Characterization of new recombinant noroviruses

Abstract

Noroviruses are important etiologic agents of acute gastroenteritis and show great genetic diversity. To characterize more fully previously detected strains that could not be assigned unequivocally to one particular genotype based on the RNA polymerase, we have sequenced a region in the capsid gene and, in some cases, in the junction between open reading frame 1 (ORF 1) and ORF 2. The results allowed us to identify several recombinant noroviruses: GGIIb viruses were detected for the first time in France in August 2000 and then spread through France and to Europe during the following winter. Here we present the characterization of three other probable GII recombinants which showed different phylogenetic positions depending on their ORF 1 and ORF 2 sequences. Analysis of the region located between ORF 1 and ORF 2 by a nucleotide identity window search showed a sudden shift in similarities. Moreover, recombination breakpoints were identified upstream and downstream of the beginning of ORF 2 by using a statistical test, thus confirming the involvement of this region in recombination. Unlike GGIIb, the three recombinants described here do not seem to have diffused widely in the community: one was found in a waterborne outbreak, and the other two were found in sporadic cases. Recombination is important for the evolution of RNA viruses and has already been described for noroviruses. Our results suggest that recombination is not a rare phenomenon among noroviruses, but not all these presumed recombinants that formed during RNA replication are able to spread widely.

FIG. 1.

Phylogenetic analysis of partial (A) RNA polymerase (145-bp) and (B) capsid (277-bp) sequences. The strains characterized in our laboratory are represented in boldface. Strains SH96 and E293 were characterized in another study; they present a good correlation between the RNA polymerase and the capsid genes, with SH96 being assigned to the Hawaii genotype and E293 being assigned to the Bristol genotype. The GenBank accession numbers for the calicivirus reference strains are Amsterdam/1998/NL, AF195848; Chiba407/1987/JP, AB042808; Hesse3/1997/GE, AF093797; Leeds/1990/UK, AJ277608; Musgrove/1989/UK, AJ277614; Norwalk/1968/US, M87661; Seacroft/1990/UK, AJ277620; Sindlesham/1995/UK, AJ277615; and Winchester/1994/UK, AJ277609. The others are given in Table 1. *, sequences not available in GenBank (the sequences were kindly provided by Marion Koopmans and Harry Vennema).

FIG. 2.

SimPlot analysis of partial RNA polymerase and capsid gene sequences of recombinant strains. Window size, 200 bp; step, 20 bp. The query sequences for panels A, B, and C are S63, L23, and E3, respectively. The vertical axis indicates the nucleotide identities between the query strain and the four reference strains, expressed as percentages. The horizontal axis indicates the nucleotide positions (in base pairs; corresponding to base pairs 4233 to 5660, 4290 to 5660, and 4233 to 5648 of the Hawaii strain with GenBank accession number U07611 for S63, L23, and E3, respectively). Vertical lines indicate the beginning of ORF2 (continuous vertical line) and putative recombination breakpoints identified by Sawyer's test at the ORF1-ORF2 junction (dotted vertical line). Gray line, Hillingdon; dashed line, Melksham genotype; bold line, Bristol; dotted line, Hawaii.

FIG. 3.

Recombination breakpoints detected in the junction region between ORF1 and ORF2 of recombinant strains by Sawyer's test. Positions according to the beginning of ORF2 are −45, −6 or −9, and 25 for the three putative recombinants L23, E3, and S63, respectively, and 26, 26, and −67 for the confirmed recombinants Arg320, L169, and E673, respectively.