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.

Epidemiological and molecular surveillance of norovirus in the Brazilian Amazon: description of recombinant genotypes and improvement of evolutionary analysis.

Noroviruses are highly infectious, genetically diverse viruses. Global outbreaks occur frequently, making molecular surveillance important for infection monitoring. This cross-sectional descriptive study aimed to monitor cases of norovirus gastroenteritis in the Brazilian Amazon. Fecal samples were tested by immunoenzymatic assay, RT-PCR and genetic sequencing for the ORF1/ORF2 and protease regions. Bayesian inference with a molecular clock was employed to construct the phylogeny. The norovirus prevalence was 25.8%, with a higher positivity rate among children aged 0-24 months. Genogroup GII accounted for 98.1% of the sequenced samples, while GI accounted for 1.9% of them. The GII.P16/GII.4 genotype was the most prevalent, with an evolution rate of 2.87x10-3 and TMRCA estimated in 2012. This study demonstrates that norovirus is a primary causative agent of gastroenteritis and provides data on viral genetic diversity that may facilitate infection surveillance and vaccine development.

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.

Detection of G3 human-like rotavirus in institutionalized dogs from Brazil.

Viral gastroenteritis is a common clinical problem in dogs and group A rotavirus (RVA) is one of the agents involved in this etiology. It mainly affects dogs in the first 6 months of life, and these animals are considered an important reservoir and potential transmitters of the virus to other susceptible hosts, such as humans. Among the different types of RVA, G3 is the most detected in dogs, and this genotype is also involved in infections in other animals, including humans. Thus, the present study aims to investigate the presence of RVA in samples of dogs from a public kennel. A total of 64 fecal samples from dogs with diarrhea were analyzed, collected from April 2019 to March 2020, from the kennel of the Zoonosis Control Center, located in Belém, a city in the North of Brazil. The extracted genetic material was subjected to reverse transcription followed by real-time PCR (RT-qPCR); the positives were tested by RT-PCR with a specific primer for the RVA VP7 gene, after nucleotide sequencing and phylogenetic analysis. One sample was subjected to high-performance sequencing. A positivity of 7.8% (5/64) was observed for RVA, all characterized as G3, grouping in the G3-III lineage, with greater similarity to human samples. Different regions of the RVA genome fragments were found. These results emphasize the need for animal health surveillance to better understand the global strain dispersion of RVA and elucidate possible interspecies transmission events, monitoring the genetic diversity of this pathogen.

Epidemiological and molecular surveillance of norovirus in the Brazilian Amazon: description of recombinant genotypes and improvement of evolutionary analysis

ABSTRACT Noroviruses are highly infectious, genetically diverse viruses. Global outbreaks occur frequently, making molecular surveillance important for infection monitoring. This cross-sectional descriptive study aimed to monitor cases of norovirus gastroenteritis in the Brazilian Amazon. Fecal samples were tested by immunoenzymatic assay, RT-PCR and genetic sequencing for the ORF1/ORF2 and protease regions. Bayesian inference with a molecular clock was employed to construct the phylogeny. The norovirus prevalence was 25.8%, with a higher positivity rate among children aged 0-24 months. Genogroup GII accounted for 98.1% of the sequenced samples, while GI accounted for 1.9% of them. The GII.P16/GII.4 genotype was the most prevalent, with an evolution rate of 2.87x10−3 and TMRCA estimated in 2012. This study demonstrates that norovirus is a primary causative agent of gastroenteritis and provides data on viral genetic diversity that may facilitate infection surveillance and vaccine development.

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.

Analysis of uncommon norovirus recombinants from Manaus, Amazon region, Brazil: GII.P22/GII.5, GII.P7/GII.6 and GII.Pg/GII.1.

Norovirus (NoV) is responsible for outbreaks and sporadic cases of nonbacterial acute gastroenteritis in humans worldwide. The virus consists of small round particles containing a single-stranded RNA genome that is divided into three Open Reading Frames. NoV evolves via mechanisms of antigenic drift and recombination, which lead to the emergence of new strains that are capable of causing global epidemics. Recombination usually occurs in the ORF1/ORF2 overlapping region and generates strains with different genotypes in the polymerase and capsid region. The primary objective of this study was to analyze recombination in positive-NoV samples. Specimens were collected during 2011, 2012 and 2014, from children under two years of age presenting gastrointestinal symptoms such as vomiting and diarrhea. The partial polymerase (B region), capsid (D region) genes and the ORF1-ORF2 overlap regions were sequenced in each sample. The recombinant analyses were performed in the Simplot software v.3.5.1 and RDP4 Beta v. 4.6 program. These analyses showed that GII.Pg/GII.1, GII.P7/GII.6, and GII.P22/GII.5 were recombinant strains. To our knowledge, this is the first time that the GII.P22/GII.5 and GII.Pg/GII.1 strains were described in South America and the GII.P7/GII.6 was detected in Northern of 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.

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.

