Molecular and evolutionary characterization of norovirus GII.17 in the northern region of Brazil.

BACKGROUND: Currently, norovirus (NoV) is associated with one-fifth of all acute gastroenteritis (AGE) cases worldwide. The NoV GII.17_2014 variant has been associated with gastroenteritis outbreaks in several Asian countries, replacing the previously dominant Sydney 2012 variant. There is limited data about circulation of this new strain in Brazil. This study aimed to describe the phylogenetic and evolutionary characteristics of the GII.17_2014 strains in the Northern region of Brazil. METHODS: NoV was detected by enzyme immunoassay (EIA) in 645 stool samples of AGE cases that were reported in Pará and Amazonas states during 2015-2016. All positive samples were tested for NoV GI and GII by reverse transcription polymerase chain reaction (RT-PCR) and the amplicons were subjected to genome sequencing. The GII.17-positive samples were retested by PCR using different sets of designed primers, which target a highly conserved capsid gene region. Next, the amplicons were sequenced and phylogenetically analyzed using Bayesian inferences. RESULTS: Of the 645 samples tested, 208 (32.2%) tested were positive for NoV by EIA, among which 95 (45.7%) were genotyped. Among the genotyped samples, 12 (12.6%) were characterized as GII.17_2014 with the first case detected in November 2015 (1/30, 3.3%) and the others in 2016 (11/65, 16.9%). All strains found in our study were clustered in clade D (epidemic strain). The uncorrelated log-normal model estimations calculated the rate of evolution for GII-17 strains as 1.95 × 10- 3 (1.28 × 10- 3-2.63 × 10- 3). In total, 36 nucleotide changes were observed after analyzing the VP1 sequence, among which 28 occurred in the P2 region. CONCLUSIONS: These data demonstrate the evolutionary dynamics in NoV GII.17_2014 strains, which indicated high mutation rates with nucleotide substitutions and indels that are related to the elevated levels of antigenic diversity. This partly explains the increase in viral prevalence.

Detection and genotyping of enteric viruses in hospitalized children with acute gastroenteritis in Belém, Brazil: Occurrence of adenovirus viremia by species F, types 40/41.

Enteric adenovirus (AdV), sapovirus (SaV), and human astrovirus (HAstV) are important pathogens involved in the gastroenteritis etiology. In this study, a total of 219 fecal samples and sera were collected from children hospitalized for acute gastroenteritis (AGE) in two large pediatric hospitals in Belém, from March 2012 to April 2015. The samples were analyzed by polymerase chain reaction (PCR) for AdV and HAstV (astrovirus) detection, and Nested-PCR and qPCR for SaV detection. AdV was detected in 50.2% (110/219) of the cases, with 42.7% (47/110) being sequenced and classified as: species F (63.9% - 30/47), A (4.2% - 2/47), B (6.4% - 3/47), C (17.1% - 8/47), D (4.2% - 2/47), and E (4.2% - 2/47). Of the 110 AdV-positive feces samples, 80 paired sera presented sufficient amounts and were also tested for this virus, of which 51 (63.7%) showed positive results and 26 (70.3%) pairs (feces plus sera) presented concordant results after sequencing being classified as: species F (21/26; 80.8%), A (1/26; 3.8%), B (1/26; 3.8%), and C (3/26; 11.5%). Overall, HAstV rate in the feces samples was 1.8% (4/219), including both HAstV-1a (2/4; 50%) and HAstV-2c (2/4; 50%). SaV was detected in 4.6% (10/219) of the fecal samples, out of which 50% (5/10) of the positive samples were characterized into the genogroups GI.1 (1), GI.2 (2), and GII.4 (2). These findings highlighted the important contributions of AdV, HAstV, and SaV in the enteric virus spectrum in our region and showed the high genetic diversity of AdV. In addition, it demonstrated for the first time in Brazil, the circulation of AdV in the serum of hospitalized children with AGE.

High prevalence of norovirus in children with sporadic acute gastroenteritis in Manaus, Amazon Region, northern Brazil.

