Differentiation between wild-type and vaccines strains of varicella zoster virus (VZV) based on four single nucleotide polymorphisms.

Varicella-zoster virus (VZV) infection (chickenpox) results in latency and subsequent reactivation manifests as shingles. Effective attenuated vaccines (vOka) are available for prevention of both illnesses. In this study, an amplicon-based sequencing method capable of differentiating between VZV wild-type (wt) strains and vOka vaccine is described. A total of 44 vesicular fluid specimens collected from 43 patients (16 from China and 27 from the UK) with either chickenpox or shingles were investigated, of which 10 had received previous vaccination. Four sets of polymerase chain reactions were set up simultaneously with primers amplifying regions encompassing four single nucleotide polymorphisms (SNPs), '69349-106262-107252-108111'. Nucleotide sequences were generated by Sanger sequencing. All samples except one had a wt SNP profile of 'A-T-T-T'. The sample collected from a patient who received vaccine 7-10 days ago, along with VZV vaccine preparations, Zostavax and Baike-varicella gave a SNP profile 'G-C-C-C'. The results show that this method can distinguish vaccine-derived virus from wt viruses from main four clades, (clades 1-4) and should be of utility worldwide.

[Vaccines against varicella-zoster virus (VZV)]. / Vacunas frente al virus de la varicela zóster.

In Western countries, two attenuated varicella vaccines derived from the OKA strain are licensed: Varilrix® GlaxoSmithKline (OKA/RIT strain) and Varivax® Merck Sharp and Dohme (OKA/Merck strain). Currently, in Spain, varicella vaccination is only included in the Ministry of Health, Social Services and Equality official vaccination calendar for administration in adolescents who have not had the disease. Given the good results obtained in Navarra and Madrid with universal administration of the vaccine in children, it would be desirable to include the vaccine in the routine immunization schedule, with the administration of two doses at 15-18 months of age in the future. The protective efficacy of the attenuated herpes zoster vaccine was evaluated in the Shingles Prevention Study, which showed that in the short term (0-4 years) the vaccine reduced the incidence of herpes zoster by 53%, post-herpetic neuralgia by 66%, and the disease burden in immunocompetent persons aged ≥60 years by 61%. Another study demonstrated protective efficacy in persons aged 50-59 years. Over time, the protective efficacy decreases, but remains at acceptable levels, especially for post-herpetic neuralgia and the disease burden. Recently, the results of a controlled clinical trial (phase III) conducted in 18 countries to assess the protective efficacy of the inactivated subunit vaccine (glycoprotein E) adjuvanted with the adjuvant AS01B were published. The study inferred that the vaccine significantly reduced the incidence of herpes zoster in the short term (3.2 years) in people aged ≥50 years. Vaccine protection did not decrease with age at vaccination, ranging between 96.8% and 97.9% in all age groups.

Detection and differentiation of wild-type and vaccine mutant varicella-zoster viruses using an Invader Plus method.

We report the use of a prototype Invader Plus method (Third Wave Technologies, Inc., Madison, WI) for the qualitative detection of varicella-zoster virus (VZV) and differentiation of wild-type and Oka vaccine VZV. The analytical sensitivity of the VZV Invader Plus reagents is at 10 copies per reaction. A total of 174 skin and mucous swab specimens were used to validate the assay's performance. The sensitivity and specificity were 98.3% and 98.1%, respectively, in comparison to a PCR-EIA assay. A perfect 100% agreement was obtained when VZV wild-type and vaccine differentiation was performed on 54 VZV-positive swab specimens against an allele-specific FRET real-time assay. The Invader Plus method provides another reliable tool for qualitative detection of VZV and differentiation of wild-type and vaccine virus.

DNA sequence variability in isolates recovered from patients with postvaccination rash or herpes zoster caused by Oka varicella vaccine.

