Genotyping of Varicella-Zoster Virus and the Discrimination of Oka Vaccine Strains by TaqMan Real-Time PCR

Single nucleotide polymorphisms (SNPs) in five genes have beenused to identify four major subtypes of wild-type varicella-zostervirus (VZV) A, B, C, and J. Additional SNPs, located in theIE62 major transactivating gene can be used to differentiatethe Oka vaccine strain (vOka) from wild-type VZV. Primer-probesets for the detection of the five polymorphic loci were designedby Applied Biosystems for the ABI 7900HT platform. Probes foreach allele were labeled with VIC or 6-carboxyfluorescein fluorogenicmarkers. Each primer-probe set was validated to establish assaysensitivity and specificity using VZV DNA of predetermined copynumber and genotype. Further evaluation was carried out usingDNA samples from the vesicle fluid or skin swab of the rashof adult patients with herpes zoster or rashes due to vOka.Assay sensitivity ranged from 10 and 10⁸ copies/ml of VZV DNA(equivalent to 2 to 20 copies per reaction). Statistical analysesshowed that for each genotype, a set of two probes clearly differentiatedthe nucleotide present (allele) at that locus (P < 0.0001).It was possible to determine the genotype of wild-type VZV usingone of four SNP assays and also to differentiate wild type fromvOka using a single SNP assay. The assay can be used for diagnosticand epidemiological studies of VZV, including the differentiationof vOka from wild-type strains, investigation of breakthroughinfections, and varicella outbreaks following immunization.

INTRODUCTION

Top

Introduction

Varicella-zoster virus (VZV) is the etiological agent of chickenpoxin childhood and shingles (herpes zoster) following the reactivationof latent virus dormant in the dorsal root ganglia (2). VZVis genetically stable, and one major serotype has been describedpreviously (2). Using single nucleotide polymorphisms (SNPs)located in five open reading frames (ORFs) of VZV, we have identifiedfour major subtypes of wild-type VZV: A (African/Asian), B,C (European), and J (Japanese) (1). Similar results have beendescribed by other groups of investigators, although nomenclatureremains to be agreed (5, 13, 18). A live attenuated VZV vaccine,the vOka strain, is derived from a Japanese wild-type genotypeJ virus. Since 1995 vOka has been licensed for toddler immunizationin the United States (4). The vaccine is effective in healthyand immunocompromised individuals, with 95% protection againstsevere infection and 75% against all infection (11). Complicationsare rare, the commonest being rashes which occur in 5% of childrenand 10% of adults (6, 19).

Establishing the vaccine origin of rashes occurring postvaccinationis important for the purposes of surveillance (15). Initialmethods exploited the geographical SNP variations between theJapanese vaccine strain and wild-type strains found in the UnitedStates (10). However, these methods did not distinguish betweenthe vaccine and some wild-type Japanese strains (16).

An Sma1 and an Nae1 site in the ORF 62 gene are present in allpreparations of the vaccine and in all vaccine virus rashesand are absent from all wild-type strains, whatever their geographicalorigin (7, 12, 14). In order to maximize detection and typingof low-copy-number vaccine virus in vaccine rashes, we reportthe development of a sensitive and discriminatory real-timePCR that will facilitate the epidemiological surveillance ofvaccine rashes.

MATERIALS AND METHODS

Top

Materials and Methods

VZV strains and DNA extraction. VZV DNA was extracted (QIAamp DNA MiniKit; QIAGEN, Crawley,United Kingdom) from eight clinical isolates, 79 vesicle fluidsamples of patients with shingles, vesicular swabs of the rashesof 14 patients with suspected zoster due to vaccine breakthrough,and commercially available VZV vaccine (MERCK and GSK). Theextracted DNA was genotyped as A, B, C, or J using sequenceanalysis of four regions of the VZV genome, ORFs 1, 21, 50,and 54 (1). The identity of the vaccine strains of VZV was confirmedas vOka by PCR and digestion across the SmaI restriction endonucleasesite in ORF 62 (14).

TaqMan SNP assays. For each of the SNPs in Table 1, primer-probe sets (Table 2)were made using the Applied Biosystems design service (California).Two fluorogenic minor groove binder probes were used for eachlocus using the dyes 6-carboxyfluorescein (FAM; excitation,494 nm) and VIC (excitation, 538 nm) which are easily differentiatedin the Applied Biosystems Prism 7900HT PCR system. Real-timePCR was performed using 7.5 µl of TaqMan 2x universalmaster mix (Applied Biosystems, CA), 0.19 µl of primer-probe(470 nM and 100 µM, respectively), 5.31 µl of RNase-and DNase-free water (Sigma), and 2 µl of sample DNA,in a total volume of 15 µl per single tube reaction. Twowells of a 384 plate (Applied Biosystems, CA) were used foreach sample or SNP. DNase-free water used as nontemplate controland DNA of known VZV genotype (appropriate to the SNP of interest)used as a positive control were included in each assay run.Assay conditions were 2 min at 50°, 10 min at 95°C,and 40 cycles of 95°C for 15 s and 60°C for 1 min. Initially,the SNP assay was set up using SDS, version 2.1, software (AppliedBiosystems, CA) as an absolute quantification assay, but afterassay completion the plate was read using the allelic discriminationsettings. Postassay analysis was performed using the SDS software.