Validação de RT-PCR multiplex para detecção de rotavírus A, D, F e G em aves

Os Rotavírus (RV) são considerados o principal patógeno que causa gastroenterite tanto em seres humanos, quanto em animais jovens de várias espécies, como as aves e os mamíferos. A transmissão dos RV ocorre principalmente por via fecal-oral, podendo ocorrer também por meio de água e alimentos contaminados. Os grupos de RV que infectam exclusivamente as aves são os RVA, RVD, RVF e RVG. No Brasil, os RV já foram detectados em aves de corte, aves domésticas e aves selvagens. No estado do Pará, já foram registrados os RVA, RVD, RVF e RVG em aves de corte e os RVD em aves silvestres, porém foram detectados em ensaios de RT-PCR separadamente, e por esse motivo é que se sugere um ensaio diagnóstico de PCR Multiplex para que esses agentes sejam detectados simultaneamente. Este estudo objetivou padronizar a técnica de RT-PCR Multiplex para detecção de Rotavírus A, D, F e G em aves. Foram utilizadas 21 amostras do banco de amostras do Instituto Evandro Chagas. Suspensões fecais a 10% em tampão Tris-Ca++ 0,01M foram realizadas, seguida da extração do genoma viral. A Reação em Cadeia mediada pela Polimerase precedida por Transcrição Reversa (RT-PCR Multiplex) foi realizada utilizando diferentes iniciadores específicos para RVA, RVD, RVF e RVG. Para confirmar a positividade das amostras, bem como a validação da RT-PCR Multiplex, os produtos da RT- PCR Multiplex foram submetidos à eletroforese em gel de agarose a 2%, em seguida purificados e sequenciados pelo método de Sanger. Os primers foram avaliados quanto a sua especificidade e sensibilidade e a técnica de RT-PCR Multiplex foi avaliada quanto a sua reprodutibilidade, repetibilidade e o coeficiente de Kappa. Dessa forma, todos os primers foram específicos e com um limiar de detecção de 5x10- 5ng/µL. A técnica apresentou uma reprodutibilidade de 91,6%, repetibilidade 93,54% e o coeficiente de Kappa igual a 0,82. Dessa forma, o teste desenvolvido foi considerado uma ferramenta específica e sensível para a detecção simultânea dos diferentes grupos de RV que infectam aves. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização genômica de rotavírus A com potencial zoonótico em Belém, Pará, Brasil

Os RVA são de particular importância epidemiológica e estão dispersos em todo o mundo, causando a maioria das gastroenterites em humanos e com alta prevalência em animais. A transmissão zoonótica pode ocorrer de animais para humanos, e as vacinas previnem a infecção por RVA, contudo novas cepas surgem e a recombinação zoonótica explica a atual diversidade antigênica e genética de RVA. No estudo, selecionou-se 83 amostras fecais de origem humana positivas para RVA de Belém, Pará, Brasil. Identificou-se 17 amostras com potencial zoonótico, destas, nove foram confirmadas como potencial zoonótico. As amostras do genótipo G4P[6] apresentaram origem suína. A amostra HSE005 apresentou perfil zoonótico e as análises filogenéticas indicaram que esta amostra agrupou na linhagem II quando analisada para a maioria dos genes, exceto para VP7, VP4 e NSP4. Foi identificado padrão de rearranjo com amostras oriundas de suínos para os genes NSP4, VP3, VP4 e VP6. Uma amostra de genótipo G3P[3] evoluiu de origem canina, felina e símia e um rearranjo com a linhagem II. As cepas G12P[9] tiveram origem em quirópteros, bovinos e felinos. O presente estudo incluiu análises filodinâmicas para elucidar os padrões evolutivos desconhecidos, principalmente em relação aos genótipos G3P[3] e G12P[9]. Esses eventos evolutivos contribuem na identificação de cepas emergentes de RVA com potencial zoonótico e escape vacinal.

Inquérito soroepidemiológico e molecular das hepatites A, B e C em escolas estaduais no município de Capanema, Pará, Brasil

Hepatite é um termo genérico que significa inflamação do fígado. Entre os principais vírus das diferentes hepatites virais humanas, destacam-se os vírus das hepatites A (VHA), B (VHB) e C (VHC). A hepatite A desenvolve doença aguda e não chega a progredir para um processo crônico; segundo a Organização Mundial de Saúde, cerca de dois bilhões de pessoas já foram infectadas pelo VHB. Para o HCV existe cerca de 71 milhões de indivíduos infectados, com 3 a 4 milhões de portadores crônicos no Brasil. O estudo é relevante por informar a realidade epidemiológica das hepatites A, B e C, e a situação vacinal para a hepatite B dos escolares, no município de Capanema, Pará, visando o planejamento da prevenção e assistência a saúde. Esta pesquisa identificou por meio de estudo soroepidemiológico e molecular a prevalência dos vírus das hepatites A, B e C, entre alunos de escolas públicas no Município de Capanema, Pará, Brasil. Participaram do estudo 505 alunos do ensino médio e Educação de Jovens e Adultos das Escolas João Santos, Professora América Leão Condurú e Professora Maria Amélia Vasconcelos, com idade 18 ± 4,84 anos e a mediana foi de 17 anos de idade. A coleta de dados foi realizada, com o consentimento voluntário dos participantes, preenchimento de ficha de inquérito individual e coleta de amostras de sangue de cada participante. Os testes sorológicos das hepatites A (anti-VHA total), B (HBsAg, anti-HBc total, anti-HBs) e C (anti-VHC) foram realizados na Seção de Hepatologia do IEC, por técnica imunoenzimática (ELISA). Nas amostras HBsAg e anti-HBc total isolado reagentes, foram pesquisados o anti-HBc IgM, HBeAg e anti-HBe. Nos resultados inconclusivos para o VHB e VHC, foi realizada detecção da carga viral por PCR em tempo real. Os resultados mostraram prevalência de 36,6% para o anti-VHA total reagente com 63,4% suscetíveis; de 0,2% para HBsAg e 47,5% anti-HBs isolado reagente, na única amostra HBsAg reagente o VHB- DNA foi detectável com baixa carga viral ; não houve resultado reagente para o anti-VHC. Observou-se que a pesquisa foi relevante para o município, uma vez que esferas governamentais de saúde pública foram provocadas a avaliarem os serviços de saúde e vigilância epidemiológica as hepatites virais, mostrando que estratégias prevenção devem ser ajustadas a realidade da população, com intensificação das campanhas de vacinação, para aumentar a cobertura vacinal contra as hepatites A e B na população de município de Capanema, Pará, Brasil.

