Restriction endonuclease analysis of varicella-zoster vaccine virus and wild-type DNAs

The DNA from several clinical isolates of varicella-zoster virus (VZV) were compared with the DNA from the vaccine strain VZV using three restriction endonucleases: BamHI, BgII, and HpaI. When electrophoresed through an agarose gel, the vaccine DNA digestion pattern was significantly different from the digestion patterns of the wild-type DNAs. Variations in the digestion pattern of the separate clinical isolates were also observed.     

Cycling probe technology to quantify and discriminate between wild-type varicella-zoster virus and Oka vaccine strains

Rapid differentiation between wild-type varicella zoster virus (VZV) and Oka-vaccine (vOka) strains is important for monitoring side reactions of varicella vaccination. To develop a high-throughput molecular diagnostic method for the differentiation of wild-type VZV and vOka strains based on cycling probe technology. The primers were designed to amplify common sequences spanning a single nucleotide polymorphism (SNP) in gene 62 of VZV. DNA–RNA chimeric probes (cycling probes) were designed to detect the SNP at nucleotide 105705. The cycling probe real-time PCR assays for VZV wild-type and vOka strains specifically amplified plasmids containing target sequences that ranged between 10 and 1 × 10⁶ copies per reaction. The inter- and intra-assay coefficients of variation were less than 5%. After initial validation studies, the clinical reliability of this method was evaluated using 38 swab samples that were collected from patients suspected of being zoster. Compared to the loop mediated isothermal amplification method, which is defined as the gold standard, cycling probe real-time PCR was highly sensitive and specific. The cycling probe real-time PCR technology is a reliable tool for differentiating between wild-type VZV and vOka strains in clinical samples.     

水痘-带状疱疹病毒临床分离株基因型别分析以及与Oka疫苗株的区别

目的 运用分子生物学方法 对从水痘或带状疱疹患者皮肤疱疹液中分离得到的水痘-带状疱疹(VZV)株进行基闪型研究,并区分感染是南野牛株还是由Oka疫苗株引起的.方法 从水痘或带状疱疹患者的皮肤水疱液中分离VZV,然后利用聚合酶链反应和限制性片段长度多态性分析对病毒株的ORF38、54、62和R5可变区基因进行分析.结果 在所分离的19株VZV中,存在PstⅠ+ Bgl Ⅰ+ R5A和Pst Ⅰ+ Bgl Ⅰ+RSB两种基因型,其中Pst Ⅰ+ Bgl Ⅰ+ R5A占52.7%,Pst Ⅰ+ Bgl Ⅰ+R5B占47.3%,没有发现与Oka疫苗株相同的基因型.结论 本研究中所分离的VZV毒株均系野生株,它们的基因型与欧洲、美国、日本的VZV分离株均不相同.利用病毒基因组中ORF38和ORF62区域的单一核苷酸多态性,能够区分疫苗株和野毒株.     

Analysis of United Kingdom wild-type strains of varicella-zoster virus: Differentiation from the Oka vaccine strain

In Japan and the United States, where vaccination against varicella-zoster virus (VZV) infection with the live attenuated Oka strain of varicella is routine, cases of chickenpox or shingles occurring in vaccinees can be caused by either wild-type or vaccine virus. Differentiating such cases is important epidemiologically and can be achieved only using molecular typing methods. In the United Kingdom, the Oka vaccine is being considered for use in groups at risk of severe primary varicella, such as seronegative immunocompromised patients and women who may be considering pregnancy. In addition, seronegative health workers who may be occupationally exposed to VZV infection might also be offered vaccination. We analysed 249 U.K. wild-type VZV strains, 105 from cases of chickenpox and 144 from shingles cases, to determine whether they could be distinguished from Oka by the genotyping systems used in Japan and the United States. Four polymorphic loci were examined, a Pst 1 restriction site in gene 38, a Bgl 1 restriction site in gene 54, the R5 repeat region, and the R2 repeat region. The results suggest that U.K. strains of VZV are more similar to U.S. strains than to Japanese strains. All the U.K. wild-type viruses were positive for the Pst 1-1 restriction site, unlike Oka, which is negative. However, one of thirty strains was indistinguishable from Oka at all other loci. J. Med. Virol. 53:60–62, 1997. © 1997 Wiley-Liss, Inc.     

