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Noroviruses are a leading cause of epidemic and pandemic acute gastroenteritis (AGE) worldwide, and contaminated food and water are important routes for its transmission. Raw sewage has been used for viral surveillance to monitor the emergence of new norovirus strains with the potential to cause epidemics. In this study, we investigated norovirus occurrence and norovirus RNA levels in 156 samples collected from May 2013 to May 2014, across three different stages (52 samples each) of a wastewater treatment plant (WWTP) in Rio de Janeiro, Brazil. We also explored norovirus GII diversity in raw sewage samples by next-sequencing generation (NGS). In addition, we examined norovirus prevalence and molecular epidemiology from acute gastroenteritis cases. Using RT-qPCR, norovirus GI and GII was detected in 38.5% and 96.1% of raw sewage samples, 40.4% and 96.1% of primary effluent samples and 1.9% and 5.8% of final effluent samples, respectively. Norovirus RNA levels varied from 4 to 6.2 log 10 genome copies per litre (gc L -1 ) for GI and from 4.4 to 7.3 log 10  gc L -1 for GII. Using MiSeq NGS, we identified 13 norovirus genotypes over the one-year period, with six dominant capsid genotypes, including GII.4, GII.17, GII.5, GII.2, GII.3 and GII.1. GII.4 noroviruses were the most prevalent in wastewater samples (68.5%), and a similar trend was observed in AGE cases (71%). The emergent GII.17 was the second most prevalent genotype (14.3%) identified in the raw sewage samples, however, it was not detected in clinical cases. Due to the high burden of norovirus outbreaks and the lack of vaccine and antiviral drugs, it is essential to understand the genotypic diversity of norovirus at the population level. Complementary data obtained from both clinical and environmental (sewage) samples proved to be an effective strategy to monitor the circulation and emergence of norovirus epidemic genotypes., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The aim of this study was to perform the molecular characterization of conserved and variable regions of feline calicivirus capsid genome in order to investigate the molecular diversity of variants in Brazilian cat population. Twenty-six conjunctival samples from cats living in five public short-term animal shelters and three multicat life-long households were analyzed. Fifteen cats had conjunctivitis, three had oral ulceration, eight had respiratory signs (cough, sneeze and nasal discharge) and nine were asymptomatic. Feline calicivirus were isolated in CRFK cells and characterized by reverse transcription PCR target to both conserved and variable regions of open reading frame 2. The amplicons obtained were sequenced. A phylogenetic analysis along with most of the prototypes available in GenBank database and an amino acid analysis were performed. Phylogenetic analysis based on both conserved and variable region revealed two clusters with an aLTR value of 1.00 and 0.98 respectively and the variants from this study belong to feline calicivirus genogroup I. No association between geographical distribution and/or clinical signs and clustering in phylogenetic tree was observed. The variants circulating in public short-term animal shelter demonstrated a high variability because of the relatively rapid turnover of carrier cats constantly introduced of multiple viruses into this location over time., (Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

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 × 10 2 to 8·3 × 10 4 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., (© 2018 The Society for Applied Microbiology.)

Genogroup (G) IV norovirus (NoV) has been described in the literature as infectious agents in humans, although there are few reports regarding the frequency and spread of this virus, resulting in insufficient epidemiological data. The aim of this study was to investigate the occurrence of GIV norovirus in the State of Rio de Janeiro, Brazil in order to evaluate frequency, concentration, and genetic diversity using clinical and environmental approaches. For this purpose, 316 stool samples were collected from acute gastroenteritis cases reported over a period of three years. Wastewater samples were also obtained from the main wastewater treatment plant (WWTP) located in Rio de Janeiro throughout one year, totalizing 156 samples. All samples were submitted to quantitative analysis by TaqMan™ real-time PCR for GIV norovirus. Three out of 316 clinical samples were positive (0.9%) for GIV, with viral load ranging from 10 4 to 10 6 genome copies (CG) per gram. Regarding wastewater samples, GIV were detected in 52% of raw sewage, with viral load ranging from 10 4 to 10 6 CG per liter. Phylogenetic analysis revealed the circulation of a new GIV genotype in both clinical and environmental samples. To our knowledge, this is the first description of GIV norovirus in clinical samples in Brazil. These results demonstrate the importance of performing laboratory surveillance of clinical and environmental samples, assisting the comprehension of the epidemiology pattern of viruses with neglected diagnosis and indefinite impact in the population.

