Detecção e genotipagem de norovírus em sangue e fezes de crianças hospitalizadas com quadro de gastrenterite em Belém, Pará, Brasil

Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Resumo: As doenças diarréicas agudas acometem anualmente milhões de indivíduos e ainda constituem uma das cinco principais causas de morte entre crianças menores de cinco anos. A gastrenterite aguda (GA) possui diversos agentes etiológicos, entretanto, recentemente os vírus vêm sendo identificados como responsáveis por cerca de 70% do total de casos. Dentre estes, destaca-se o norovírus (NoV), um dos principais responsáveis por casos esporádicos e surtos de GA. Este vírus é normalmente excretado nas fezes dos pacientes, em grande quantidade, o que facilita sua disseminação e novas contaminações. Mais recentemente o NoV foi detectado também no soro de pacientes com sintomas extra-intestinais, como: coagulação intravascular disseminada, enterocolite necrosante e convulsões. Logo, o objetivo desta pesquisa foi investigar a presença de NoV no soro de crianças hospitalizadas com quadro de GA que apresentavam o vírus nas fezes e correlacionar este achado com o quadro clínico apresentado pelo paciente. De março/2012 a junho/2015, foram obtidos dados clínicos e amostras de fezes/ soro de crianças com até nove anos de idade, hospitalizadas por GA em duas clínicas pediátricas de Belém, Pará, Brasil. Para a detecção de NoV nas amostras fecais utilizou-se o ensaio imunoenzimático (EIE) (kit RIDASCREEN®), e no soro a técnica de reação em cadeia da polimerase quantitativa precedida de transcrição reversa (RT-qPCR), sistema TaqMan, iniciadores COG2F e COG2R, sonda RING-2 e uma curva-padrão plasmidial. As amostras positivas foram submetidas à reação em cadeia da polimerase precedida de transcrição reversa (RT-PCR) com iniciadores específicos para a região do capsídeo ou da junção entre a polimerase e o capsídeo, visando o sequenciamento parcial do genoma viral. Amostras com suspeita de recombinação foram analisadas por meio do programa Simplot. Os dados clínicos foram tratados estatisticamente, utilizando os testes do qui-quadrado e de Mann-whitney. Uma positividade de 24,3% (108/445) foi obtida para NoV nas amostras fecais, dentre as quais 20,4% (22/108) também foram positivas nos soros. A carga viral fecal foi maior no grupo de crianças que também apresentou o vírus no soro em comparação ao que tinha o vírus apenas nas fezes (p<0,0001), sendo este mesmo padrão observado com relação ao tempo de hospitalização (p= 0,0252). A faixa etária de 0 a 36 meses foi a mais acometida por NoV em relação as fezes, enquanto que no soro foi entre >6 até 24 meses, havendo diminuição no número de casos em ambos os grupos conforme o aumento da idade (p=0,0059; p=0,0211). Foram encontrados nove diferentes genótipos do genogrupo GII sendo deles três cepas recombinantes (GII.2, GII.4, GII.6, GII.7, GII.8, GII.17, GII.P6/GII.7, GII.P22/GII.5 e GII.P13/GII.17) e dois do genogrupo GI, sendo um recombinante (GI.2 e GI.Pb/GI.6). O GII.4 variante Sydney foi o prevalente. Nesses três anos de pesquisa, verificou-se que os NoV foram responsáveis por quase 1/4 dos casos de GA entre as crianças hospitalizadas, dentre as quais a circulação do RNA viral nos soros foi expressiva, considerando outros estudos já realizados, e influenciou o tempo de hospitalização destas crianças. Além disso, observou-se ampla diversidade genética, o que contribui para estudos futuros visando uma vacina preventiva

Norovirus RNA in serum associated with increased fecal viral load in children: Detection, quantification and molecular analysis.

Worldwide, norovirus (NoV) is a major cause of acute gastroenteritis (AGE) responsible for pandemics every ~3 years, and over 200,000 deaths per year, with the majority in children from developing countries. We investigate the incidence of NoV in children hospitalized with AGE from Belém, Pará, Brazil, and also correlated viral RNA levels in their blood and stool with clinical severity. For this purpose, paired stool and serum samples were collected from 445 pediatric patients, ≤9 years between March 2012 and June 2015. Enzyme-linked immunosorbent assay (EIA) was used to detect NoV in stool and reverse transcription quantitative PCR (RT-qPCR) used to quantify NoV RNA levels in sera (RNAemia) and in the positive stool. Positives samples were characterized by the partial ORF1/2 region sequence of viral genome. NoV antigen was detected in 24.3% (108/445) of stool samples, with RNAemia also present in 20.4% (22/108). RNAemia and a high stool viral load (>107 genome copies/gram of faeces) were associated with longer hospitalizations. The prevalent genotypes were GII.4 Sydney_2012 (71.6%-58/81) and New Orleans_2009 (6.2%-5/81) variants. Eight other genotypes belonging to GII were detected and four of them were recombinant strains. All sera were characterized as GII.4 and shared 100% similarity with their stool. The results suggest that the dissemination of NoV to the blood stream is not uncommon and may be related to increased faecal viral loads and disease severity

Evolutionary and molecular analysis of complete genome sequences of norovirus from Brazil: emerging recombinant strain GII.P16/GII.4