New method of differentiating wild-type varicella-zoster virus (VZV) strains from Oka varicella vaccine strain by VZV ORF 6-based PCR and restriction fragment length polymorphism analysis.

A new method was developed to distinguish accurately wild-type varicella-zoster virus (VZV) strains from the Oka vaccine strain. Several DNA fragments covering open reading frame (ORF) 1-37 were amplified from wild-type VZV strains including the Oka parent strain and from the Oka vaccine strain. Restriction fragment length polymorphisms of these regions were compared, and nucleotide differences between the vaccine virus and other wild-type VZV strains were noted in ORFs 6, 10, and 35. In addition, variations of the R2 and R4 reiterated structures of the vaccine and its parent strains were examined. The Oka vaccine strain used in Japan was shown to be a mixture of viruses with different nucleotide sequences that had variations in at least three nucleotide positions in ORF 1-37 and had variable polymorphisms at R2 and R4 repeat regions (two and three patterns, respectively). The Oka parent strain on the other hand showed a single sequence and had only one reiterated structure at these regions. When VZV ORF 6 was amplified and its product was digested with AluI, the Oka vaccine strain could be precisely differentiated from its parent and from 56 other Japanese clinical isolates.     

Recombination in tissue culture between varicella-zoster virus strains

Several clinical varicella-zoster virus isolates obtained during testing of a live varicella vaccine had DNA restriction fragment patterns resembling neither vaccine nor wild-type virus [Gelb et al., J Infect. Dis. 155, 633-640, 1987]. One explanation for these isolates was recombination in vivo. To determine if such recombination is likely, two strains of varicella-zoster virus, distinguishable by restriction endonuclease fragment size differences (wild-type strain EF and the OKA vaccine strain), were grown together in tissue culture. After three passages, the mixed infection virus was plaque-purified. DNA from about 13% of the plaque-purified isolates had one or more BglI fragments found in neither parental virus. Hybridization studies showed that isolates containing one of the new BglI fragments were recombinants of the two parental strains. The BglI restriction fragment pattern of these recombinants resembled those of the unusual varicella isolates from individuals either vaccinated with the live attenuated OKA varicella vaccine and later exposed to natural varicella, or simultaneously exposed to both a recent recipient of the vaccine and natural varicella.     

Differentiation between vaccine and wild-type varicella-zoster virus genotypes by high-resolution melt analysis of single nucleotide polymorphisms

BACKGROUND: The analysis of single nucleotide polymorphisms (SNPs) of varicella-zoster virus (VZV) has enabled differentiation between wild-type genotypes from the Oka vaccine strain (V-Oka). OBJECTIVES: To genotype VZV strains in Australia using high-resolution melt (HRM) analysis of SNPs in five gene targets. STUDY DESIGN: Extracted DNA from 78 samples obtained from patients with chickenpox and zoster were genotyped by HRM analysis of SNPs in five open reading frames (ORFs): 1 (685 G>A), 21 (33725 C>T), 37 (66288 G>A), 60 (101464 C>A) and 62 (106262 T>C) using a double-stranded (ds) DNA saturating dye, LC Green Plus. RESULTS: For each genotype, melt curve temperature (T(m)) shifts differentiated the nucleotide present at that locus (P<0.0001) with melting curve shifts between alleles ranging from 0.56 degrees C (ORF 37) to 3.34 degrees C (ORF 62). The most common genotypes detected were the European Type C (59%) and B (18%) strains. This was followed by the African/Asian Type A (14%) and Japanese J1 (9%), strains, both prevalent in the Northern Territory and Western Australia. CONCLUSIONS: HRM analysis of SNPs showed that the European B and C genotypes were most prevalent in Australia, with genotypes A and J strains also present. HRM analysis using a dsDNA dye provides a useful tool in classifying varicella-zoster viruses.     

