Enteric viruses' dissemination in a private reserve of natural heritage.

This study aimed to assess anthropogenic impact of surrounding population in the Private Reserve of Natural Heritage at Pantanal, the world's largest freshwater wetland ecosystem located in the centre of South America. Viral aetiological agents of acute gastroenteritis as rotavirus A (RVA), noroviruses, human adenoviruses, klassevirus and of hepatitis, as hepatitis A virus, were investigated in different aquatic matrices. Annual collection campaigns were carried out from 2009 to 2012, alternating dry and rainy seasons. Viral particles present in the samples were concentrated by the adsorption-elution method, with negatively charged membranes, and detected by qualitative and quantitative PCR. From a total of 43 samples at least one virus was detected in 65% (28) of them. Viruses were detected in all matrices with concentrations ranging from 2 × 102 to 8·3 × 104 genome copies per litre. A significant higher RVA frequency was observed in the dry season. Our data revealing dissemination of human enteric viruses in water matrices both inside and outside the reserve could be useful to trace faecal contamination in the environment and to minimize the risk of infection by exposure of susceptible individuals. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is part of a collaborative project designed to investigate the environmental and health conditions of the Private Reserve of Natural Heritage at Pantanal, the largest seasonally flooded wetland in the world. The project aimed to promote health and quality of human and wildlife extending technical-scientific knowledge about pathogens present in the region. By assessing the occurrence of human enteric viruses in different water matrices we demonstrated the anthropogenic impact of surrounding population and pointed out the potential risk of infection by exposure of susceptible individuals.

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.

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.

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.

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.

Evaluation of the swab sampling method to recover viruses from fomites.

The monitoring of virus contamination on fomites, especially at hospitals has been used for a more effective evaluation of the microbiological quality of surfaces. Swab sampling is the method used currently, although the use of an internal control process (ICP) has not yet been assessed. The aim of this study is to determine the recovery rate of murine norovirus 1 (MNV-1) and bacteriophage PP7 on different surfaces in order to assess their potential use as an ICP. For this purpose both viruses were spiked experimentally both on porous and non-porous formic as well as on rubberized surfaces. Quantitative PCR (qPCR) showed a variable efficiency with a percentage recovery ranging from 0.6 to 77% according to viruses and surfaces. A global analysis suggested that MNV-1 could be used as a potential ICP for the swab sampling method.

Gastroenteric virus dissemination and influence of rainfall events in urban beaches in Brazil.

AIMS: This study was conducted to assess rotavirus A (RV-A), genogroup II (GII) norovirus (NoV), and human adenovirus (HAdV) dissemination in recreational water in an urban beach located in the city of Rio de Janeiro and their persistence during rainfall events. METHODS AND RESULTS: Viruses, including bacteriophage (PP7), used as internal control, were concentrated, reverse transcribed and quantified by a low-cost method based on organic flocculation with skimmed milk coupled with quantitative polymerase chain reaction protocols. The analysis of 74 superficial water samples obtained during 6 months of monitoring detected HAdV (66%), RV-A (37%) and GII NoV (14%), with a mean viral load of 4·1 log10 genome copies l(-1) (g.c. l(-1) ), 4·3 log10 g.c l(-1) and 3·8 log10 g.c. l(-1) , respectively. Investigation of those viruses during two rainfall events showed a longer permanence after rainfall events compared with bacterial indicators. CONCLUSIONS: The results highlight the need for further monitoring using viral parameters to determine the microbiological quality of recreational waters to allow bath in these waters, especially during rainy events. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on virus contamination in recreational waters on tourist beaches frequented throughout the year, emphasizing the importance of viral parameters for assessing microbiological quality of water, as well as the potential risk of waterborne infections.

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.

