A full-genome phylogenetic analysis of varicella-zoster virus reveals a novel origin of replication-based genotyping scheme and evidence of recombination between major circulating clades

Abstract

Varicella-zoster virus (VZV) is a remarkably stable virus that until recently was thought to exhibit near-universal genetic homogeneity among circulating wild-type strains. In recent years, the expanding knowledge of VZV genetics has led to a number of groups proposing sequence-based typing schemes, but no study has yet examined the relationships between VZV genotypes at a full-genome level. A central hypothesis of this study is that VZV has coevolved with humankind. In this study, 11 additional full VZV genomic sequences are presented, bringing the current number of complete genomic sequences publicly available to 18. The full-genome alignment contained strains representing four distinct clades, but the possibility exists that a fifth clade comprised of African and Asian-like isolates was not represented. A consolidated VZV genotyping scheme employing the origin-associated region between reiteration region R4 and open reading frames (ORFs) 63 and 70 is described, one which accurately categorizes strains into one of four clades related to the geographic origin of the isolates. The full-genome alignment also provided evidence for recombination having occurred between the major circulating VZV clades. One Canadian clinical isolate was primarily Asian-like in origin, with most of the genome showing strong sequence identity to the Japanese-like clade B, with the exceptions being two putative recombination regions, located in ORFs 14 to 17 and ORFs 22 to 26, which showed clear similarity to the European/North American clade A. The very low rate of single-nucleotide polymorphisms scattered across the genome made full-genome sequencing the only definitive method for identifying specific VZV recombination events.

FIG. 1.

Phylogenetic trees of VZV strains based on full-genome sequence (A), aligned sequences of five glycoprotein genes and IE62 (13, 57) (B), and origin of replication region (C). Strains in boldface type are those represented in the full-genome alignment, while strains in italics were taken from previous studies.

FIG. 2.

Variable locations within the VZV origin-associated region located between reiteration region R4 and ORF 63/70. As there was noted variability between the copy numbers of TA and GA subunits within the origin of replication between virus sequences within a clade, the differences in copy numbers are not shown. Dumas Location refers to the coordinates of the features in the VZV-Dumas IR_S copy of the origin of replication; these locations are duplicated in TR_S. The nucleotide at each of these variable locations is listed below the coordinates for the four clades examined. Poly-G, Poly-A, and Poly-C refer to homopolymer stretches that have displayed variability in length both between and within clades. A dash (—) indicates that the clade does not have a base at this location due to an insertion or deletion event. The bases listed for clade C are based on the sequence of VZV-8 only. *, VZV-11 from clade D was the only strain reported to contain a G at location 109994.

FIG. 3.

Evidence for recombination in VZV. (A) Alignment of variable sites. The full-genome alignment was condensed to only the variable sites by removing the high-passage isolates derived from a single strain (i.e., 32p22, 32p72, vOka, VarilRix, and VariVax) as well as by removing noninformative variable sites, such as singletons and other strain-specific variations. The Dumas coordinates of the variable sites are indicated above the alignment to provide a relative reference. The boxed regions indicate the segments proposed to be involved in the recombination events and correspond to Dumas coordinates bp 20656 to 25067 for the first segment and bp 38714 to 44835 for the second. Positions in these regions which are conserved between the clade B and D strains are highlighted in red, and the locations in which clade A strains are proposed to have developed mutations after the recombination event are highlighted in yellow. (B) SimPlot analysis of the variable site alignment. Analysis was conducted by grouping the strains based on their phylogenetic designations. Clade A was selected as the query group using a 20-bp window and a 10-bp step value. Similarities to clade D are indicated in green, those to clade C are indicated in blue, and those to clade B are indicated in red. (C) Phylogenetic analysis of the recombination region. The segment of the full-genome alignment between Dumas coordinates bp 13000 to 52000 was assessed using the neighbor-joining algorithm with 500 bootstrap replicates (bootstrap values are indicated).

FIG. 4.

Schematic diagram depicting the recombination events proposed to have shaped the genetic profile of the Canadian clinical isolate VZV-8. Open arrows depict ORFs, and reiteration regions R2 and R3 are depicted by solid black boxes. The superimposed vertical red lines depict locations where VZV-8 shares an SNP with either the clade A or clade B progenitor strain. SNPs which are specific to a single clade, as indicated in Fig. 3A, are not shown.