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

Recently, three distinct genotypes of clinical herpes simplexvirus type 1 (HSV-1) isolates were identified based on DNA sequenceinformation and phylogenetic analysis of clinical isolates andlaboratory strains. We utilized single-nucleotide polymorphismwithin the genes coding for glycoproteins G and I for rapidgenotype classification by PCR and restriction enzyme cleavage.The method is suitable for high-scale genotyping of clinicalHSV-1 isolates and for the detection of recombinants.

INTRODUCTION

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Introduction

Herpes simplex virus type 1 (HSV-1) is a DNA virus that belongsto the Herpesviridae family and is one of eight herpesvirusesthat infect humans (9). HSV-1 causes lifelong infections andestablishes latency in the sensory ganglia. Although the mostcommon clinical symptoms are oral or genital lesions, more severesymptoms, such as keratitis, neonatal infection, facial palsy,and encephalitis, may occur (3, 5, 8, 14). Little informationis available whether these conditions are results of geneticmakeup of the host and/or of the virus or a result of randomevents.

The HSV-1 genome consists of a 152-kb linear duplex DNA molecule,and characterization of genetic variability of clinical HSV-1isolates has become an area of growing interest. Restrictionfragment length polymorphism analyses have been used earlierin an attempt to classify HSV-1 into different genotypes (10,12, 13). However, these methods are not based on HSV-1 DNA sequencingdata and phylogenetic relationships. Recently, three genotypes,clearly separated in phylogenetic trees, were described forclinical HSV-1 isolates (7). The genotypes were arbitrarilydesignated A, B, and C, and the classification was based onDNA sequencing of the US4, US7, and US8 genes coding for theglycoproteins G (gG), I (gI), and E (gE), all localized in theunique short region of the genome. In light of this newly describedgenetic diversity of HSV-1, the search for associations betweenspecific 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. Theaim of the present study was to develop a rapid and accuratemethod for genotyping of clinical HSV-1 isolates.

MATERIALS AND METHODS

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Materials and Methods

The genotyping strategy was based on DNA sequence informationpresented recently on the gG and gI genes of 28 clinical HSV-1isolates and the laboratory strains F, 17, and KOS321 (7). Forthe gG gene, more than 180 additional sequences were availablefrom the GenBank and included in the selection of primers andrestriction enzymes.

Viral DNA was prepared by using QIAmp blood kit (QIAGEN) asdescribed recently (7). PCR primers were designed to targethighly conserved regions within each gene amplifying regionscontaining single-nucleotide point mutations specific for eachgenotype. The selected PCR primers are listed in Table 1. ThePCR 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°C, and elongation for 45 s at 72°C,with a 3-s extension per cycle. The amplicons were cleaved byrestriction enzymes according to the manufacturer's recommendation(90 min at 37°C) and separated at 80 V for 2 h on a 3% Metaphoragarose gel (Cambrex). Restriction enzymes and recognition sequencesare summarized in Table 1. To simplify the protocol for cleavageof the PCR products, the enzymes were selected for simultaneoususe in the same buffer and temperature.

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TABLE 1. PCR primers and recognition sites for the restriction enzymes

RESULTS

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Results

Genotyping patterns. Primers amplifying the gG gene generated a 269-nucleotide (nt)PCR product, which was cleaved by the enzymes PflMI and DdeI(New England Biolabs). For isolates belonging to genotype Athe amplicon was not cleaved, for isolates belonging to genotypeB two fragments were generated (97 and 172 nt), and for isolatesbelonging to genotype C the amplicon was cleaved into threefragments (57, 97, and 115 nt). The classification of genotypeswas based on the size of the two largest fragments since thesmallest fragment (57 nt) was only visible as a faint band.The restriction enzymes SacI and PleI were used for cleavageof a 410-nt amplicon of the gI gene. Isolates belonging to thegenotype A were not cleaved by either enzyme. Isolates belongingto genotype B were cleaved into two fragments (55 and 355 nt),and isolates belonging to genotype C were cleaved into threefragments (55, 133, and 222 nt), where the classification wasbased on the two longer fragments (Fig. 1).

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FIG. 1. Genotyping of HSV-1 based on the US4 and the US7 genes localized in the unique short (US) segment of the genome using restriction enzymes. Nucleotide positions are within parenthesis and refer to HSV-1 strain 17. The genotypes cleaved by each restriction enzyme are depicted below the respective enzyme. The cleavage products were separated on a 3% Metaphor agarose gel. UL, unique long segment.

For the 28 clinical isolates and the three laboratory strainsthe following classification of genotypes was achieved. Basedon the genotyping target in the gG gene, 8 isolates were classifiedas genotype A, 15 isolates were classified as genotype B, and8 isolates were classified as genotype C. Based on the gI gene,9 isolates were classified as genotype A, 12 isolates were classifiedas genotype B, and 10 isolates were classified as genotype C.Seven of the clinical isolates presented different genotypeidentities in the two genotyping targets and were thereforeclassified as recombinants. Two isolates belonged to genotypeB in the gG gene and to genotype C in the gI gene, three isolateswere classified into genotype B in the gG gene and switchedto genotype C in the gI gene, and finally two isolates belongedto genotype A in the gG gene and to genotype B in the gI gene.The classification of these recombinants corresponded to resultsfrom HSV-1 DNA sequence analyses (7). Hence, recombinants withat least one recombination point located between the two genotypingtargets and that belong to different genotypes in the gG andgI genes can be detected by these typing systems. Phylogenetictrees based on complete gene sequences, as well as the cleavagepattern for 15 of the clinical isolates and the three laboratorystrains, are presented in Fig. 2.