Gemycircularvirus (GemyCV) is a group of viruses which has been recently proposed as a new viral genus detected in fecal and environmental samples around the world. GemyCVs have been detected in human blood, brain tissue, cerebrospinal fluid, and stool sample. In the present study, we demonstrate for the first time, through molecular detection and characterization, the presence of GemyCVs in environmental samples from Brazil. Our results show a percentage of positivity ranging from 69 (25/36) to 97 % (35/36) in river water samples collected in Manaus, Amazon region, and wastewater from a wastewater treatment plant located in Rio de Janeiro, respectively, revealing GemyCVs as an important environmental contaminant.

Aim: To determine the prevalence and molecular epidemiology of human sapovirus (SaV) in both wastewater and stool samples in a 3-year (2012-2014) surveillance study performed in Rio de Janeiro State, Brazil., Methods and Results: A total of 156 wastewater and 341 stool samples were analysed using quantitative real-time PCR. SaV was detected in 3·5% (12/341) in stool samples with virus load concentrations ranging from 10(4) to 10(9) genome copies per gram (gc g(-1) ), and in 33·0% (51/156) wastewater samples, with range concentration varying from 10(4) to 10(6)  gc l(-1) . Partial genome sequencing of wastewater and stool samples revealed the circulation of genotypes GI.1, GI.2, GI.6, GII.1 and GV.1., Conclusion: This study demonstrated the prevalence of human SaV in acute gastroenteritis (AGE) cases and revealed, for the first time, the environmental dissemination of those viruses in Brazil., Significance and Impact of the Study: SaV diagnosis should be considered in hospitalized children with AGE and the higher positive rate detection in environmental samples suggests that SaV infection could be underestimated or associated with asymptomatic cases., (© 2016 The Society for Applied Microbiology.)

Rotavirus A (RVA) and noroviruses (NoV) are the major viral agents of acute gastroenteritis (AGE) worldwide. In the present study, we aimed to evaluate the performance of a one-step duplex quantitative RT-PCR (dRT-qPCR) assay, established for detection and quantification of RVA and NoV genogroup II (GII) using a single DNA standard curve (SC), as well as to investigate the association between fecal viral load and optical density (OD) values, and viruses' genotyping. The results obtained by dRT-qPCR in 530 fecal samples from AGE cases were compared with methods employed for the diagnosis of those viruses as follows: enzyme immunoassay (EIA) and polyacrylamide gel electrophoresis (PAGE) for RVA; and qualitative PCR for NoV. By using dRT-qPCR, we detected RVA and NoV in 353 (66%), increasing the positivity rate by 22.5% for RVA and 11.5% NoV, comparing the number of positive samples. RVA and NoV GII were detected in a range of 5.17 × 10(3) to 6.56 × 10(9) and 3.76 × 10(3) to 9.13 × 10(10) genome copies per gram of feces, respectively. We observed a significant direct correlation between genome copies values and optical density, using dRT-qPCR and EIA assays, respectively (Spearman ρ=0.41; p<0.0001). Viruses characterization demonstrated a predominance of NoV GII.4 Sidney 2012 variant during October 2013 to February 2014, followed by the emergence of RVA genotype G12P[8] in 2014. The established assay using a single SC provides an early feedback concerning detection and quantification, with the advantage of detecting simultaneously RVA and NoV GII, reducing time and reagent costs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Acute gastroenteritis norovirus (NoV) in a country of continental dimensions like Brazil has resulted in under-reporting of the number of outbreaks, as well as the genotypes associated., Objectives: To demonstrate the role of NoV in outbreaks occurring in the State of Rio Grande do Sul, Southern Brazil, we determined its prevalence, as well as the genotypes associated, and evaluated clinical and epidemiological aspects., Study Design: NoV investigation was carried out in rotavirus group A negative stool samples from 2265 patients from 741 outbreaks that occurred in the State of Rio Grande do Sul, Brazil, during a period of eight years (2004-2011). NoV detection and nucleotide sequencing for genotype characterization was carried by using sets of primers targeting a conservative Rd-Rp polymerase genome region and the viral capsid gene, respectively., Results: NoVs were detected in 817 stool samples (36.1%) and associated with 327 outbreaks (44.1%). NoV GII.2, GII.3, GII.4, GII.6, GII.12, GII.13, GII.14, GII.15, GII.17, GII.21; and GI.1 and GI.3 were characterized. GII.4 was the most frequently detected (72.3%), with five variants identified (Asia&#95;2003, Hunter&#95;2004, Yerseke&#95;2006a, Den&#95;Haag&#95;2006b, New Orleans&#95;2009). This study describes the first detection of GI.1 and GII.13 and GII.15 in Brazil and demonstrates NoV winter-spring seasonality in this region of the country., Conclusions: NoVs were responsible for almost 50% of outbreaks, with about 70% of them resulting from genotype GII.4 and its variants. The seasonality observed could help health authorities to establish a system of active surveillance in order to reduce NoV impact especially in congregate settings., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Noroviruses (NoVs) are the major cause of acute gastroenteritis outbreaks, and, despite a wide genetic diversity, genotype II.4 is the most prevalent strain worldwide. Mutations and homologous recombination have been proposed as mechanisms driving the epochal evolution of the GII.4, with the emergence of new variants in 1-3-year intervals causing global epidemics. There are no data reporting the dynamics of GII.4 variants along a specific period in Brazil. Therefore, to improve the understanding of the comportment of these variants in the country, the aim of this study was to evaluate the circulation of NoV GII.4 variants during a 9-year period in 3 out of 5 Brazilian regions. A total of 147 samples were sequenced, and a phylogenetic analysis of subdomain P2 demonstrated the circulation of six GII.4 variants, Asia&#95;2003, Hunter&#95;2004, Den Haag&#95;2006b, Yerseke&#95;2006a, New Orleans&#95;2009, and Sydney&#95;2012, during this period. The most prevalent variant was Den Haag&#95;2006b, circulating in different Brazilian regions from 2006 to 2011. A Bayesian coalescent analysis was used to calculate the mean evolutionary rate of subdomain P2 as 7.3 × 10(-3) (5.85 × 10(-3) -8.82 × 10(-3)) subst./site/year. These analyses also demonstrated that clade Den Haag&#95;2006b experienced a rapid expansion in 2005 and another in 2008 after a period of decay. The evaluation of the temporal dynamics of NoV GII.4 in Brazil revealed a similar pattern, with few exceptions, to the worldwide observation. These data highlight the importance of surveillance for monitoring the emergence of new strains of NoV GII.4 and its impact on cases of acute gastroenteritis.

