Molecular analysis of norovirus in specimens from children enrolled in a 1982-1986 study in Belém, Brazil: A community-based longitudinal study

Abstract

Fecal specimens were collected during a longitudinal, community-based study in the city of Belém, North Brazil, that was conducted over 3 years (October 1982 to March 1986), in which 20 children were included from birth to 3 years of age. A total of 229 fecal samples were screened by real time RT-PCR targeting the junction region (ORF 1/2) of the norovirus (NoV) genome. NoV-positive samples were subjected to PCR and sequencing of the viral polymerase (ORF1) and viral protein 1 (VP1) genes (ORF2). The junction region was also sequenced to assess for recombination when ORF1 and ORF2 genotyping results were dissimilar. Samples classified as GII.P4/GII.4 were further characterized by sequencing the P2 subdomain of the viral capsid to determine possible alterations. An overall positivity of 16.1% (37/229) was observed, including GI (16.2%-6/37) and GII (83.8%-31/37) genogroups. Cases of NoV reinfection in at least 2-month intervals were observed, and 12 children developed at least one case of asymptomatic NoV infection. In total, 48.6% (18/37) NoV-positive samples were subjected to nucleotide sequencing analysis targeting the following polymerase genes: GI.P3 (n = 1), GII.Pa (n = 1), GII.Pc (n = 1), GII.P4 (n = 5), GII.P6 (n = 5), GII.P7 (n = 3), GII.P12 (n = 1), and GII.P22 (n = 1). For the VP1 gene, characterization was performed in 14 (77.8%) samples: GI.3 (n = 1), GII.2 (n = 1), GII.4 (n = 4), GII.6 (n = 4), GII.7 (n = 1), GII.12 (n = 1), GII.14 (n = 1), and GII.23 (n = 1). Recombination events were confirmed in three cases (GII.P12/GII.2, GII.P7/GII.14, and GII.Pa/GII.12), and four samples genotyped as GII.P4/GII.4 were analyzed to identify variants. None had contemporary counterparts. Three children developed consecutive NoV infections by different genotypes. The present report documents the importance of NoV as a cause of childhood infection during a longitudinal study conducted more than 30 years ago.

Keywords: children; diarrhea; gastroenteritis; norovirus.