Partial VP2 sequencing of canine parvovirus (CPV) strains circulating in the state of Rio de Janeiro, Brazil: detection of the new variant CPV-2c

Canine parvovirus (CPV) is the most important enteric virus for dogs and it seems to be undergoing continuous evolution, generating new genetic and antigenic variants throughout the world. The aim of this study was to analyze the distribution of CPV variants from 1995 to 2009 and to investigate the circulation of the new variant CPV-2c in Rio de Janeiro, Brazil. In addition, the clinical features of CPV infection were also reported. After CPV laboratorial confirmation by HA/HI and PCR, thirty-two fecal samples were analyzed by sequencing a 583-bp fragment of the VP2 gene. One sample, collected in 2008 was typed as the new type CPV-2c. All samples from 1995 to 2003 were identified as "new CPV-2a". From 2004 to 2006, both "new CPV-2a" and CPV-2b were observed. From 2006 to 2009, most of the samples were characterized as CPV-2b. The classical signs of CPV enteritis were observed in 16/18 CPV-2a and 5/13 CPV-2b infected puppies. These results show that continuous epidemiological surveillance of CPV strain distribution is essential for studying the patterns of CPV-2a and 2b spread and for determining whether the new variant CPV-2c has become permanently established in Brazilian canine population.

Outbreak of acute gastroenteritis in young children with death due to rotavirus genotype G9 in Rio Branco, Brazilian Amazon region, 2005.

BACKGROUND: An epidemic of acute gastroenteritis occurred in Rio Branco City, Acre State, in Brazil's Amazon region in 2005. An investigation was conducted to confirm the etiology and identify possible risk factors for death. METHODS: Rio Branco municipality surveillance data for the period May to October 2005 were reviewed. In a case-control study, children who died following acute gastroenteritis were compared to age-matched controls with acute gastroenteritis who survived. Rotavirus A (RV-A) was investigated in 799 stool samples and genotyped by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The cumulative incidence of diarrhea in children aged <5 years was 21%. A fatal outcome was significantly associated with uncovered household water storage containers. RV-A was identified in 88% of samples and G9 was the prevalent genotype (71%). CONCLUSIONS: Oral rehydration solution and boiling or chlorinating drinking water likely limited mortality. This epidemic was caused by RV-A genotype G9. After the outbreak, a rotavirus vaccine was introduced into the official childhood immunization schedule in Brazil.

Detection of uncommon rotavirus A strains P[8]G8 and P[4]G8 in the city of Rio de Janeiro, 2002.

Group A rotaviruses (RV-A) are the major cause of gastroenteritis in infants and young children around the world. Each year RV-A causes approximately 11 million episodes of severe diarrhea, with an estimated of 611,000 deaths. Epidemiologic surveys have identified P[8]G1, P[4]G2, P[8]G3, P[8]G4, and P[8]G9 as the most common global genotypes associated with diarrhea in children up to 5-year old. Surveillance studies and documentation of RV-A G and P genotypes is necessary for a comprehensive evaluation of the evolution of new strains, and assessing the capability of vaccines to provide heterotypic protection. It is known that reassortments are the driving force for genetic diversity through sudden changes in RV-A genome. In this study, we identified two unusual P/G combinations, P[8]G8 and P[4]G8, occurring in Rio de Janeiro during 2002. Results obtained in this study suggest that P[8]G8 RV-A strain originated from a reassortment event that occurred between RV-A P[4]G8 and P[8]G9 strains circulating in Rio de Janeiro in the same year. G8 strains identified in this study, as well as G8 strains detected in Recife by Montenegro et al. [Montenegro et al. (2007) J Med Virol 79: 335-340], showed a close genetic relationship with strains from Africa, where this genotype have become prevalent.

Surveillance of norovirus infections in the state of Rio De Janeiro, Brazil 2005-2008.