BACKGROUND: Norovirus (NoV) is a major cause of acute gastroenteritis (AGE) worldwide, especially in children under five years. Studies involving the detection and molecular characterisation of NoV have been performed in Brazil, demonstrating its importance as an etiological agent of AGE. OBJECTIVES: The objectives of this study were to investigate the frequency of human NoV and to genotype the strains isolated from 0-14-year-old patients of AGE in Manaus, Brazil, over a period of two years. METHODS: A total of 426 faecal samples were collected between January 2010 and December 2011. All samples were tested for the presence of NoV antigens using a commercial enzyme immunoassay kit. RNA was extracted from all faecal suspensions and reverse transcription-polymerase chain reaction (RT-PCR) for the NoV-polymerase partial region was performed as a trial test. Positive samples were then subjected to PCR with specific primers for partial capsid genes, which were then sequenced. FINDINGS: NoV was detected in 150 (35.2%) faecal samples, for at least one of the two techniques used. NoV was detected in children from all age groups, with the highest positivity observed among the group of 1-2 years old. Clinically, fever was verified in 43% of the positive cases and 46.3% of the negative cases, and vomiting was observed in 75.8% and 70.8% cases in these groups, respectively. Monthly distribution showed that the highest positivity was observed in January 2010 (81.2%), followed by February and April 2010 and March 2011, when the positivity rate reached almost 50%. Phylogenetic analyses performed with 65 positive strains demonstrated that 58 (89.2%) cases of NoV belonged to genotype GII.4, five (7.7%) to GII.6, and one (1.5%) each to GII.7 and GII.3. MAIN CONCLUSIONS: This research revealed a high circulation of NoV GII.4 in Manaus and contributed to the understanding of the importance of this virus in the aetiology of AGE cases, especially in a region with such few studies available.

High prevalence of norovirus in children with sporadic acute gastroenteritis in Manaus, Amazon Region, northern Brazil

BACKGROUND Norovirus (NoV) is a major cause of acute gastroenteritis (AGE) worldwide, especially in children under five years. Studies involving the detection and molecular characterisation of NoV have been performed in Brazil, demonstrating its importance as an etiological agent of AGE. OBJECTIVES The objectives of this study were to investigate the frequency of human NoV and to genotype the strains isolated from 0-14-year-old patients of AGE in Manaus, Brazil, over a period of two years. METHODS A total of 426 faecal samples were collected between January 2010 and December 2011. All samples were tested for the presence of NoV antigens using a commercial enzyme immunoassay kit. RNA was extracted from all faecal suspensions and reverse transcription-polymerase chain reaction (RT-PCR) for the NoV-polymerase partial region was performed as a trial test. Positive samples were then subjected to PCR with specific primers for partial capsid genes, which were then sequenced. FINDINGS NoV was detected in 150 (35.2%) faecal samples, for at least one of the two techniques used. NoV was detected in children from all age groups, with the highest positivity observed among the group of 1-2 years old. Clinically, fever was verified in 43% of the positive cases and 46.3% of the negative cases, and vomiting was observed in 75.8% and 70.8% cases in these groups, respectively. Monthly distribution showed that the highest positivity was observed in January 2010 (81.2%), followed by February and April 2010 and March 2011, when the positivity rate reached almost 50%. Phylogenetic analyses performed with 65 positive strains demonstrated that 58 (89.2%) cases of NoV belonged to genotype GII.4, five (7.7%) to GII.6, and one (1.5%) each to GII.7 and GII.3. MAIN CONCLUSIONS This research revealed a high circulation of NoV GII.4 in Manaus and contributed to the understanding of the importance of this virus in the aetiology of AGE cases, especially in a region with such few studies available.

Norovirus genogroups I and II in environmental water samples from Belém city, Northern Brazil.

This study investigated the presence of norovirus (NoV) GI and GII in environmental samples from the northern region of Brazil. Water samples were collected monthly (November 2008/October 2010) from different sources and sewage and concentrated by the adsorption-elution method. The NoV investigation used molecular methods followed by sequencing reactions. The general positivity for NoV was 33.9% (57/168). Considering the results obtained only in the semi-nested RT-PCR (reverse transcription polymerase chain reaction) and only in the TaqMan® real-time PCR, the rates were 26.8% (45/168) and 27.4% (46/168), respectively, being for NoV GI 22.2% (10/45) and 19.6% (9/46); for GII 17.8% (8/45) and 15.2% (7/46); and for GI + GII 60% (27/45) and 65.2% (30/46), respectively. Different GI (GI.1, GI.4, GI.7 and GI.8) and GII (GII.4, GII.6, GII.9, GII.12 and GII.14) genotypes were detected. These results demonstrated the NoV was disseminated in the waters of Belém city due to a lack of sanitation that allowed the discharge of contaminated effluents into these aquatic ecosystems.

Detection and genetic characterization of the emergent GII.17_2014 norovirus genotype among children with gastroenteritis from Northern Brazil.

Norovirus is the most important cause of viral gastroenteritis outbreaks worldwide. Recently, a novel GII.17 norovirus variant emerged and caused epidemics in Asian countries, replacing the GII.4 Sydney 2012 strain in hospitalized cases. In this study we describe the emergence of this novel NoV GII.17_2014 strain in Brazil.

