Astrovirus infection in hospitalized children: Molecular, clinical and epidemiological features.

BACKGROUND: Astrovirus (HAstV) is a common viral pathogen that causes gastroenteritis worldwide. It is classified into eight classical human types (HAstV-1/8) and seven other less prevalent types, described as HAstV VA1, VA2, VA3, VA4, MLB-1, MLB-2 and MLB-3. During outbreaks, the elderly and children are the most affected, and the spread of the virus is associated with person-to-person contact, food ingestion and contaminated water. OBJECTIVES: The aim of the present study was to investigate the prevalence of infection and genetic diversity of HAstV strains. Samples were collected from children with acute gastroenteritis admitted to a large pediatric hospital during a surveillance period of three years (2008-2011) in Belém city, Pará State, Amazon Region, Northern Brazil. STUDY DESIGN: Screening and genotyping tests were conducted using RT-PCR to detect the classical and non-classical HAstV types using specific primers. A semi-nested RT-PCR protocol was developed to improve viral detection in samples with a low viral load. RESULTS: The overall positivity observed in this study was 3.9% (19/483). The age distribution showed a high prevalence of positive cases in children under one year old (5.3%). We found vomiting associated with 75% of the positive cases, fever with 82.3%, and dehydration with 76.9%. Most patients with positive cases demonstrated two to five days of diarrhea, two to three episodes of vomiting during hospitalization, and three bowel movements per day. Co-infection with HAstV and norovirus was observed in three cases (15.8%), and no pattern of seasonality or any relationship between the HAstV positivity rate and climate variables was observed. Eighteen positive samples (94.7%-18/19) were genotyped based on the ORF 2 region, and the greatest prevalence was of HAstV-1a (66.6%-12/18), followed by HAstV-2 (22.2%-4/18, comprising two type-2b and two type-2c genotypes), HAstV-3c (5.6%-1/18) and HAstV-4c (5.6%-1/18). No non-classical types were detected in the clinical samples analyzed. CONCLUSIONS: The present study showed that although HAstV infections occur at low frequency, they are involved in severe pediatric cases of acute gastroenteritis presenting with a high diversity of strains, including the lineages 3c and 4c, which were never before detected in Brazil.

Genotype diversity and molecular evolution of noroviruses: A 30-year (1982-2011) comprehensive study with children from Northern Brazil.

A chronologically comprehensive 30-year study was conducted that involved children living in Belém, in the Amazon region of Northern Brazil, who participated in eight different studies from October 1982 to April 2011. The children were followed either in the community or in health units and hospitals in order to identify the norovirus genotypes involved in infections during this time. A total of 2,520 fecal specimens were obtained and subjected to RT-PCR and nucleotide sequencing for regions A, B, C, D and P2 of the viral genome. An overall positivity of 16.9% (n = 426) was observed, and 49% of the positive samples were genotyped (208/426), evidencing the presence of several genotypes as follows: Polymerase gene (GI.P4, GII.Pa, GII.Pc, GII.Pe, GII.Pg, GII.Pj, GII.P3, GII.P4, GII.P6, GII.P7, GII.P8, GII.P12, GII.P13, GII.P14, GII.P21, GII.P22), and VP1 gene (GI.3, GI.7, GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.8, GII.10, GII.12, GII.14, GII.17, GII.23). The GII.P4/GII.4 genotype determined by both open reading frames (ORFs) (partial polymerase and VP1 genes) was found for 83 samples, and analyses of the subdomain P2 region showed 10 different variants: CHDC (1970s), Tokyo (1980s), Bristol_1993, US_95/96, Kaiso_2003, Asia_2003, Hunter_2004, Yerseke_2006a, Den Haag_2006b (subcluster "O") and New Orleans_2009. Recombination events were confirmed in 47.6% (n = 20) of the 42 samples with divergent genotyping by ORF1 and ORF2 and with probable different breakpoints within the viral genome. The evolutionary analyses estimated a rate of evolution of 1.02 x 10-2 and 9.05 x 10-3 subs./site/year using regions C and D from the VP1 gene, respectively. The present research shows the broad genetic diversity of the norovirus that infected children for 30 years in Belém. These findings contribute to our understanding of noroviruses molecular epidemiology and viral evolution and provide a baseline for vaccine design.

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.

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.

Occurrence of Norovirus GIV in Environmental Water Samples from Belém City, Amazon Region, Brazil.