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FIG. 2. Phylogenetic trees based on US4 and US7 genes, as well as cleavage patterns for the two genotyping targets for 15 clinical HSV-1 isolates and the three laboratory strains 17, F, and KOS321. The trees were calculated by using the maximum-likelihood method applied to 100 bootstrap replicates, and bootstrap values greater than 90 are shown. The US4 gene was analyzed in two segments due to a suggested ancient recombination event (7). The different genotypes—A, B, and C—are marked in red, blue, and green, respectively. One of the isolates (no. 3355) was detected as a recombinant and is underlined in the phylogenetic trees.

Performance of the PCR typing systems. Recently, Namvar et al. (6) described a real-time TaqMan PCRsystem for the detection of HSV-1 based on a type-specific segmentin the gB-1 gene. A plasmid (pUC57) containing the target sequencewas constructed (GenScript) and amplified in Escherichia coliXL1-Blue, purified by HiSpeed Plasmid Maxi Kit (QIAGEN) andquantified by spectrophotometer analysis. Plasmid DNA and HSV-1DNA from clinical isolates belonging to the genotypes A, B,and C were extracted in a MagnaPure LC robot (Roche) and amplifiedin a real-time PCR instrument ABI 7900 (Applied Biosystems).From the generated standard curve, the detection limits forthe typing systems were calculated. To achieve clearly visiblefragments in the gel, the sensitivity varied between differentisolates in the range of 500 to 5,000 HSV-1 genome copies forboth the gG and the gI systems.

The primers were selected not to amplify the homologous gG andgI gene sequences of HSV-2. Accordingly, five clinical HSV-2isolates were negative in the PCRs (data not shown). The capacityof the method is highly dependent on the facilities of the laboratory.We easily processed 50 clinical HSV-1 isolates per day manually.By automation using robots, several hundreds of isolates canbe processed simultaneously. Although DNA sequencing of theisolates can also be automated, the method described here isless expensive and faster since sequence analysis and the identificationof informative sites specific for each genotype are not necessary.

DISCUSSION

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Discussion

We present here a rapid and accurate method for genotyping clinicalHSV-1 isolates into three genotypes. The method may be a usefulscreening tool in searching for associations between genotypeidentity and a wide range of manifestations of HSV-1 infectionsuch as clinical symptoms, immune responses of the host, thetransmission of virus, virulence, and tropism. Furthermore,the method might also be of interest for epidemiological studiesfrom different geographical regions to determine the genotypeidentity for a large number of clinical isolates. The gE andgI proteins have been shown to form a complex that is involvedin cell-to-cell spread (2) and the binding of immunoglobulinG via the Fc receptor (4). The gG protein has been describedas important for virus entry through the apical surfaces ofpolarized cells (11). The method can be used to study possibleassociations between genotype identity and the functions ofthe gG and gI proteins.

Although the typing method used here is based only on 31 HSV-1gI gene sequences (7), we have since used the method for morethan 200 clinical HSV-1 isolates. For a single isolate an atypicalcleavage pattern was recognized. This isolate contained a pointmutation T₅₁₃ $->$ C (the nucleotide position refers to strain 17)within the restriction enzyme site (PleI) in the gI gene. Thisrare mutation created a typical PCR fragment of 222 nt thatcorrectly classified the isolate to genotype C and an atypicaluncleaved fragment of 188 nt instead of two fragments containing55 and 133 nt (see Fig. 1). The genotyping system of the gG-1gene is based on more than 180 HSV-1 sequences derived fromWestern and East Asia countries, and no ambiguous cleavage patternswere identified for the additional HSV-1 isolates examined.We and others have shown that homologous recombination is commonin the HSV-1 genome (1, 7). Although we found that 7 of 28 clinicalHSV-1 isolates were recombinants using a relatively short distancebetween the genotyping targets, it is possible that more recombinantscould be detected if a longer distance between the genotypingtargets is selected. Hence, based on phylogenetic and recombinationanalysis of other parts of the genome, e.g., the unique longsegment, the use of such additional targets might be of interest.

ACKNOWLEDGMENTS

This study was supported by grants from The Swedish Societyof Medicine, the LUA Foundation at Sahlgrenska University Hospital,and The Swedish Research Council (grant 11225).

FOOTNOTES

* Corresponding author. Mailing address: Department of Virology, University of Göteborg, Guldhedsgatan 10 B, S-413 46 Göteborg, Sweden. Phone: (46) 31 3424615. Fax: (46) 31 3424960. E-mail: peter.norberg{at}microbio.gu.se.

Published ahead of print on 11 October 2006.

REFERENCES

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