Static and Evolving Norovirus Genotypes: Implications for Epidemiology and Immunity

Abstract

Noroviruses are major pathogens associated with acute gastroenteritis worldwide. Their RNA genomes are diverse, with two major genogroups (GI and GII) comprised of at least 28 genotypes associated with human disease. To elucidate mechanisms underlying norovirus diversity and evolution, we used a large-scale genomics approach to analyze human norovirus sequences. Comparison of over 2000 nearly full-length ORF2 sequences representing most of the known GI and GII genotypes infecting humans showed a limited number (≤5) of distinct intra-genotypic variants within each genotype, with the exception of GII.4. The non-GII.4 genotypes were comprised of one or more intra-genotypic variants, with each variant containing strains that differed by only a few residues over several decades (remaining “static”) and that have co-circulated with no clear epidemiologic pattern. In contrast, the GII.4 genotype presented the largest number of variants (>10) that have evolved over time with a clear pattern of periodic variant replacement. To expand our understanding of these two patterns of diversification (“static” versus “evolving”), we analyzed using NGS the nearly full-length norovirus genome in healthy individuals infected with GII.4, GII.6 or GII.17 viruses in different outbreak settings. The GII.4 viruses accumulated mutations rapidly within and between hosts, while the GII.6 and GII.17 viruses remained relatively stable, consistent with their diversification patterns. Further analysis of genetic relationships and natural history patterns identified groupings of certain genotypes into larger related clusters designated here as “immunotypes”. We propose that “immunotypes” and their evolutionary patterns influence the prevalence of a particular norovirus genotype in the human population.

Sequential Gastroenteritis Outbreaks in a Single Year Caused by Norovirus Genotypes GII.2 and GII.6 in an Institutional Setting

“…Our data are consistent with recent reports that reinfection with noroviruses from different genotypes occurs often in children [16, 22, 36] and that norovirus reinfections can occur within a single year in both children and adults [14, 15]. Similar to our previous report that infection with 2 different GII genotypes (GII.4 followed by GII.6) could occur within a single year in a young child [15], we show here that yet another GII genotype pairing (GII.2 followed by GII.6) could cause sequential outbreaks in adults within a single year.…”

“…Phylogenetic comparisons of the norovirus capsid regions (encoded in ORF2) with reference GI and GII sequences showed that each Henryton norovirus clustered closely with a specific genotype, GII.2 (highlighted in rust), or GII.6 (highlighted in blue) (Figure 1A). The GII.6 viruses form 3 genetic clusters, designated here as A, B, and C. The Henryton GII.6 norovirus grouped within cluster C, which includes viruses detected in the United States and Japan during 2008–2012 [22, 30]. The Henryton GII.2 norovirus grouped with the single cluster of the GII.2 genotype represented by the prototype Snow Mountain virus, which has remained highly conserved for approximately 40 years [22].…”

“…The GII.6 viruses form 3 genetic clusters, designated here as A, B, and C. The Henryton GII.6 norovirus grouped within cluster C, which includes viruses detected in the United States and Japan during 2008–2012 [22, 30]. The Henryton GII.2 norovirus grouped with the single cluster of the GII.2 genotype represented by the prototype Snow Mountain virus, which has remained highly conserved for approximately 40 years [22]. Alignment of the VP1 amino acid sequences revealed that the GII.2 and GII.6 Henryton viruses differed by 28.3% (157/554), with the capsid P domain having the greatest amino acid variation (36.6%; 120/328) and the S domain having the least (16.4%; 37/226).…”

“…The amplicons were sequenced using a Big Dye terminator cycle sequencing ready reaction kit (Applied Biosystems), and the norovirus typing tool was used to assign genotype [10, 21]. The near full-length genome sequences were determined as described previously [22]. In addition, the authentic 5’- and 3’-end genomic sequences were verified using the Roche 5’/3’ RACE Kit, 2nd Generation (Roche Diagnostics Corporation, Indianapolis, IN) according to the manufacturer’s protocol.…”

“…Phylogenetic analyses of ORF2 were performed as previously described using the neighbor-joining and Tajima-Nei methods [22] in the Molecular Evolutionary Genetics Analysis program, version 7 [23]. …”