A 4-year (2005-2008) norovirus (NoV) surveillance study was conducted in the state of Rio Janeiro, Brazil, to demonstrate the role of these viruses in outbreaks and sporadic cases of acute gastroenteritis. A cohort of 1,687 fecal samples was obtained from patients with gastroenteritis; 324 were rotavirus-positive. Of the remainder 1,363 rotavirus-negative samples, 1,087 samples were tested for NoV RNA in this study. The study enrolled 267 outpatients from Municipal Public Health Centers and 820 inpatients, whose samples were obtained by active surveillance in Public Hospitals. Fecal samples were tested by reverse transcription (RT) followed by polymerase chain reaction (PCR) using the MON 431-434 set of degenerate primers for NoV GI and GII detection, and there were 35.1% (381/1,087) positive samples for NoV, consisting of 30.2% (248/820) and 49.8% (133/267) from inpatient and outpatient, respectively. Children infected by NoV had significantly more frequent mucus in feces, vomiting and fever. No seasonal pattern in NoV infections was observed in patients admitted to hospital; however, two peaks of NoV infections were observed from ambulatory cases, suggesting that there was an occurrence of outbreaks in those time periods. Molecular characterization revealed GII to be the most prevalent genogroup, totaling 96.3% (104/108) of all sequences analyzed, and GII.4 was the genotype detected most frequently (80.7%), followed by GII.6, 3, 14, 7, and 8. Two GI strains, GI.2 and GI.3, were also observed. The number of outbreaks and sporadic cases described in this study highlights the need to implement diagnosis of NoV in surveillance laboratories.

Assessment of norovirus contamination in environmental samples from Florianópolis City, Southern Brazil.

AIMS: To assess norovirus (NoV) contamination in aquatic ecosystems in the city of Florianópolis, in Southern Brazil, to provide epidemiological data that can support actions for environmental contamination control. METHODS AND RESULTS: An adsorption-elution method, followed by ultrafiltration, was performed to concentrate the viruses. NoV were detected using semi-nested PCR and quantified by real-time PCR. From June 2007 to May 2008, NoV were detected in 23% (22/94) of the samples analysed, including seawater, drinking water, superficial water (creek and brackish lagoon) and treated sewage. The mean viral loads for genogroups (G)I and GII in treated sewage samples were 297 and 440 genomic copies (gc) l(-1) , respectively, whereas creek water samples contained 2603 and 1361 gc l(-1) , respectively. Six samples were sequenced: two samples were GII.4, two were GII.2 and two were GI.3. CONCLUSIONS: NoV were detected in all water types analysed, demonstrating the widespread contamination of this geographical area with several cocirculating strains belonging to GI and GII. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the environmental spread of NoV in environmental waters and highlights the potential hazard for human health following the consumption of or contact with these waters, which could result in waterborne or foodborne acute gastroenteritis.

First detection of porcine norovirus GII.18 in Latin America.

Human (Hu) noroviruses (NoVs) circulate worldwide infecting people of all ages in developing and developed countries. Animal NoVs present some antigenic and genetic relationship to HuNoVs, although their zoonotic potential has not been established yet. Among animal NoVs, porcine (Po) NoVs are the most genetically related to HuNoVs. PoNoVs have only been detected in healthy finisher pigs in a few developed countries. Information about them lacks in developing countries. In this study 96 fecal samples from pigs of different ages from five farms in Rio de Janeiro State, Brazil were tested for NoVs. We report detection and genotyping by RT-PCR, nucleotide sequencing and phylogenetic analysis of partial polymerase and capsid regions of viral genome PoNoV genogroup II genotype 18 (GII.18) in one stool sample from a healthy finisher pig. This is the first report of PoNoV detection in Latin America and it supports the assumption that PoNoVs present a worldwide distribution.

Partial VP2 sequencing of canine parvovirus (CPV) strains circulating in the state of Rio de Janeiro, Brazil: detection of the new variant CPV-2c.

Canine parvovirus (CPV) is the most important enteric virus for dogs and it seems to be undergoing continuous evolution, generating new genetic and antigenic variants throughout the world. The aim of this study was to analyze the distribution of CPV variants from 1995 to 2009 and to investigate the circulation of the new variant CPV-2c in Rio de Janeiro, Brazil. In addition, the clinical features of CPV infection were also reported. After CPV laboratorial confirmation by HA/HI and PCR, thirty-two fecal samples were analyzed by sequencing a 583-bp fragment of the VP2 gene. One sample, collected in 2008 was typed as the new type CPV-2c. All samples from 1995 to 2003 were identified as "new CPV-2a". From 2004 to 2006, both "new CPV-2a" and CPV-2b were observed. From 2006 to 2009, most of the samples were characterized as CPV-2b. The classical signs of CPV enteritis were observed in 16/18 CPV-2a and 5/13 CPV-2b infected puppies. These results show that continuous epidemiological surveillance of CPV strain distribution is essential for studying the patterns of CPV-2a and 2b spread and for determining whether the new variant CPV-2c has become permanently established in Brazilian canine population.