Little is known about the pathogenic potential of individual strains in the varicella vaccine. We analyzed genomic variation among specimens obtained from vaccine recipients with postvaccination rash or herpes zoster (HZ), focusing on polymorphisms between live attenuated varicella vaccine virus and wild-type varicella-zoster virus. Eleven of 18 postvaccination HZ specimens contained >1 strain, and 7 of 18 appeared to be clonal. All 21 postvaccination rash specimens contained mixtures of vaccine strains. Four single-nucleotide polymorphisms (SNPs) consistently occurred in every isolate; all were polymorphisms in open-reading frame (ORF) 62, and 2 confer amino acid substitutions in the immediate-early protein 62. Four wild-type SNPs occurred in every isolate: one each occurred in ORF 10, ORF 21, ORF 62, and a noncoding region upstream of ORF 64. The frequencies of the remaining wild-type SNPs were variable, with the SNPs uniformly expressed (even in mixtures) in 20.5%-97.4% of isolates (mean frequency, 67.7%). No 2 clinical isolates had identical SNP profiles; as such, vaccine latency usually involves >1 strain.

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 been used to identify four major subtypes of wild-type varicella-zoster virus (VZV) A, B, C, and J. Additional SNPs, located in the IE62 major transactivating gene can be used to differentiate the Oka vaccine strain (vOka) from wild-type VZV. Primer-probe sets for the detection of the five polymorphic loci were designed by Applied Biosystems for the ABI 7900HT platform. Probes for each allele were labeled with VIC or 6-carboxyfluorescein fluorogenic markers. Each primer-probe set was validated to establish assay sensitivity and specificity using VZV DNA of predetermined copy number and genotype. Further evaluation was carried out using DNA samples from the vesicle fluid or skin swab of the rash of adult patients with herpes zoster or rashes due to vOka. Assay sensitivity ranged from 10 and 10(8) copies/ml of VZV DNA (equivalent to 2 to 20 copies per reaction). Statistical analyses showed that for each genotype, a set of two probes clearly differentiated the nucleotide present (allele) at that locus (P < 0.0001). It was possible to determine the genotype of wild-type VZV using one of four SNP assays and also to differentiate wild type from vOka using a single SNP assay. The assay can be used for diagnostic and epidemiological studies of VZV, including the differentiation of vOka from wild-type strains, investigation of breakthrough infections, and varicella outbreaks following immunization.

Molecular diagnosis of zoster post varicella vaccination.

BACKGROUND: Herpes zoster can be caused by endogenous reactivation of both wild-type virus or vaccine varicella-zoster virus (VZV) becoming latent within sensory ganglia after natural primary infection or varicella vaccination. OBJECTIVES: To demonstrate molecular biological methods for reliable discrimination between wild-type VZV and vaccine strain Oka by an example of zoster in a vaccinated girl. STUDY DESIGN: VZV was isolated from zoster occurring 16 months after varicella vaccination in a 2-year-old infant. Including VZV wild-type and different Oka strains as controls, viral DNA fragments located in the open reading frames (ORF) 38, 54, 62 and the R5 variable repeat region were characterized by amplification and restriction fragment length polymorphisms (RFLP) analysis. RESULTS: VZV vaccine strain Oka was definitely proven to be the causative virus in this case of zoster post vaccination. CONCLUSIONS: Molecular procedures for characterization of ORF 38 allow reliable discrimination between Oka-like and wild-type VZV outside Japan and Japanese communities. To distinguish Oka vaccine virus from Oka-like wild strains, analysis of DNA fragments located in the ORF 62 should be included.

Varicella vaccine.

Varicella vaccine is safe, effective, and cost-effective in healthy children, adolescents, and adults. Breakthrough cases of MVLS are significantly milder than wild-type varicella infection. No severe adverse events have been reported following vaccination, and the incidence of herpes zoster is less in vaccinees than in individuals who have had natural varicella infections. To date, there is no evidence waning immunity following vaccination. "New and improved" varicella vaccines that may be more effective than the current vaccine and can be stored at refrigerator temperatures may soon become available in the United States.