Noroviruses are the major cause of non-bacterial acute gastroenteritis outbreaks in humans, with few reports about the occurrence of the norovirus GIV strain. We investigated the presence of norovirus GIV in surface water (river, bay, and stream) and untreated sewage, and we determined a positivity rate of 9.4% (9/96). The strains genotyped were GIV.1. To our knowledge, this is the first report of GIV in Brazil.

Genogroup I avian picobirnavirus detected in Brazilian broiler chickens: a molecular epidemiology study.

Picobirnavirus (PBV) belongs to the family Picobirnaviridae. Picobirnaviruses contain a bisegmented dsRNA genome that is non-enveloped. A total of 85 pooled faecal samples were collected from the poultry of 37 farms from the Metropolitan Mesoregion of Belém (MMB), Pará state, Brazil. The viral RNA from each sample was analysed by PAGE and reverse transcriptase PCR (RT-PCR). For each county affected, at least one positive sample was selected, cloned and sequenced. The samples showed a positivity of 15.3 % (13/85) by PAGE and 49.4 % (42/85) by RT-PCR. Sequencing of these strains demonstrated a considerable RdRp gene heterogeneity that ranged from 56.1 to 100 % at the nucleotide level compared with prototypes of different species and water sewage, and from 50.3 to 100 % among themselves. Avian picobirnavirus (AvPBV) was detected in MMB broiler farms and showed a heterogeneous relationship with the prototypes used. This report includes what is believed to be the first gene sequencing of AvPBV in Brazilian broiler chickens.

Norovirus diversity in diarrheic children from an African-descendant settlement in Belém, Northern Brazil.

Norovirus (NoV), sapovirus (SaV) and human astrovirus (HAstV) are viral pathogens that are associated with outbreaks and sporadic cases of gastroenteritis. However, little is known about the occurrence of these pathogens in relatively isolated communities, such as the remnants of African-descendant villages ("Quilombola"). The objective of this study was the frequency determination of these viruses in children under 10 years, with and without gastroenteritis, from a "Quilombola" Community, Northern Brazil. A total of 159 stool samples were obtained from April/2008 to July/2010 and tested by an enzyme immunoassay (EIA) and reverse transcription-polymerase chain reaction (RT-PCR) to detect NoV, SaV and HAstV, and further molecular characterization was performed. These viruses were detected only in the diarrheic group. NoV was the most frequent viral agent detected (19.7%-16/81), followed by SaV (2.5%-2/81) and HAstV (1.2%-1/81). Of the 16 NoV-positive samples, 14 were sequenced with primers targeting the B region of the polymerase (ORF1) and the D region of the capsid (ORF2). The results showed a broad genetic diversity of NoV, with 12 strains being classified as GII-4 (5-41.7%), GII-6 (3-25%), GII-7 (2-16.7%), GII-17 (1-8.3%) and GI-2 (1-8.3%), as based on the polymerase region; 12 samples were classified, based on the capsid region, as GII-4 (6-50%, being 3-2006b variant and 3-2010 variant), GII-6 (3-25%), GII-17 (2-16.7%) and GII-20 (1-8.3%). One NoV-strain showed dual genotype specificity, based on the polymerase and capsid region (GII-7/GII-20). This study provides, for the first time, epidemiological and molecular information on the circulation of NoV, SaV and HAstV in African-descendant communities in Northern Brazil and identifies NoV genotypes that were different from those detected previously in studies conducted in the urban area of Belém. It remains to be determined why a broader NoV diversity was observed in such a semi-isolated community.

Norovirus infection in children admitted to hospital for acute gastroenteritis in Belém, Pará, Northern Brazil.

