Norovirus recombination in ORF1/ORF2 overlap

Abstract

Norovirus (NoV) genogroups I and II (GI and GII) are now recognized as the predominant worldwide cause of outbreaks of acute gastroenteritis in humans. Three recombinant NoV GII isolates were identified and characterized, 2 of which are unrelated to any previously published recombinant NoV. Using data from the current study, published sequences, database searches, and molecular techniques, we identified 23 recombinant NoV GII and 1 recombinant NoV GI isolates. Analysis of the genetic relationships among the recombinant NoV GII isolates identified 9 independent recombinant sequences; the other 14 strains were close relatives. Two of the 9 independent recombinant NoV were closely related to other recombinants only in the polymerase region, and in a similar fashion 1 recombinant NoV was closely related to another only in the capsid region. Breakpoint analysis of recombinant NoV showed that recombination occurred in the open reading frame (ORF)1/ORF2 overlap. We provide evidence to support the theory of the role of subgenomic RNA promoters as recombination hotspots and describe a simple mechanism of how recombination might occur in NoV.

Figure 1

Phylogenetic analysis of the nucleotide sequences of capsid and polymerase regions of 9 identified recombinant norovirus genogroup II strains in relation to 26 known strains and prototype strains. The left tree analyzes the relationship of a 420-bp region of the 3´ end of the polymerase region. The right tree shows the relationship of 550 bp of the 5´ end of the capsid sequence. Suspected recombinants are underlined to emphasize their different phylogenetic groupings, and strains described in this study are represented in boldface. The percentage bootstrap values in which the major groupings were observed among 100 replicates are indicated. The branch lengths are proportional to the evolutionary distance between sequences and the distance scale, in nucleotide substitutions per position, is shown. The capsid clustering is shown in bold and is based on the classification of Vinjé et al. (23) (Table).

Figure 2

Similarity plot for Sydney 2212. The graph represents as a percentage the identity of the 2 putative parental strains, New Orleans/279 (black line) and Lordsdale (short dash), with the recombinant strain Sydney 2212. The window size was 100 bp with a step size of 10 bp. The site where the 2 parental strains have equal identity to the recombinant (i.e., where the lines cross) is the predicted site of recombination. By varying the window from 20 to 200 bp, the average recombination site was nucleotide 5081 with reference to Lordsdale. The percentage identity of Arg320 (long dash) to Sydney 2212 is also plotted. ORF, open reading frame.

Figure 3

A simple mechanism for recombination in norovirus. 1) RNA transcription by the RNA-dependent RNA polymerase (RdRp) (gray circle) generates a negative-stranded intermediate (dashed line). 2) Binding of the RdRp to the almost identical RNA promoter sequences (filled boxes) generates positive-stranded (straight line) genomes and subgenomic RNA. 3) These templates direct RNA synthesis from the 3´ end that leads to the generation of both a full-length negative genome and a negative subgenomic RNA species. 4) Recombination occurs when the enzyme initiates positive-strand synthesis at the 3´ end of the full-length negative strand, stalls at the subgenomic promoter, and then template switches to an available negative subgenomic RNA species generated by a co-infecting virus. The net result is a recombinant virus that has acquired new open reading frame (ORF)2 and ORF3 sequences.