Novel norovirus recombinants and of GII.4 sub-lineages associated with outbreaks between 2006 and 2010 in Belgium

Abstract

Background: Noroviruses (NoVs) are an important cause of acute gastroenteritis in humans worldwide. To gain insight into the epidemiologic patterns of NoV outbreaks and to determine the genetic variation of NoVs strains circulating in Belgium, stool samples originating from patients infected with NoVs in foodborne outbreak investigations were analysed between December 2006 and December 2010.

Results: NoVs were found responsible of 11.8% of all suspected foodborne outbreaks reported in the last 4 years and the number of NoV outbreaks reported increased along the years representing more than 30% of all foodborne outbreaks in 2010. Genogroup II outbreaks largely predominated and represented more than 90% of all outbreaks. Phylogenetic analyses were performed with 63 NoV-positive samples for the partial polymerase (N = 45) and/or capsid gene (N = 35) sequences. For 12 samples, sequences covering the ORF1-ORF2 junction were obtained. A variety of genotypes was found among genogroups I and II; GII.4 was predominant followed in order of importance by GII.2, GII.7, GII.13, GI.4 and GI.7. In the study period, GII.4 NoVs variants 2006a, 2006b, 2007, 2008 and 2010 were identified. Moreover, phylogenetic analyses identified different recombinant NoV strains that were further characterised as intergenotype (GII.e/GII.4 2007, GII.e/GII.3 and GII.g/GII.1) and intersub-genotype (GII.4 2006b/GII.4 2007 and GII.4 2010/GII.4 2010b) recombinants.

Conclusions: NoVs circulating in the last 4 years in Belgium showed remarkable genetic diversity either by small-scale mutations or genetic recombination. In this period, GII.4 2006b was successfully displaced by the GII.4 2010 subtype, and previously reported epidemic GII.b recombinants seemed to have been superseded by GII.e recombinants in 2009 and GII.g recombinants in 2010. This study showed that the emergence of novel GII.4 variants together with novel GII recombinants could lead to an explosion in NoV outbreaks, likewise to what was observed in 2008 and 2010. Among recombinants detected in this study, two hitherto unreported strains GII.e/GII.3 and GII.g/GII.1 were characterised. Surveillance will remain important to monitor contemporaneously circulating strains in order to adapt preventive and curative strategies.

Figure 1

Characteristics of noroviruses detected in acute gastroenteritis outbreaks reported in Belgium. A) Monthly distribution of norovirus (NoV)-associated outbreaks in Belgium from December 2007 to December 2010. Months corresponding to the primer detection of novel NoV genotypes (GII.e and GII.g) or sub-lineages (GII.4 variants 2007, 2008 and 2010) are indicated by an arrow; Diversity of NoV genogroups (B), genotypes and GII.4 sub-lineages (C) detected in gastro-enteritis outbreaks in Belgium during 2006 and 2010. Percentages of genogroup prevalence are indicated in the vertical bars. Typing results were obtained by phylogenetic clustering with reference strains (http://www.noronet.nl) either for the partial polymerase gene sequence (grey) or the partial capsid gene sequence (black).

Figure 2

Phylogenetic analyses of the partial polymerase region of the detected norovirus genomes. Phylogenetic trees were inferred under a maximum-likelihood framework from the nucleic acid sequences aligned at the protein level (GTR model with aLRT node support, see Material and Methods for details). The aLRT node supports were only indicated when superior to 0.8 and relevant to the genotype identification. Reference NoV strains identified as highly related to the norovirus (NoV) samples were highlighted in bold, italic blue font. NoV samples originating from the same outbreak and co-localized in the phylogenetic tree were also highlighted in color. Identical NoV sequences were represented on the same node (in color if originating from the same outbreak, in black if originating from different outbreaks). Details on the genotyping of each NoV sample can be found in Table 1.

Figure 3

Phylogenetic analyses of the partial capsid region of the detected norovirus genomes. Phylogenetic trees were inferred under a maximum-likelihood framework from the nucleic acid sequences aligned at the protein level (GTR model with aLRT node support, see Material and Methods for details). The aLRT node supports were only indicated when superior to 0.8 and relevant to the genotype identification. Reference NoV strains identified as highly related to the norovirus (NoV) samples were highlighted in bold, italic blue font. NoV samples originating from the same outbreak and co-localized in the phylogenetic tree were also highlighted in color. Identical NoV sequences were represented on the same node (in color if originating from the same outbreak, in black if originating from different outbreaks). Details on the genotyping of each NoV sample can be found in Table 1.

Figure 4

Similarity plots of norovirus GII recombinants IPH2700/08 (A), IPH0163/10 (B), IPH2172/09 (C) and IPH0143/09 (D). SimPlot analyses were conducted with partial polymerase and capsid gene sequence alignments. Sequences, either a concatemer of partial polymerase and capsid sequences (A) or sequences covering the ORF1/2 overlap (B-D) were analyzed for recombination. Graphs show the similarity of the putative parental reference strains with the recombinant GII NoVs detected in our study relative to the genomic position (bp). Dashed vertical lines indicate the start of the capsid gene. Genbank accession numbers from parental NoV strains are as follows: Miami81/1986/US (GII.1): AF414416; NSW390I/2008/AU (GII.4 2007): GQ845369; Paris Island/2003/US (GII.b/GII.3): AY652979; Riviera1635/2008/US (GII.4 1996/GII.4 2007): GQ413969; Seoul0448/2009/KR (GII.g/GII.12): HM635104; Seoul0520/2007/KR (GII.4 2006b): FJ913975.