Noroviruses are the leading cause of epidemic, non-bacterial outbreaks of acute gastroenteritis, and are also a major cause of sporadic acute gastroenteritis in infants. The aim of the present study was to identify norovirus infections in children not infected by rotavirus admitted to hospital for acute gastroenteritis in Belém. A total of 348 fecal specimens were obtained from children with diarrhea aged less than 5 years, all of whom had tested negative for rotavirus, between May 2008 and April 2010. Fecal samples were screened for norovirus antigen using enzyme-immunoassay (EIA). Specimens were subjected to reverse-transcription polymerase chain reaction (RT-PCR) using the primers Mon432/434-Mon431/433 for detection of the GI and GII norovirus strains, respectively. Based on both methods, the overall norovirus positivity rate was 36.5% (127/348). Of the 169 samples collected in the first year, 44.4% (n = 75) tested positive for norovirus using both methods, 35.5% (n = 60) by EIA and 40.8% (n = 69) by RT-PCR. Using RT-PCR as a reference standard, a sensitivity of 78.3%, specificity of 94%, and agreement of 87.6% were recorded. Genome sequencing was obtained for 22 (31.9%) of the 69 positive samples, of which 90.9% (20/22) were genotype GII.4d and 9.1% (2/22) were genotype GII.b. Norovirus infection was most frequent in children under 2 years of age (41.5%-115/277). The peak incidence (62.1%) of norovirus-related acute gastroenteritis in these patients (not infected by rotavirus) was observed in February 2010. These findings emphasize the importance of norovirus as a cause of severe acute gastroenteritis among children in Belém, Pará, Northern Brazil.

Phylogenetic analysis of probable non-human genes of group A rotaviruses isolated from children with acute gastroenteritis in Belém, Brazil.

Rotaviruses (RVs) are the main cause of acute viral gastroenteritis in both humans and young animals of various species such as calves, horses, pigs, dogs, cats, and birds. The genetic diversity of RVs is related to a variety of evolutionary mechanisms, including point mutation, and genome reassortment. The objective of this study was to characterize molecularly genes that encode structural and nonstructural proteins in unusual RV strains. The clinical specimens selected for this study were obtained from children and newborn with RV gastroenteritis, who participated in research projects on viral gastroenteritis conducted at the Evandro Chagas Institute. Structural (VP1-VP4, VP6, and VP7) and nonstructural (NSP1-NSP6) genes were amplified from stool samples by the polymerase chain reaction and subsequently sequenced. Eight unusual RV strains isolated from children and newborn with gastroenteritis were studied. Reassortment between genes of animal origin were observed in 5/8 (62.5%) strains analyzed. These results demonstrate that, although rare, interspecies (animal-human) transmission of RVs occurs in nature, as observed in the present study in strains NB150, HSP034, HSP180, HST327, and RV10109. This study is the first to be conducted in the Amazon region and supports previous data showing a close relationship between genes of human and animal origin, representing a challenge to the large-scale introduction of RV vaccines in national immunization programs.

Identification of lineage III of G12 rotavirus strains in diarrheic children in the Northern Region of Brazil between 2008 and 2010.

This study reports on the surveillance for rotavirus genotypes and the identification of G12 human rotavirus in the Northern Region of Brazil. Rotavirus-positive samples were collected from children <5 years of age with acute diarrhea from January 2008 to October 2010. G2P[4] was the most prevalent genotype, accounting for 45.6% (126/303) of cases. Five rotavirus strains bearing G12P[6] genotype specificity were detected. Phylogenetic analysis of the VP7 gene showed that G12 strains clustered into lineage III. This is the first detection of G12 strains from lineage III in Latin America, broadening the current evidence for the worldwide emergence of this genotype.

Evaluation of third-generation RIDASCREEN enzyme immunoassay for the detection of norovirus antigens in stool samples of hospitalized children in Belém, Pará, Brazil.

Noroviruses (NoVs) are major agents of gastroenteritis outbreaks and hospitalization worldwide. This study evaluated the sensitivity and specificity of the commercially available third-generation RIDASCREEN® Norovirus Enzyme Immunoassay (EIA) kit in comparison to the reverse transcription-polymerase chain reaction (RT-PCR) to detect NoVs in hospitalized children with gastroenteritis. An agreement of 88% (81/92) was observed when comparing EIA with RT-PCR. A sensitivity of 92% and a specificity of 83.3% were demonstrated. Eleven samples were positive by 1 method only (4 RT-PCR/7 EIA). Fourteen samples were sequenced and all classified as NoV genogroup GII-4. The 7 positive only by EIA were also evaluated by electron microscopy, and in 3 (42.9%) samples viral particles with a suggestive morphology of NoVs were visualized. These same samples were tested by seminested-RT-PCR with a positivity of 85.7%. The results obtained in this study demonstrated a significant improvement in the sensitivity and specificity of this updated assay.

