Phylogenetic analysis of clinical herpes simplex virus type 1 isolates identified three genetic groups and recombinant viruses

Abstract

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen which establishes lifelong infections. In the present study, we determined the sequence diversity of the complete genes coding for glycoproteins G (gG), I (gI), and E (gE), comprising 2.3% of the HSV-1 genome and located within the unique short (US) region, for 28 clinical HSV-1 isolates inducing oral lesions, genital lesions, or encephalitis. Laboratory strains F and KOS321 were sequenced in parallel. Phylogenetic analysis, including analysis of laboratory strain 17 (GenBank), revealed that the sequences were separated into three genetic groups. The identification of different genogroups facilitated the detection of recombinant viruses by using specific nucleotide substitutions as recombination markers. Seven of the isolates and strain 17 displayed sequences consistent with intergenic recombination, and at least four isolates were intragenic recombinants. The observed frequency of recombination based on an analysis of a short stretch of the US region suggests that most full-length HSV-1 genomes consist of a mosaic of segments from different genetic groups. Polymorphic tandem repeat regions, consisting of two to eight blocks of 21 nucleotides in the gI gene and seven to eight repeats of 3 nucleotides in the gG gene, were also detected. Laboratory strain KOS321 displayed a frameshift mutation in the gI gene with a subsequent alteration of the deduced intracellular portion of the protein. The presence of polymorphic tandem repeat regions and the different genogroup identities can be used for molecular epidemiology studies and for further detection of recombination in the HSV-1 genome.

FIG. 1.

Schematic representation of sequence data for the US4, US7, and US8 genes of 28 clinical isolates as well as laboratory strains F, KOS321, and 17 (GenBank). The sequences were compared to the consensus sequence of each gene, and synonymous (s) and nonsynonymous (n) substitutions are indicated. The sequences are presented in the same order for all three genes. The genetic groups indicated by the phylogenetic analysis (see Fig. 2) are arbitrarily designated A, B, and C, and the isolates are color coded based on genetic group identity. Isolates not clustering distinctly with any genetic group are shown in black. The asterisk in the US7 gene of strain KOS321 represents an insertion of an extra nucleotide. Nucleotide substitutions in the noncoding region upstream of the US4 gene are marked (x). The tandem repeat regions (TR) are shown in light blue, and the numbers of repeats are denoted.

FIG. 2.

Phylogenetic analysis with the maximum-likelihood method of the gI and gE gene sequences, excluding the tandem repeat regions, for 28 clinical isolates and laboratory strains F, KOS321, and 17 (GenBank). The trees were constructed from 100 bootstrap replicates by using the Phylip package. Bootstrap values of >70 are shown. The trees clearly separate the isolates into three genetic groups (A to C). Isolates which cluster with different groups in the trees are considered intergenic recombinants. Isolates with a recombination point located between the gI and gE genes are shown in bold type and are connected by broken lines, while isolates with a recombination point located between the gG and gI genes are shown in bold type and are underlined (see Fig. 4). Intragenic recombinants (see Fig. 5) are marked with an asterisk.

FIG. 3.

Organization of the tandem repeat regions in the gI genes of 28 clinical isolates and laboratory strains F, KOS321, and 17 (GenBank), which are shown in bold type. The second type of block, which is present only in isolates belonging to genetic group C, is underlined. Single nucleotide substitutions are shown in bold type and are underlined.

FIG. 4.

Phylogenetic analysis of two gG gene segments, from nt −26 to nt 234 and from nt 256 to nt 717, separated by the tandem repeat region. The isolates are color coded, and intragenic recombinants with a recombination point located between the gG and gI genes are underlined (Fig. 2). The informative sites are displayed, and the genetic groups (A to C) are represented by the laboratory strains.

FIG. 5.

Bootscan analysis of the gE and gI genes with the SimPlot program. A sliding window of 250 nt with a 20-nt step size was used. The neighbor-joining algorithm was applied to 100 bootstrap replicates. The consensus sequences for genetic groups A, B, and C were matched to four putative intragenic recombinant isolates—993615 (A) and E4 (B) for the gE gene and 25 (C) and 7682 (D) for the gI gene. The corresponding phylogenetic trees with bootstrap values of >70 are shown above the plots.

FIG. 6.

Schematic phylogenetic illustration of a suggested evolutionary history of the gG, gI, and gE genes of HSV-1. Nucleotide positions are for laboratory strain 17. The straight broken line between genogroups B and C displays the possible earlier recombination event between the genogroups in the gG gene. The curved broken lines illustrate recombination of genomic segments between the different genogroups described for present-day clinical HSV-1 isolates.