Análise genotípica de cepas de noravírus provenientes de Manaus, Amazonas, no periódo de 2010-2014

Resumo: Mundialmente, os norovírus (NoV) constituem uma das principais causas de gastrenterite aguda (GA). Durante os últimos anos, têm-se observado a ascensão desses vírus como agentes causadores de diarreia, sendo atualmente a segunda maior causa de GA viral em crianças e o principal responsável por surtos de diarreia não-bacterianos. O objetivo deste estudo foi realizar a genotipagem e caracterização filogenética dos NoV detectados em amostras fecais de crianças com GA provenientes da cidade de Manaus, Amazonas, durante o período de janeiro de 2010 a dezembro de 2014. Neste trabalho, foram analisadas amostras fecais positivas para NoV pelo ensaio imunoenzimático (EIE). Primeiramente, foi realizada a Reação em Cadeia da Polimerase Precedida de Transcrição Reversa (RT-PCR) para as regiões B da polimerase e D do capsídeo viral, utilizando os iniciadores Mon 431/432/433/434 e Cap C, D1 e D3, respectivamente. Vinte por cento das amostras caracterizadas como genótipo GII.4 foram testadas e sequenciadas também com os iniciadores EVP2F/EVP2R específicos para a região P2 do capsídeo viral. Nas amostras com genótipos discordantes quando comparadas as regiões da polimerase e do capsídeo (D), sugestivas de recombinação, foi realizada uma PCR para sequenciamento da região de junção da ORF1-ORF2 utilizando os iniciadores Mon 431 e G2SKR. As análises filogenéticas foram realizadas por MáximaVerossimilhança utilizando-se o programa IQTree v.1.3.0 com 1000 réplicas de bootstrap. Edições nas árvores filogenéticas foram feitas com o programa FigTree v.1.4.2 e as análises dos epítopos da região P2 no programa MEGA 6. As análises de recombinação foram realizadas com as configurações padrões do programa SimPlot (v.3.5.1). Durante os anos de 2010 a 2014, os NoV foram detectados em 33,1% (349/1053) das amostras testadas pelo EIE. Dentre estas, 250 foram testadas por RT-PCR e 75,6% (189) amplificaram por pelo menos uma região do genoma, permitindo a genotipagem. A análise filogenética demonstrou os seguintes genótipos: GII.Pe/GII.4 Sydney_2012, GII.P4/GII.4 New Orleans_2009, GII.P8/GII.8, GII.P12/GII.12, GII.P15/GII.15, GI.P5/GI.5, GII.P4_US95_96/NG, além das cepas recombinantes GII.P7/GII.6, GII.P22/GII.5, GII.Pg/GII.1, GII.Pg/GII.12. O genótipo mais frequente foi o GII.4, na forma de suas variantes Sydney_2012 e New Orleans_2009, as quais foram responsáveis por 52,4% (99/189) dos casos. Nos anos de 2010 e 2011, observouse a circulação da cepa GII.P4/GII.4 New Orleans_2009 em maior frequência. A partir de junho de 2012, essa cepa foi substituída pela cepa Sydney_2012, a qual predominou até o final do estudo em 2014. A análise da região P2 demonstrou que nove cepas New Orleans_2009 apresentavam mudanças aminoacídicas no aminoácido (AA) 294 localizado no epítopo A e duas no AA 413 no epítopo E. Nas cepas Sydney_2012 foram identificadas cinco mudanças nos AA nos epítopos antigênicos, são elas: 297 e 372 (epítopo A), 340 (epítopo C), 393 (epítopo D) e 412 (epítopo E). Ressalta-se a importância de dar continuidade a estudos epidemiológicos e moleculares como este, a fim de se monitorar a incidência desse patógeno na população, bem como identificar os genótipos circulantes, uma vez que os NoV sofrem frequentemente eventos de mutação e recombinação.