Molecular characterization of norovirus, sapovirus and astrovirus in children with acute gastroenteritis from Belém, Pará, Brazil / Caracterização molecular de norovírus, sapovírus e astrovírus em crianças com gastroenterite aguda em Belém, Pará, Brasil

The importance of norovirus (NoVs), sapovirus (SaVs) and human astrovirus (HAstVs) as causes of gastroenteritis outbreaks are already well-defined, but a few studies have described sporadic cases of acute gastroenteritis caused by these viral entities. The aim of this study was to determine the role of these viruses in the etiology of acute gastroenteritis in children enrolled to participate in hospital – and emergency department – based intensive surveillance carried out in Belém, Brazil, from March to September 2003. A total of 305 stool specimens from patients with severe gastroenteritis were collected and screened by reverse transcription followed by polymerase chain reaction (RT-PCR), using the specific primers Mon 269 and Mon 270 for HAstVs, p289 and p290 for human calicivirus (HuCVs), and Mon 431/433 and Mon 432/434 for NoVs. Sequencing of RT-PCR HAstV, HuCVs and NoVs amplicons was carried out using the same primers. Of the 305 samples tested, 96 (31.5 percent) were positive, with 51 diagnosed as HuCVs, 40 as HAstVs and five as mixed infections. Of the 56 (18.4 percent) HuCVs sequenced, 30 were NoVs (9.8 percent) of genogroups GI-4 and GII-4, and 15 (4.9 percent) were SaVs of types GI-1, GI-2 and GII-1. HAstVs, including genotypes 1, 8 and 2, were detected in 45 (14.7 percent) samples. This study has highlighted the importance of these viruses as causes of acute gastroenteritis and established the circulation of different genotypes during the study period. These results reinforce the need for establishing an intensive surveillance for gastroenteritis caused by these viruses to assess the burden of disease and to monitor the circulation of genotypes.

A importância dos norovírus (NoVs), sapovírus (SaVs) e astrovírus humanos (HAstVs) como causa de surtos de gastroenteritis já está bem definida. Entretanto, poucos estudos têm descrito casos esporádicos de gastroenterites aguda causados por esses agentes. O objetivo deste estudo foi determinar o papel destes vírus na etiologia da gastroenterite aguda em crianças atendidas durante uma vigilância intensiva realizada em hospitais e ambulatórios de Belém, Brasil, de março a setembro de 2003. Um total de 305 espécimes fecais de pacientes com gastrenterite grave foram coletados e testados por reação em cadeia da polimerase precedida de transcrição reversa (RT-PCR), utilizando iniciadores específicos Mon 269 e Mon 270 para os HAstVs; p289 e p290 para os calicivírus humanos (HuCVs); e Mon 431/433 e Mon 432/434 para os NoVs. Sequenciamento dos amplicons de HAstV, HuCVs e NoVs, obtidos por RT-PCR, foi realizado usando os mesmos iniciadores. Das 305 amostras testadas, 96 (31,5 por cento) apresentaram resultados positivos, sendo que 51 diagnosticadas como HuCVs, 40 como HAstVs e cinco infecções mistas. Das 56 (18,4 por cento) amostras de HuCVs sequenciadas, 30 foram NoVs (9,8 por cento) pertencentes aos genogrupos GI-4 e GII-4, e 15 (4,9 por cento) SaVs dos grupos GI-1, GI-2 e GII-1. HAstVs foram detectados em 45 (14,7 por cento) das amostras, incluindo os genótipos 1, 8 e 2. Esta pesquisa ressalta a importância destas viroses como causa de gastrenterite aguda e demonstra a circulação de diferentes genótipos durante o período de estudo. Estes resultados reforçam a necessidade de se estabelecer uma vigilância intensiva das gastrenterite causadas por estes vírus, de forma a poder avaliar o impacto da doença e monitorar os genótipos circulantes.

The "pressure pan" evolution of human erythrovirus B19 in the Amazon, Brazil.

To understand the evolutionary dynamics of human parvovirus B19, we analyzed VP1 and VP2 gene sequences of B19 sampled from Belém (Amazon), the city of São Paulo, Brazil and globally. Our analysis revealed a strikingly different pattern of evolutionary change for those viral lineages introduced into Belém, which exhibited a higher rate of nonsynonymous substitutions compared to those viruses sampled from other locations. We propose that difference this is due to the high prevalence of B19 in Belém (up to 85%) compared to other locations (prevalences of approximately 50%), which imposes a more intense selection pressure. Hence, those B19 lineages introduced into Belém experienced an elevated rate of amino acid change, driven by positive selection, in order to generate serial re-infections in a small web of transmission, which can be thought of as an evolutionary "pressure pan".