Norovirus (NoV) infections are a major cause of acute gastroenteritis outbreaks around the world. In Brazil, the surveillance system for acute diarrhoea does not include the diagnosis of NoV, precluding the ability to assess its impact on public health. The present study assessed the circulation of NoV genotypes in different Brazilian states by partial nucleotide sequencing analysis of the genomic region coding for the major capsid viral protein. NoV genogroup II genotype 4 (GII.4) was the prevalent (78%) followed by GII.6, GII.7, GII.12, GII.16 and GII.17, demonstrating the great diversity of NoV genotypes circulating in Brazil. Thus, this paper highlights the importance of a virological surveillance system to detect and characterize emerging strains of NoV and their spreading potential.

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., ((c) 2010 Wiley-Liss, Inc.)

Noroviruses (NoV) are a leading cause of outbreaks of nonbacterial acute gastroenteritis in humans worldwide and have become an important cause of hospitalization of children in South America. NoV belong to the family Caliciviridae and are non-enveloped single stranded, positive sense, RNA viruses. NoV of genotype GII/4 have emerged worldwide, causing four epidemic seasons of viral gastroenteritis during which four novel variants emerged. Despite the importance of NoV outbreaks, little is known about the evolutionary rates, viral spread and population dynamics of NoV populations. In order to gain insight into these matters, a Bayesian Markov chain Monte Carlo (MCMC) approach was used to analyze region D or full-length VP1 gene sequences of GII/4 NoV populations isolated in Brazil or Japan, respectively. The results of these studies revealed that the expansion population growth model was the best to fit the data in both datasets. The dates of the most common recent ancestors revealed that these viruses can quickly emerge in a geographical location. A mean evolutionary rate of 1.21 x 10(-2) nucleotide substitution/site/year (s/s/y) was obtained for the VP1 gene using full-length sequences. This rate is higher than the rates reported for other rapidly evolving RNA. Roughly similar rates (1.44 x 10(-2)s/s/y) were found using region D sequences, revealing the suitability of this region for evolutionary studies, in agreement with previous reports. High evolutionary rates and fast population growth may have contributed to the vigorous initial transmission dynamics of the GII/4 NoV populations studied.