SAPOVIRUSES IN CHILDREN WITH ACUTE GASTROENTERITIS FROM MANAUS , AMAZON REGION, BRAZIL, 2010-2011.

Sapoviruses (SaVs) are responsible for acute gastroenteritis in humans, especially children and the elderly. In Brazil, data on SaVs infections are very limited, especially in Northern Brazil. Here, we investigated the occurrence of SaVs in samples from hospitalized children under ten years old that presented acute gastroenteritis. Positive samples were genotyped and phylogenetic analysis was performed using prototype strains sequences obtained from GenBank database. In total, 156 fecal samples were screened by RT-PCR for SaVs. A positivity rate of 3.8% (6/156) was found in children under three years of age. Four genotypes were detected: GI.I, GI.2 and GII.2?-GII.4?/GII.4, suggesting a possible inter-genotypes recombination. Most infections (83.3%) occurred between August and September. The positivity was similar to that found in other countries and genotyping demonstrated the presence of distinct genotypes. To our knowledge, this is the first study reporting the circulation of SaVs in Manaus, state of Amazonas, Amazon region, Brazil.

Caliciviruses in hospitalized children, São Luís, Maranhão, 1997-1999: detection of norovirus GII. 12

ABSTRACT Gastroenteritis is one of the most common diseases during childhood, with norovirus (NoV) and sapovirus (SaV) being two of its main causes. This study reports for the first time the incidence of these viruses in hospitalized children with and without gastroenteritis in São Luís, Maranhão. A total of 136 fecal samples were tested by enzyme immunoassays (EIA) for the detection of NoV and by reverse transcription-polymerase chain reaction (RT-PCR) for detection of both NoV and SaV. Positive samples for both agents were subjected to sequencing. The overall frequency of NoV as detected by EIA and RT-PCR was 17.6% (24/136) and 32.6% (15/46), respectively in diarrheic patients and 10.0% (9/90) in non-diarrheic patients (p < 0.01). Of the diarrheic patients, 17% had fever, vomiting and anorexia, and 13% developed fever, vomiting and abdominal pain. Of the 24 NoV-positive samples, 50% (12/24) were sequenced and classified as genotypes GII.3 (n = 1), GII.4 (6), GII.5 (1), GII.7 (2), GII.12 (1) and GII.16 (1). SaV frequency was 9.8% (11/112), with 22.6% (7/31) in diarrheic patients and 4.9% (4/81) in nondiarrheic (p = 0.04) ones. In diarrheic cases, 27.3% had fever, vomiting and anorexia, whereas 18.2% had fever, anorexia and abdominal pain. One SaV-positive sample was sequenced and classified as GII.1. These results show a high genetic diversity of NoV and higher prevalence of NoV compared to SaV. Our data highlight the importance of NoV and SaV as enteropathogens in São Luís, Maranhão.

Caliciviruses in hospitalized children, São Luís, Maranhão, 1997-1999: detection of norovirus GII.12.

Gastroenteritis is one of the most common diseases during childhood, with norovirus (NoV) and sapovirus (SaV) being two of its main causes. This study reports for the first time the incidence of these viruses in hospitalized children with and without gastroenteritis in São Luís, Maranhão. A total of 136 fecal samples were tested by enzyme immunoassays (EIA) for the detection of NoV and by reverse transcription-polymerase chain reaction (RT-PCR) for detection of both NoV and SaV. Positive samples for both agents were subjected to sequencing. The overall frequency of NoV as detected by EIA and RT-PCR was 17.6% (24/136) and 32.6% (15/46), respectively in diarrheic patients and 10.0% (9/90) in non-diarrheic patients (p<0.01). Of the diarrheic patients, 17% had fever, vomiting and anorexia, and 13% developed fever, vomiting and abdominal pain. Of the 24 NoV-positive samples, 50% (12/24) were sequenced and classified as genotypes GII.3 (n=1), GII.4 (6), GII.5 (1), GII.7 (2), GII.12 (1) and GII.16 (1). SaV frequency was 9.8% (11/112), with 22.6% (7/31) in diarrheic patients and 4.9% (4/81) in nondiarrheic (p=0.04) ones. In diarrheic cases, 27.3% had fever, vomiting and anorexia, whereas 18.2% had fever, anorexia and abdominal pain. One SaV-positive sample was sequenced and classified as GII.1. These results show a high genetic diversity of NoV and higher prevalence of NoV compared to SaV. Our data highlight the importance of NoV and SaV as enteropathogens in São Luís, Maranhão.

The prevalence of norovirus, astrovirus and adenovirus infections among hospitalised children with acute gastroenteritis in Porto Velho, state of Rondônia, western Brazilian Amazon.

