Molecular epidemiology and temporal evolution of norovirus associated with acute gastroenteritis in Amazonas state, Brazil

Abstract

Background: Globally, Norovirus (NoV) is considered the most common cause of diarrheal episodes across all age groups. Despite its wide genetic diversity, the GII.4 strain is the most predominant and has been associated with epidemics worldwide. In this study, we characterized sporadic cases of diarrhea from NoV-positive children, during a five-year period (2010-2014).

Methods: A total of 250 NoV-positive samples identified by an enzyme immunoassay (EIA) were subjected to RT-PCR and partial nucleotide sequencing for polymerase and capsid genes. Phylogenetic analysis was performed to identify NoV genotypes using the binary classification. In addition, sequences from the P2 subdomain (capsid) gene of GII-4 variants were characterized by evolutionary analyses, using the MCMC method implemented in the BEAST package. A 3D structure was built using protein modeling.

Results: Phylogenetic analysis demonstrated a predominance of genotype GII.4 (52.4% - 99/189), variants New Orleans_2009 and Sydney_2012 followed by GII.P7/GII.6 with 6.3% (12/189). Amino acid analyses of the GII.4 strains showed several important amino acid changes. A higher evolutionary rate was found, 7.7 × 10^{- 3} in the Sydney variant and 6.3 × 10^{- 3} in the New Orleans. Based in evolutionary analysis the time to the most recent common ancestor (TMRCA) has been calculated as estimates of the population divergence time. Thus, TMRCA for New Orleans and Sydney variant were 2008.7 and 2010.7, respectively. Also, we observed a lineage of transition between New Orleans and Sydney.

Conclusion: This study describes the different strains of norovirus isolated from Amazonas state in Brazil during a five-year period. Considering that NoV are capable of changing their antigenic epitopes rapidly, a continuous surveillance is important to monitor the occurrence and changes of the NoV in the community through epidemiological studies. These results contribute to the understanding of NoV molecular epidemiology and its evolutionary dynamics in Amazonas state, Brazil.

Keywords: Acute gastroenteritis; Genetic diversity; Norovirus; Phylogenetic.

Fig. 1

Maximum likelihood clade tree, based on the RNA polymerase gene (200 bp) of 143 partial nucleotide sequences of norovirus from children with acute gastroenteritis in Manaus city, Amazon, Brazil. The color of the branches is based on the NoV-genotype. All samples that were studied begin with the initials AM, and 35 identical sequences of the same are not shown

Fig. 2

Maximum likelihood phylogenetic analysis based on the capsid gene (D region of VP1) (188 bp) of 64 partial nucleotide sequences of norovirus from children with acute gastroenteritis in Manaus city, Amazon, Brazil. The color of the branches is based on the NoV-genotype. All samples that were studied begin with the initials AM, and 67 identical sequences of the same are not shown

Fig. 3

Monthly distribution of NoV-genotypes associated with acute gastroenteritis in Manaus city, Amazon, Brazil, between 2012 and 2014

Fig. 4

Alignment of antigenic residues of capsid protein (P2 region) in strains circulating in Brazil, 2011-2014. Antigenic residues are divided in five epitopes (A-E). The prototype strains representative of the epidemic strain variants that emerged between 2009 and 2012 are highlighted in blue. Amino acid residues that differ from those of the prototype are highlighted in yellow and green. a Structure modeling of the VP1 protein (PDB 4OP7) showing the amino acid replacement in NoV GII.4 New Orleans_2009 lineages from Manaus, Brazil. Antigenic epitopes are colored in yellow (A), blue (B), green (C), red (D), and purple (E). b Structure modeling of the VP1 protein (PDB 4OP7) showing the amino acid replacement in NoV GII.4 Sydney_2012 lineages from Manaus, Brazil

Fig. 5

Molecular clock phylogeny based on the 58 nucleotide sequence (627 bp) of P2 region (VP1), estimated by uncorrelated log-normal model using the Coalescent Piecewise Bayesian Skyride Plot method with 100 million replicates, from 2010 to 2014 in Amazon, Brazil. The taxa were represented by colored circles according to the GII.4 variant. The most recent common ancestor (TMRCA) of each variant is indicated next to the clade. The black circle represents supported clades (> 95%). Three-dimensional VP1 structure of the GII.4 predicted by homologous modeling based on the crystal structure (PDB number 4OP7) (*) Surface-exposed electrostatic charges showing differences within the Sydney lineage. (**) Surface-exposed electrostatic charges showing a sample clustering with Sydney variant but demonstrating more similarities in the surface protein with New Orleans variant. Boxplot of evolutionary rates of Sydney and New Orleans variants