Environmental dissemination of group A rotavirus: P-type, G-type and subgroup characterization.

Rotaviruses A (RV-A) infection is the most common cause of acute diarrheal diseases in infants and the dissemination of these viruses in the environment represents a public health hazard. The present study aims to evaluate reverse transcription-polymerase chain reaction (RT-PCR) based protocols for the detection of RV-A genes in different types of environmental samples. RV-A were concentrated by the adsorption-elution method using negatively charged membranes associated with a Centriprep Concentrator 50. The RV-A VP4, VP7 and VP6 genes were detected using RT-PCR in river water from the Amazon Hydrographic basin (Northern region) and from wastewater in a sewage treatment plant in Rio de Janeiro (Southeast region), Brazil. RV-A were successfully detected in water environmental samples by the methods used. The detection of the VP6 gene by RT-PCR was the most sensitive for detecting RV-A in environmental samples (44.0%), when compared to the detection of the VP4 (33.3%) and VP7 (25.3%) genes. Based on nucleotide sequence and phylogenetic analysis of the partial VP6 gene, 22 environmental samples were determined to be subgroup II (Wa-like). These results indicate that analysis of environmental samples could possibly make a valuable contribution to studies on the epidemiology of RV-A.

Detection of caliciviruses associated with acute infantile gastroenteritis in Salvador, an urban center in Northeast Brazil

Acute gastroenteritis caused by viruses is one of the leading causes of infantile morbidity. The aim of the present study was to investigate the presence of human caliciviruses of the genera norovirus and sapovirus in children up to 3 years of age with acute gastroenteritis from low-income communities in the city of Salvador, Brazil. This study is an extension of previous work carried out to establish the profile of the most prevalent enteric pathogens present in these communities. In this report, 139 fecal samples, collected from July 2001 to January 2002 were analyzed by RT-PCR and 13 (9 percent) were positive for human caliciviruses. By sequencing, seven isolates were characterized as norovirus genogroup GII and one as sapovirus genotype GII/1. Sequencing of the previously detected group-A rotaviruses and human astroviruses was also performed and revealed the circulation of rotavirus group A genotypes G1P[8] and G9P[8], and human astrovirus genotypes 6, 7, and 8. No mixed infection was observed. Community-based studies provide geographically representative information on disease burden. However, there are only a few reports in developing countries concerning the genotypes of the most important gastroenteric viruses detected in such communities. The present findings demonstrate the wide diversity of genotypes of the most important viruses responsible for acute gastroenteritis circulating in low-income communities.

Detection of caliciviruses associated with acute infantile gastroenteritis in Salvador, an urban center in Northeast Brazil.

Acute gastroenteritis caused by viruses is one of the leading causes of infantile morbidity. The aim of the present study was to investigate the presence of human caliciviruses of the genera norovirus and sapovirus in children up to 3 years of age with acute gastroenteritis from low-income communities in the city of Salvador, Brazil. This study is an extension of previous work carried out to establish the profile of the most prevalent enteric pathogens present in these communities. In this report, 139 fecal samples, collected from July 2001 to January 2002 were analyzed by RT-PCR and 13 (9%) were positive for human caliciviruses. By sequencing, seven isolates were characterized as norovirus genogroup GII and one as sapovirus genotype GII/1. Sequencing of the previously detected group-A rotaviruses and human astroviruses was also performed and revealed the circulation of rotavirus group A genotypes G1P[8] and G9P[8], and human astrovirus genotypes 6, 7, and 8. No mixed infection was observed. Community-based studies provide geographically representative information on disease burden. However, there are only a few reports in developing countries concerning the genotypes of the most important gastroenteric viruses detected in such communities. The present findings demonstrate the wide diversity of genotypes of the most important viruses responsible for acute gastroenteritis circulating in low-income communities.