Although viruses are well-established causes of acute gastroenteritis, few data on the circulation of these pathogens in Porto Velho, state of Rondônia, Brazil, are available. Thus, faecal samples from hospitalised diarrhoeic children, under six years of age, were collected and tested for the presence of norovirus (NoV), adenovirus (AdV) and astrovirus (AstV) from February 2010-February 2012. Specimens were screened by reverse-transcription polymerase chain reaction and viruses were found in 10.7% (63/591) of the cases. NoV, AdV and AstV were detected in 7.8%, 2% and 0.8% of the samples, respectively. NoV infection was observed at all ages and was most prevalent in zero-18-month-old children (84.7%; p = 0.002). A higher incidence of NoV was detected from February-April 2010, when it was found in 52.2% of the cases. Co-infections involving these viruses, rotavirus and enteropathogenic bacteria were detected in 44.4% (28/63) of the children with viral diarrhoea. Nosocomial infections were demonstrated in 28.6% (18/63) of the cases in which viruses were detected. The present paper reports, for the first time, the circulation of NoV and AstV among the paediatric population of Porto Velho and it contributes to our understanding of the roles of these pathogens in gastrointestinal infections.

The prevalence of norovirus, astrovirus and adenovirus infections among hospitalised children with acute gastroenteritis in Porto Velho, state of Rondônia, western Brazilian Amazon

Although viruses are well-established causes of acute gastroenteritis, few data on the circulation of these pathogens in Porto Velho, state of Rondônia, Brazil, are available. Thus, faecal samples from hospitalised diarrhoeic children, under six years of age, were collected and tested for the presence of norovirus (NoV), adenovirus (AdV) and astrovirus (AstV) from February 2010-February 2012. Specimens were screened by reverse-transcription polymerase chain reaction and viruses were found in 10.7% (63/591) of the cases. NoV, AdV and AstV were detected in 7.8%, 2% and 0.8% of the samples, respectively. NoV infection was observed at all ages and was most prevalent in zero-18-month-old children (84.7%; p = 0.002). A higher incidence of NoV was detected from February-April 2010, when it was found in 52.2% of the cases. Co-infections involving these viruses, rotavirus and enteropathogenic bacteria were detected in 44.4% (28/63) of the children with viral diarrhoea. Nosocomial infections were demonstrated in 28.6% (18/63) of the cases in which viruses were detected. The present paper reports, for the first time, the circulation of NoV and AstV among the paediatric population of Porto Velho and it contributes to our understanding of the roles of these pathogens in gastrointestinal infections.

Enteropathogens detected in a daycare center, Southeastern Brazil: bacteria, virus, and parasite research.

INTRODUCTION: The objective of this study was to determine the prevalence and etiological profile of enteropathogens in children from a daycare center. METHODS: From October 2010 to February 2011 stool samples from 100 children enrolled in a government daycare center in the municipality of São José do Rio Preto, in the state of São Paulo, were collected and analyzed. RESULTS: A total of 246 bacteria were isolated in 99% of the fecal samples; 129 were in the diarrheal group and 117 in the non-diarrheal group. Seventy-three strains of Escherichia coli were isolated, 19 of Enterobacter, one of Alcaligenes and one of Proteus. There were 14 cases of mixed colonization with Enterobacter and E. coli. Norovirus and Astrovirus were detected in children with clinical signs suggestive of diarrhea. These viruses were detected exclusively among children residing in urban areas. All fecal samples were negative for the presence of the rotavirus species A and C. The presence of Giardia lamblia, Entamoeba coli, Endolimax nana and hookworm was observed. A significant association was found between food consumption outside home and daycare center and the presence of intestinal parasites. CONCLUSIONS: For children of this daycare center, intestinal infection due to pathogens does not seem to have contributed to the occurrence of diarrhea or other intestinal symptoms. The observed differences may be due to the wide diversity of geographical, social and economic characteristics and the climate of Brazil, all of which have been reported as critical factors in the modulation of the frequency of different enteropathogens.