Discriminating between varicella-zoster virus vaccine and wild-type strains by loop-mediated isothermal amplification

The loop-mediated isothermal amplification (LAMP) method was developed to distinguish between the varicella-zoster virus (VZV) vaccine (vOka) strain and wild-type strains. Two single nucleotide polymorphisms (SNPs) (nucleotide [nt] 105705 for VR-1 VZV LAMP and nt 106262 for VR-2 VZV LAMP) located in the open reading frame 62 gene were selected as LAMP targets. Amplified vOka DNA demonstrated a typical ladder pattern; however, no LAMP product was detected in reactions performed with DNAs from other human herpesviruses by either VR-1 VZV LAMP or VR-2 VZV LAMP. This result was confirmed by a turbidity assay. The sensitivities of both VR-1 and VR-2 VZV LAMP determined by either the turbidity assay or agarose gel electrophoresis were 100 copies per reaction. To discriminate the vOka strain from wild-type strains, VR-1 and VR-2 VZV LAMP products were digested with the appropriate restriction enzymes (SacII for VR-1 LAMP and SmaI for VR-2 LAMP). The digested products were clearly different in the vOka strain and wild-type strains. To evaluate the utility of the LAMP methods for rapid differentiation, viral DNA (without DNA extraction) in swab samples was directly tested. Wild-type VZV DNA was detected in 20 swab samples by either VR-1 VZV LAMP or VR-2 VZV LAMP. Sequence analysis confirmed the expected SNPs in the LAMP products amplified from the vOka strain and the five wild-type strains.     

Construction of Oka varicella vaccine expressing human immunodeficiency virus env antigen

Oka varicella vaccine has been used to confer active immunity to varicella-zoster virus (VZV) in healthy and immunocompromised hosts. Based on its attenuated nature, Oka varicella vaccine expressing human immunodeficiency virus (HIV) env antigen was constructed by inserting the HIVenv gene into the viral genome and its immunogenicity was assessed in guinea pigs. The HIVenv gene encoding 296-463 amino acids was inserted between the sequences of the hepatitis B surface antigen and the thymidine kinase gene of the cloned plasmid and the recombinant virus was isolated by cotransfection of the chimeric plasmid with viral DNA. Insertion of the HIVenv gene into the viral genome was confirmed by PCR and sequencing of the viral genome of the recombinant virus. The recombinant virus expressed 30k HIVenv fusion protein in its infected cells. In guinea pigs, immunization with the recombinant virus induced an antibody response to both the HIV antigen and the V3 peptide of gp120 as well as VZV gE:gI. Cell-mediated immunity to the HIV antigen and gE:gI was assessed by the cutaneous reaction representing delayed type hypersensitivity. Immunized guinea pigs responded well to both the HIV antigen and gE:gI. Thus the recombinant Oka varicella vaccine expressing the HIVenv antigen induced both a humoral and cell-mediated immunity to the HIV antigen similar to VZV as Oka varicella vaccine induces humoral and cell-mediated immunity to VZV in the vaccinees. This recombinant Oka varicella vaccine expressing the HIVenv antigen may be evaluated for its immunogenicity as one of the AIDS vaccine candidates.     

Analysis of varicella zoster virus attenuation by evaluation of chimeric parent Oka/vaccine Oka recombinant viruses in skin xenografts in the SCIDhu mouse model

Varicella-zoster virus (VZV) is the only human herpes virus for which a vaccine has been licensed. A clinical VZV isolate, designated the parent Oka (pOka) strain was passed in human and non-human fibroblasts to produce vaccine Oka (vOka). The pOka and vOka viruses exhibit similar infectivity in cultured cells but healthy susceptible individuals given vaccines derived from vOka rarely develop the cutaneous vesicular lesions characteristic of varicella. Inoculation of skin xenografts in the SCIDhu mouse model of VZV pathogenesis demonstrated that vOka had a reduced capacity to replicate in differentiated human epidermal cells in vivo (Moffat, J.F., Zerboni, L., Kinchington, P.R., Grose, C., Kaneshima, H., Arvin A.M., 1998a. Attenuation of the vaccine Oka strain of varicella-zoster virus and role of glycoprotein C in alphaherpesvirus virulence demonstrated in the SCID-hu mouse. J Virol. 72:965-74). In order to investigate the attenuation of vOka in skin, we made chimeric pOka and vOka recombinant viruses from VZV cosmids. Six chimeric pOka/vOka viruses were generated using cosmid sets that incorporate linear overlapping fragments of VZV DNA from cells infected with pOka or vOka. The cosmid sets consist of pOka and vOka DNA segments that have identical restriction sites. As expected, the growth kinetics and plaque morphologies of the six chimeric pOka/vOka viruses were indistinguishable in vitro. However, the chimeric viruses exhibited varying capacities to replicate when evaluated in skin xenografts in vivo. The presence of ORFs 30-55 from the pOka genome was sufficient to maintain wild-type infectivity in skin. Chimeric viruses containing different vOka components retained the attenuation phenotype, suggesting that vOka attenuation is multi-factorial and can be produced by genes from different regions of the vOka genome.     