PAPQ/PROPESP ; PPG-BAIP/UFPA ; Evandro Chagas Institute, Secretary of Health Surveillance, Ministry of Health (IEC/SVS/MS)Federal University of Pará. Institute of Biological Sciences. Program in Biology of Infectious and Parasitic Agents. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Federal University of Pará. Institute of Biological Sciences. Program in Biology of Infectious and Parasitic Agents. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Noroviruses (NoVs) are enteric viruses that cause acute gastroenteritis, and the pandemic GII.4 genotype is spreading and evolving rapidly. The recombinant GII.P16/GII.4_Sydney strain emerged in 2016, replacing GII.P31/GII.4_Sydney (GII.P31 formerly known as GII.Pe) in some countries. We analyzed the complete genome of 20 NoV strains (17 GII.P31/GII.4_ Sydney and 3 GII.P16/GII.4_Sydney) from Belém and Manaus, Brazil, collected from 2012 to 2016. Phylogenetic trees were constructed by maximum likelihood method from 191 full NoV-VP1 sequences, demonstrated segregation of the Sydney lineage in two larger clades, suggesting that GII.4 strains associated with GII.P16 already have modifications compared with GII.P31/GII.4. Additionally, the Bayesian Markov Chain Monte Carlo method was used to reconstruct a time-scaled phylogenetic tree formed by GII.P16 ORF1 sequences (n = 117) and three complete GII.P16 sequences from Belém. The phylogenetic tree indicated the presence of six clades classified into different capsid genotypes and locations. Evolutionary rates of the ORF1 gene of GII.P16 strains was estimated at 2.01 × 10–3 substitutions/site/year, and the most recent common ancestors were estimated in 2011 (2011–2012, 95% HPD). Comparing the amino acid (AA) sequence coding for ORF1 with the prototype strain GII.P16/GII.4, 36 AA changes were observed, mainly in the non-structural proteins p48, p22, and RdRp. GII.P16/GII.4 strains of this study presented changes in amino acids 310, 333, 373, and 393 of the antigenic sites in the P2 subdomain, and ML tree indicating the division within the Sydney lineage according to the GII.P16 and GII.P31 polymerases. Notably, as noroviruses have high recombination rates and the GII.4 genotype was prevalent for a long time in several locations, additional and continuous evolutionary analyses of this new genotype should be needed in the future

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

SUMMARY 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

Universidade do Estado do Pará. Programa de Pós-Graduação em Biologia Parasitária na Amazônia. Belém, PA, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Virologia Comparada e Ambiental. Rio de Janeiro, RJ, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Programa de Pós-Graduação em Virologia. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Programa de Pós-Graduação em Virologia. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.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 RNA in serum associated with increased fecal viral load in children: Detection, quantification and molecular analysis - Fig 6

<p>Simplot analysis of the ORF1-ORF2 overlap sequence (530 bp) of the strains VIR613F (MG023180)/ VIR699F (MG023186)/ VIR715F (MG023183) (a), VIR554F (MG023188) /VIR693F (MG023184) (b), VIR 138F (MG023190) (c), VIR 560 (MG023187) (d). The assay was performed using standards parameters of the program with a window size of 200 bp, a step size of 20 bp and with the Kimura (2-parameter) model. The accession numbers of the prototypes used in the analyses were the following: GII.P13/GII.13 (EU921354.2), GII.17/GII.17 (AY502009.1), GII.P22/GII.22 (AB233471), GII.P5/GII.5 (AF397156), GII.P7/GII.7 (JQ751043), GII.P6/GII.6 (AB039778), GI.Pb/GI.6 (AB081723), GI.6/GI.6 (AF093797), GI.Pb/GI.6 (AB354289). The y-axis indicates the nucleotide sequence similarity between the recombinant sequence and reference strains. The y-axis indicates nucleotide position.</p

Phylogenetic cladogram generated by the maximum likelihood test using partial nucleotide sequences (360 bp) from the C region of norovirus strains from 10 paired samples of sera and feces from children hospitalized for acute gastroenteritis at two clinics in the city of Belém, Brazil, from March/2012 to June/2015.

<p>Phylogenetic cladogram generated by the maximum likelihood test using partial nucleotide sequences (360 bp) from the C region of norovirus strains from 10 paired samples of sera and feces from children hospitalized for acute gastroenteritis at two clinics in the city of Belém, Brazil, from March/2012 to June/2015.</p

Phylogenetic cladogram generated by the maximum likelihood test using partial nucleotide sequences (360 bp) of the C region of the capsid of norovirus strains obtained from 47 stools of children hospitalized for acute gastroenteritis at two clinics in the city of Belém, Brazil, from March / 2012 to June / 2015.

<p>Phylogenetic cladogram generated by the maximum likelihood test using partial nucleotide sequences (360 bp) of the C region of the capsid of norovirus strains obtained from 47 stools of children hospitalized for acute gastroenteritis at two clinics in the city of Belém, Brazil, from March / 2012 to June / 2015.</p

Distribution by age group of norovirus-positive patients in serum/stool samples collected from children hospitalized for acute gastroenteritis in the city of Belém, Pará, Brazil, from March/2012 to June/2015.

<p>Distribution by age group of norovirus-positive patients in serum/stool samples collected from children hospitalized for acute gastroenteritis in the city of Belém, Pará, Brazil, from March/2012 to June/2015.</p