were subject to ultracentrifugation methodology in order to concentrate the viruses. RT-PCR directed to the ORF2 genome region was performed followed by sequencing and phylogenetic analysis. Forty-three (45 %) out of 96 analyzed samples were positive for HAstV (Mamastrovirus 1) and 31 of them were successfully sequenced being 21 (49 %) of them classified as HAstV-1 genotype (1a lineage) and 10 (23 %) as HAstV-2 genotype (eight strains belonging to the 2d lineage and two strains to the 2c lineage). The 1a lineage circulated throughout the year, while the 2d lineage only in the coldest months (June to October). Strikingly, the 2c lineage was detected only in Salto city during March 2011. In this city it was observed the highest frequency of HAstV and the greatest genetic diversity, probably due to its role as high touristic spot with an important influx of visitants from others regions of Uruguay and also from other countries. This study constitutes the first report in Uruguay that describes the phylogenetic diversity and genotype distribution of HAstV strains circulating in the Northwestern region evidencing a high frequency and also the presence of several different lineages.

Assessment of gastroenteric viruses from wastewater directly discharged into Uruguay River, Uruguay.

The aim of this study was to assess the viral contamination of group A rotavirus (RVA), norovirus (NoV), and human astrovirus (HAstV) in sewage directly discharged into Uruguay River and to characterize RVA genotypes circulating in Uruguay. For this purpose, sewage samples (n = 96) were collected biweekly from March 2011 to February 2012 in four Uruguayan cities: Bella Unión, Salto, Paysandú, and Fray Bentos. Each sample was concentrated by ultracentrifugation method. Qualitative and quantitative RT-PCR for RVA, NoV, and HAstV were performed. A wide dissemination of gastroenteric viruses was observed in the sewage samples analyzed with 80% of positivity, being NoV (51%) the most frequently detected followed by RVA with a frequency of 49% and HAstV with 45%. Genotypes of RVA were typed using multiplex semi-nested RT-PCR as follows: P[8] (n = 15), P[4] (n = 8), P[10] (n = 1), P[11] (n = 1), G2 (n = 29), and G3 (n = 2). The viral load ranged from 10(3) to 10(7) genomic copies/liter, and they were detected roughly with the same frequency in all participant cities. A peak of RVA and HAstV detection was observed in colder months (June to September), whereas no seasonality was observed for NoV. This study demonstrates for the first time, the high degree of gastroenteric viral contamination in the country; highlighting the importance of developing these analyses as a tool to determine the viral contamination in this hydrographic boundary region used by the local populations for recreation and consumption, establishing an elevated risk of gastroenteric diseases for human health.