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

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

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

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

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

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

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

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 and genotyping of human adenovirus and sapovirus in children with acute gastroenteritis in Belém, Pará, between 1990 and 1992: first detection of GI.7 and GV.2 sapoviruses in Brazil

<div><p>Abstract INTRODUCTION: Acute gastroenteritis (AGE) is one of the most common causes of morbidity and mortality, especially among children from developing countries. Human adenovirus (HAdV) and sapovirus (SaV) are among the agents that cause AGE. The present study aimed to detect and genotype HAdV and SaV in 172 fecal samples from children with AGE, collected during a surveillance study carried out in a low-income community in Belém, Pará, between 1990 and 1992. METHODS: HAdV was detected by nested PCR, using primers Hex1deg/Hex2deg and NeHex3deg/NeHex4deg. SaV was assayed by reverse transcription PCR (RT-PCR), nested PCR, and quantitative PCR. The nucleotide sequence was determined by direct cycle sequencing. RESULTS: Overall, 43% (74/172) of samples were positive for HAdV, of which 70.3% (52/74) were sequenced and classified as belonging to five different species, mostly A and F. For SaV, positivity was 5.2% (9/172) and genotypes GI.1, GI.7, GII.1, and GV.2 were detected. CONCLUSIONS: The present results reinforce the need for further studies to obtain epidemiological data about the circulation of these viruses in Brazil, especially in the Amazon Region, where data from the early 1990’s are scarce. Furthermore, the study describes for the first time the detection of SaV genotypes GI.7 and GV.2 in Brazil, showing that these types circulated in the region more than 25 years ago.</p></div