Enteropathogensddetected in a daycare center, sutheastern Brazil: bacteria, viirus, and parasite research / Enteropatógenos detectados em crianças de creche no Sudeste do Brasil: pesquisa de bactérias, vírus e parasitos

Introduction: The objective of this study was to determine the prevalence and etiological profile of enteropathogens in children from a daycare center. Methods: From October 2010 to February 2011 stool samples from 100 children enrolled in a government daycare center in the municipality of São José do Rio Preto, in the state of São Paulo, were collected and analyzed. Results: A total of 246 bacteria were isolated in 99% of the fecal samples; 129 were in the diarrheal group and 117 in the non-diarrheal group. Seventy-three strains of Escherichia coli were isolated, 19 of Enterobacter, one of Alcaligenes and one of Proteus. There were 14 cases of mixed colonization with Enterobacter and E. coli. Norovirus and Astrovirus were detected in children with clinical signs suggestive of diarrhea. These viruses were detected exclusively among children residing in urban areas. All fecal samples were negative for the presence of the rotavirus species A and C. The presence of Giardia lamblia, Entamoeba coli, Endolimax nana and hookworm was observed. A significant association was found between food consumption outside home and daycare center and the presence of intestinal parasites. Conclusions: For children of this daycare center, intestinal infection due to pathogens does not seem to have contributed to the occurrence of diarrhea or other intestinal symptoms. The observed differences may be due to the wide diversity of geographical, social and economic characteristics and the climate of Brazil, all of which have been reported as critical factors in the modulation of the frequency of different enteropathogens.

Introdução: O objetivo deste estudo foi determinar a prevalência e o perfil etiológico de enteropatógenos em crianças de uma creche. Métodos: No período de outubro de 2010 a fevereiro de 2011 foram coletadas e analisadas amostras de fezes de 100 crianças matriculadas em creche do governo no município de São José do Rio Preto, Estado de São Paulo. Resultados: Um total de 246 bactérias foram isoladas em 99% das amostras de fezes; 129 eram diarreicas e 117 não-diarreicas. Foram isoladas setenta e três cepas de Escherichia coli, 19 de Enterobacter, uma de Alcaligenes e uma de Proteus. Foram detectados 14 casos de colonização mista com Enterobacter e de E. coli. Norovírus e Astrovirus foram detectados em crianças com sinais clínicos sugestivos de diarréia. Estes vírus foram detectados exclusivamente entre as crianças residentes em áreas urbanas. Todas as amostras fecais foram negativas para a presença das espécies de rotavírus A e C. Foi observada a presença de Giardia lamblia, Entamoeba coli, Endolimax nana e ancilostomídeos. Foi encontrada associação significativa entre o consumo de alimentos fora do centro da casa e creche e a presença de parasitos intestinais. Conclusões: Para as crianças desta creche, a infecção intestinal por patógenos não parece ter contribuido para a ocorrência de diarreia ou outros sintomas intestinais. As diferenças observadas podem ser atribuídas à grande diversidade de características geográficas, sociais e econômicas e o clima do Brasil, as quais tem sido relatadas como fatores críticos para a modulação da frequência de diferentes enteropatógenos.

Detection of the pandemic norovirus variant GII.4 Sydney 2012 in Rio Branco, state of Acre, northern Brazil.

Noroviruses (NoVs) are important cause of gastroenteritis in humans worldwide. Genotype GII.4 is responsible for the majority of outbreaks reported to date. This study describes, for the first time in Brazil, the circulation of NoV GII.4 variant Sydney 2012 in faecal samples collected from children aged less than or equal to eight years in Rio Branco, state of Acre, northern Brazil, during July-September 2012.

Identification of two sublineages of genotype G2 rotavirus among diarrheic children in Parauapebas, Southern Pará State, Brazil.

On a world scale, group A human rotaviruses are the most common cause of severe acute gastroenteritis during infancy and childhood, including five (G1, G2, G3, G4, and G9) epidemiologically important genotypes. Among these, G2 denotes a different genogroup which appears to have a cyclic pattern of occurrence and yet little information is available about its genetic variability. The aim of this report was to characterize the emergence of G2 genotype in Paraupebas, Southern Pará State, Brazil, some of which detected after introduction of rotavirus vaccine. A total of 241 fecal specimens from young children with acute gastroenteritis were collected from the "Yutaka Takeda Hospital," a Municipality Hospital, and at the Parauapebas' Health Unit, Pará, from January to September 2006 and during March to November 2008. All samples were tested for rotavirus using immunochromatography, polyacrylamide gel electrophoresis (PAGE), and RT-PCR, yielding an overall positivity of 12.45% (30/241). Rotavirus G2P[4] was identified in 27 of 30 samples (90%), followed by G1P[8] (2/30, 6.67%) and G9P[8] (1/30, 3.33%). Phylogenetic analysis was performed in 15 of the G2 strains, all of which grouped into lineage II. Four of these strains clustered into sublineage II-a (year 2006) and 11 into one possible new sublineage named II-c (year 2008, except SAL-1920-C). The recent re-emergence of G2 genotype associated with lineage II in Brazil warrants the continuous monitoring of circulating rotavirus strains following the nationwide universal use of rotavirus vaccine.