Genotyping of Clinical Herpes Simplex Virus Type 1 Isolates by Use of Restriction Enzymes

Abstract

Recently, three distinct genotypes of clinical herpes simplex virus type 1 (HSV-1) isolates were identified based on DNA sequence information and phylogenetic analysis of clinical isolates and laboratory strains. We utilized single-nucleotide polymorphism within the genes coding for glycoproteins G and I for rapid genotype classification by PCR and restriction enzyme cleavage. The method is suitable for high-scale genotyping of clinical HSV-1 isolates and for the detection of recombinants.Herpes simplex virus type 1 (HSV-1) is a DNA virus that belongs to the Herpesviridae family and is one of eight herpesviruses that infect humans (9). HSV-1 causes lifelong infections and establishes latency in the sensory ganglia. Although the most common clinical symptoms are oral or genital lesions, more severe symptoms, such as keratitis, neonatal infection, facial palsy, and encephalitis, may occur (3,5,8,14). Little information is available whether these conditions are results of genetic makeup of the host and/or of the virus or a result of random events.The HSV-1 genome consists of a 152-kb linear duplex DNA molecule, and characterization of genetic variability of clinical HSV-1 isolates has become an area of growing interest. Restriction fragment length polymorphism analyses have been used earlier in an attempt to classify HSV-1 into different genotypes (10, 12, 13). However, these methods are not based on HSV-1 DNA sequencing data and phylogenetic relationships. Recently, three genotypes, clearly separated in phylogenetic trees, were described for clinical HSV-1 isolates (7). The genotypes were arbitrarily designated A, B, and C, and the classification was based on DNA sequencing of the US4, US7, and US8 genes coding for the glycoproteins G (gG), I (gI), and E (gE), all localized in the unique short region of the genome. In light of this newly described genetic diversity of HSV-1, the search for associations between specific viral genetic markers and clinical symptoms is of interest. Although DNA sequencing is the most accurate method for genotyping, the technique is time-consuming in the high-scale format. The aim of the present study was to develop a rapid and accurate method for genotyping of clinical HSV-1 isolates. MATERIALS AND METHODSThe genotyping strategy was based on DNA sequence information presented recently on the gG and gI genes of 28 clinical HSV-1 isolates and the laboratory strains F, 17, and KOS321 (7). For the gG gene, more than 180 additional sequences were available from the GenBank and included in the selection of primers and restriction enzymes. Viral DNA was prepared by using QIAmp blood kit (QIAGEN) as described recently (7). PCR primers were designed to target highly conserved regions within each gene amplifying regions containing single-nucleotide point mutations specific for each genotype. The selected PCR primers are listed in Table 1. The PCR for US4 and US7 started with denaturation for 5 min at 96°C, followed by 40 cycles as follows: denaturation for 45 s at 95°C, annealing for 45 s at 58...

Sequence Variability in Clinical and Laboratory Isolates of Herpes Simplex Virus 1 Reveals New Mutations

“…3) and can be categorized as changes where strains F and H129 share the same amino acid residue with each other, but differ from strain 17, versus those positions where only strain H129 or strain F has a unique amino acid relative to the other two strains (see Table S2 for a full list of all amino acid differences for each protein). In a prior analysis using a limited number of genes, Norberg and colleagues found that strain 17 and strain F were divergent enough to fall into distinct clades (47,48). Since these clades are distinguishable based on restriction digest patterns in the US4 and US7 genes (48), we applied this approach and found that strain F and strain H129 fall into the same clade, while strain 17 does not (data not shown).…”

Recombination of Globally Circulating Varicella-Zoster Virus

“…In addition, variant calls showing a directional strand bias of Ն0.85 were excluded from further analyses. Consensus sequences were generated for each rash sample, but iterative repeat regions R1, R2, R3, R4, and R5 (19,20) as well as the terminal repeat region were trimmed prior to tree-building analyses.…”

“…Several studies have demonstrated that homologous recombination contributes significantly to the evolution of many herpesviruses, including HSV-1 (12,(18)(19)(20)(21). Recent complete genome analysis of HSV-1 strains suggests that all or most wild-type strains have recombinant mosaic genomes (22,23).…”

Diversity of the a sequence of herpes simplex virus type 1 developed during evolution

“…Journal of General Virology 89 Nahmias et al, 2006;Norberg et al, 2004;Sakaoka et al, 1994;Umene, 1998;Umene & Kawana, 2003;Umene & Sakaoka, 1999;Umene et al, 2007). Variations in restriction endonuclease cleavage patterns between HSV-1 isolates have been analysed and a number of restriction fragment length polymorphisms (RFLPs) detected (Norberg et al, 2006;Sakaoka et al, 1994;Umene & Yoshida, 1993). An RFLP is usually due to a nucleotide substitution that causes the gain or loss of a restriction endonuclease cleavage site.…”