Infection of human fetal dorsal root neurons with wild type varicella virus and the Oka strain varicella vaccine

The relative ability of a varicella-zoster virus (VZV) clinical isolate and a live attenuated VZV vaccine strain (Oka) to infect human neurons was determined in vitro. VZV infection of neurons prepared in culture from dorsal root ganglia of fetuses was assessed using an infectious center assay. Cultures were infected with 50–5,000 pfu of either VZV and assayed at either 24 or 48 hours post-VZV infection. Cultures infected with the clinical VZV isolate had seven-fold more infected neurons than cultures infected with the vaccine strain VZV. © 1993 Wiley-Liss, Inc.     

Molecular characterisation of varicella-zoster virus strains in Germany and differentiation from the Oka vaccine strain

With the introduction of varicella vaccination, surveillance of varicella-zoster virus (VZV) strains occurring in cases of chickenpox or zoster should be considered. Differentiating Oka vaccine strain from wild-type VZV can be achieved only using molecular genotyping. In the present study, the VZV genotype was examined in 53 VZV strains isolated from patients with varicella or zoster and in 73 samples from skin eruptions, cerebrospinal fluid, and throat swabs obtained from patients with VZV infections in Germany. The polymerase chain reaction and restriction fragment length polymorphisms analysis using DNA fragments of the open reading frames 38, 54, 62, and the R5 repeat region were used. Whereas all VZV isolates could be typed, direct genotyping of viral DNA in patients' samples was achieved in 63 of 73 cases (86.3%). The dominant genotype of VZV found in 88.8% of 116 patients had the wild-type pattern PstI(+) BglI(-) R5A followed by the wild-genotype PstI(+) BglI(+) R5A in 6.0%, the wild-genotype PstI(+) BglI(-) R5B in 3.4%, the wild-genotype PstI(+) BglI(-) R5C and the Oka vaccine genotype PstI(-) BglI(+) R5B in 0.9% of patients each. BglI(-) wild-types were found in 90.7% of patients with zoster and in 9.3% of patients with varicella. By contrast, the BglI(+) wild-type was diagnosed in five patients with varicella and in two patients with zoster. In conclusion, VZV strains found in Germany are similar to strains circulating in the United States and the United Kingdom. VZV wild-type strains containing a BglI restriction site in ORF 54 as well as Oka vaccine strains can rarely be detected.     

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.     

Clinical and molecular aspects of the live attenuated Oka varicella vaccine

VZV is a ubiquitous member of the Herpesviridae family that causes varicella (chicken pox) and herpes zoster (shingles). Both manifestations can cause great morbidity and mortality and are therefore of significant economic burden. The introduction of varicella vaccination as part of childhood immunization programs has resulted in a remarkable decline in varicella incidence, and associated hospitalizations and deaths, particularly in the USA. The vaccine preparation, vOka, is a live attenuated virus produced by serial passage of a wild‐type clinical isolate termed pOka in human and guinea pig cell lines. Although vOka is clinically attenuated, it can cause mild varicella, establish latency, and reactivate to cause herpes zoster. Sequence analysis has shown that vOka differs from pOka by at least 42 loci; however, not all genomes possess the novel vOka change at all positions, creating a heterogeneous population of genetically distinct haplotypes. This, together with the extreme cell‐associated nature of VZV replication in cell culture and the lack of an animal model, in which the complete VZV life cycle can be replicated, has limited studies into the molecular basis for vOka attenuation. Comparative studies of vOka with pOka replication in T cells, dorsal root ganglia, and skin indicate that attenuation likely involves multiple mutations within ORF 62 and several other genes. This article presents an overview of the clinical aspects of the vaccine and current progress on understanding the molecular mechanisms that account for the clinical phenotype of reduced virulence. Copyright © 2014 John Wiley & Sons, Ltd.     