View this table:
[in this window]
[in a new window]
TABLE 1. VZV SNP for TaqMan allelic discrimination assay

View this table:
[in this window]
[in a new window]
TABLE 2. TaqMan primers and probes evaluated for VZV genotyping

SNP assay evaluation. DNA from clinical isolates of known genotype and vaccine strainswere tested to assess the ability of each SNP assay to differentiateone allele from another, as well as background and cross talksignals. SNP assays that produced good discrimination were furtherevaluated using the zoster patient samples, representative ofthe range of quality and quantity of VZV DNA expected in clinicalspecimens.

Assay sensitivity. DNA from VZV isolates genotyped as A, B, C, or J by sequencingwere quantified using a validated in-house TaqMan viral loadassay (8), and serial dilutions from 10 to 10⁸ copies/ml wereused to assess the sensitivity of each SNP assay.

Statistical methods. An unpaired t test (GraphPad QuickCalcs; Prism, California)was used to compare the mean and standard deviation of the reactivityfor each probe at each locus.

RESULTS

Top

Results

The results of an assessment of the sensitivity of the fiveprobes for each particular genotype at each locus using seriallydiluted samples of known copy number are shown in Fig. 1. Assayreactivity was directly proportional to copy number (R² of >0.95)for all probes. All but one probe was capable of detecting between2 to 20 copies/reaction. One probe (33725FAM non-genotype C)was relatively insensitive and failed to detected DNA in sampleswith fewer than 2,000 copies/reaction.

View larger version (30K):
[in this window]
[in a new window]
FIG. 1. Efficiency of each TaqMan SNP assay. C_T, cycle threshold.

Each SNP assay showed excellent discrimination between genotypesusing clinical isolates and vaccine (Fig. 2). The adjusted meanfluorescence (absolute value minus background template control)and standard deviation for each probe at each locus was calculatedfor all the zoster and vaccine strains tested (Table 3). Statisticalcomparison of these values (unpaired t test) showed highly significantdiscrimination (P < 0.0001) between probes at each locus.No false-positive results occurred, although for locus 33725discrimination of genotype C and non-genotype C viral DNA wasbased solely on the level of reactivity of the VIC (genotypeC specific) probe unless the sample contained high copy numbersof non-genotype C DNA owing to the lower sensitivity of theFAM probe.

View larger version (15K):
[in this window]
[in a new window]
FIG. 2. Allelic discrimination for each genotype at each locus. Results are based on adjusted mean fluorescence of paired wells (absolute value minus background template control).

View this table:
[in this window]
[in a new window]
TABLE 3. Analysis of the TaqMan genotype discrimination results of DNA samples derived from vesicle fluid taken from zoster patients

DISCUSSION

Top

Discussion

Real-time PCR assays are characterized by a wide dynamic rangeof quantification and offer a high degree of technical sensitivityand precision (9). No post-PCR steps are required, which avoidsthe possibility of cross-contamination, and these plate-basedassays are easily adaptable for rapid, high-throughput screening.The use of fluorogenic probes for the detection of PCR productduring the extension phase of the PCR provides empirical datayielding quantitative results that can be manipulated by statisticalprocedures to allow accurate quality assurance and control.We used TaqMan real-time allelic discrimination to genotypeVZVs using specific primer and probe sets designed around informativepolymorphic loci. The resulting assays are sensitive, detectingbetween 2 and 20 copies per reaction with good genotype specificity.The SNP 22725 assay was able to discriminate genotype C virusbut only when used in conjunction with at least one other SNPassay owing to the lack of sensitivity of the non-genotype C(FAM) probe.

The sensitivity of the SNP assays compares with a previouslypublished TaqMan-based assay method for which the limit of detectionwas 100 copies per reaction for the 107252 SNP of VZV (Oka versuswild type) (3). Five SNPs (SNP 101464 for genotype A, SNP 66288for genotype B, SNP 33725 for genotype C, SNP 685 for genotypeJ, and SNP 106262) and the Sma1 restriction endonuclease sitein ORF 62 of VZV can be used to distinguish vaccine and wild-typeVZV. The sensitivity and discrimination of the assay allow genotypingeven when copy number is low, as in the case of vaccine virusrashes.

Two groups have described melting curve assays based on real-timePCR using a LightCycler (Roche Applied Science) for the differentiationof vaccine from wild type using SNP 69649 in ORF 38 (17) andSNP 106262 in ORF 62 (12). The geographical SNPs used in ourassay provide additional genotype information that will be especiallyimportant for monitoring the origin of varicella outbreaks incountries that have good vaccine coverage and low levels ofendogenous varicella. In addition, the method provides proofthat recombination of vaccine and wild type is not occurringas vaccine is introduced.

In summary, we have described a highly discriminatory and sensitivemethod for the detection and genotyping of virus from post-Okavaccine rashes. The method is simple and reproducible and willbe useful for postvaccination surveillance.

ACKNOWLEDGMENTS

We acknowledge the help of Fiona Scott in collecting the viralstrains and Anne Gershon for the gift of the vaccine rash strains.We also thank Rachael Nugent and Mahmoud Al-Bassam for technicalhelp.

This work was supported by grants from Barts and the LondonSpecial Trustees and by Sanofi Pasteur.

FOOTNOTES

* Corresponding author. Mailing address: Skin Virus Laboratory, Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and the London school of Medicine and Dentistry, 4 Newark St., London E1 2AT, England. Phone: 44 207 882 2308. Fax: 44 207 882 2181. E-mail: j.breuer{at}qmul.ac.uk.

Present address: Abbot-Dartford, Murex Biotech Ltd., CentralRoad, Dartford, Kent DA1 5LR, United Kingdom.

Present address: HPA Laboratory, Institute of Pathology, NewcastleGeneral Hospital, Westgate Road, Newcastle upon Tyne, NE4 6BE,United Kingdom.

REFERENCES

Top