Flow chart of study samples demonstrating a global view of the study developed during a 30-year period of study (1982–2011) in Belém, Brazil.

<p>¹ Total of samples genotyped by at least one of the genomic regions analyzed. ² Total of sequences used for recombination analysis. ³ Total of sequences used for phylogenetic analysis.</p

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

<div><p>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.</p></div

Summary information for eight studies conducted from 1982 to 2011, the fecal samples of which were included in the present study.

<p>Summary information for eight studies conducted from 1982 to 2011, the fecal samples of which were included in the present study.</p

Norovirus genotypes detected in infected children during a 30-year period (1982–2011) in different studies conducted in Belém, Brazil.

<p>(a) (I) Samples genotyped only by a partial sequence of the polymerase gene (Regions A or B); (II) Samples genotyped only by a partial sequence of the VP1 gene (Regions C or D). (b) Binary genotyping targeted two regions, polymerase (A or B) and capsid (C or D). It is noteworthy that this study did not account for samples genotyped by only one nucleotide fragment (i.e., the polymerase or the capsid region). An asterisk represents samples confirmed as recombinants (more details are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178909#pone.0178909.t004" target="_blank">Table 4</a>).</p

Description of 20 recombinant norovirus strains circulating in the pediatric population of Belém, Brazil over a period of 30 years (1982–2011).

<p>Description of 20 recombinant norovirus strains circulating in the pediatric population of Belém, Brazil over a period of 30 years (1982–2011).</p

Samples genotyped by maximum likelihood analysis of partial genome sequences from noroviruses detected in infected children in various collections during a 30-year period of study (1982–2011) in Belém, Brazil.

<p>Samples genotyped by maximum likelihood analysis of partial genome sequences from noroviruses detected in infected children in various collections during a 30-year period of study (1982–2011) in Belém, Brazil.</p

Maximum likelihood phylogenetic tree based on the P2 region of 83 partial genome sequences from noroviruses of different GII.4 variants detected in infected children during various collection periods over 30 years (1982–2011) in Belém, Brazil.

<p>Asterisks in the tree represent bootstrap values greater than 70% with 1000 replicates. Groupings of samples from the present study are in red. A dendrogram was constructed using model test GTR+G4. Variant reference strains used in the analyses were submitted to the GenBank database under the accession numbers CHDC_1970s (JX023286), Tokyo_1980s (AB684720), US_95/96 (DQ078829), Bristol_1993 (X86557), Kaiso_2003 (AB294779), Asia_2003 (AJ844476), Hunter_2004 (HM802544), Yerseke_2006a (EF126963), Den_Haag_2006b “O” (EF126965), Den_Haag_2006b “Y” (JX975571), and New_Orleans_2009 (GU445325).</p

Positivity rates of norovirus observed in each of the eight studies conducted between 1982 and 2011 in Belém, Brazil.

<p>Positivity rates of norovirus observed in each of the eight studies conducted between 1982 and 2011 in Belém, Brazil.</p

Temporal distribution of 124 samples classified as norovirus GII.4 variants according to the capsid region that were detected in infected children during a 30-year period (1982–2011) in Belém, Brazil.

<p>Of note, samples from Study C (nosocomial [1992–1994]) were not tested based on capsid region due to the depletion of these stools.</p

Detection of Epstein-Barr virus in gastric adenocarcinoma: qPCR and fish comparison

Scientifc initiation scholarships were paid by National Council for Scientifc and Technological Development (CNPq). Salaries were paid by Evandro Chagas Institute (IEC) and Federal University of Pará. Equipment and supplies were provided by the IECMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, BrasilFederal University of Pará. Institute of Biological Sciences. Laboratory of Human Cytogenetics. Belém, PA, BrazilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, BrasilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, BrasilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, BrasilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, BrasilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, BrasilFederal University of Pará. Institute of Biological Sciences. Laboratory of Human Cytogenetics. Belém, PA , Brazil / Ophir Loyola Hospital. Molecular Biology Laboratory. Belém, PA, BrazilEBV-associated gastric cancer accounts for about 10% of all gastric carcinomas worldwide. We aimed to verify the prevalence of EBV in gastric adenocarcinoma samples using FISH and qPCR and comparing the results obtained by both techniques. Gastric cancer samples from 191 cases were analyzed. The FISH assay was performed to detect small EBV RNAs (EBER1) and qPCR was performed to detect the EBV-EBNA-1 gene region. Cohen's kappa index and the chi-square test were used to compare the methodologies and investigate correlations with the clinical-pathological data of the gastric adenocarcinoma patients. Most of the patients were men, and the average age was 60 years. The intestinal subtype cancer presented more aggressive stages with 90% of patients having a reactive FISH for EBV (EBV+), although the virus infection frequency in epithelial gastric tissue was only 1%. No positive association with clinicopathological features and EBV+ was found by FISH. Using qPCR analysis, the percentage of positive samples was lower (52.4%), and a positive association was found in samples from older patients (> 60 years). Interestingly, 71 qPCR-negative cases were detected by FISH in the presence of non-epithelial cells and in 10 qPCR-positive cases with no evidence of EBV according to FISH. The concordance between the two techniques was low, with only 57.6%. FISH is more informative for associating the gastric carcinoma with EBV positivity in tumor/epithelial cells; however, qPCR can provide relevant information regarding the progression and characteristics of neoplasia