The genome of choristoneura fumiferana nuclear polyhedrosis virus: Molecular cloning and mapping of the EcoRI, BamHI, SmaI, XbaI and Bg1II restriction sites

The DNA genome of Choristoneura fumiferana nuclear polyhedrosis virus (CfMNPV) was extracted from purified virus preparations and analyzed with restriction endonucleases EcoRI, BamHI, HindIII, SmaI, XbaI and Bg1II. The sizes of the fragments were calculated with respect to mobilities of standard adenovirus-2 DNA fragments. The total size of the genome was calculated to be about 125 kb. Most of the CfMNPV genome was then cloned as EcoRI fragments in the plasmid vector pBR322. The clones were authenticated by electrophoretic analysis in the presence of EcoRI-digested CfMNPV DNA and by hybridization of ³²P nick-translated clones to Southern blots of different enzyme digests of the genome. Data from such hybridization experiments complemented with other mapping procedures were used to construct restriction maps for BamHI, EcoRI, XbaI, Bg1II and SmaI enzymes.     

Rapid genotyping of varicella-zoster virus vaccine and wild-type strains with fluorophore-labeled hybridization probes

We developed a single-tube rapid method for the detection and differentiation of varicella-zoster virus (VZV) vaccine and wild-type strains that combines rapid-cycle PCR with wild-type-specific fluorescent probe melting profiles for product genotyping. A region including the polymorphic site in VZV open reading frame (ORF) 62 was amplified in the presence of two fluorescence-labeled hybridization probes. During the annealing step of the thermal cycling, both probes bound to their complementary sequences in the amplicon, resulting in resonance energy transfer, thus providing real-time fluorescence monitoring of PCR. Continuous acquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in a strain-specific manner as the detection probe, which was fully complementary to the wild-type VZV ORF 62 region, melted off the template. Use of this method allowed genotyping of samples within minutes after the completion of PCR, eliminating the need for post-PCR sample manipulation. In addition to reducing the time required to produce a result, this method substantially reduces the risk of contamination of the final product as well as the risk of sample tracking errors. The genotypes of 79 VZV-positive samples determined by this fluorescent resonance energy transfer (FRET) method were identical to the genotypes obtained by conventional PCR and restriction fragment length polymorphism analysis. The genotyping of VZV strains by the FRET method is a rapid and reliable method that is suitable for typing and that is also practical for use for the processing of large numbers of specimens.     

Characterization of the genome of a vaccine strain of canine adenovirus type 1

Restriction endonuclease cleavage maps have been constructed for the genome of a canine adenovirus type 1 (CAV-1) vaccine strain (CLL; Connaught Laboratories, Ltd., Willowdale, Ontario). Restriction enzyme analyses were also carried out on CAV-1 (CLL) genomes isolated from viral stocks over 8 serial passages in a dog kidney cell line (DK 6722). The right hand 20% of the genome became more heterogeneous in size with increasing passage in DK 6722 cells due to deletions up to 3–4 kb, whereas the left terminal region was stable throughout these passages. A comparative study of CAV-1(CLL) and a virulent strain of CAV-1, Glaxo, revealed that the genome of CAV-1(CLL) was the shorter, by about 480 bp, within the region covering 0.83–0.91 map units. By virtue of its location within the genome and its dispensable nature for viral growth, this region would appear to encompass a genetic sequence corresponding to the E3 region of human adenoviruses. In terms of viral attenuation, the possible importance of the observed differences between CAV-1(CLL) and CAV-1(Glaxo) is discussed.     

Seroprevalence of varicella-zoster virus antibody and immunogenicity of live attenuated varicella vaccine in healthcare workers in Taiwan

BackgroundHealthcare workers (HCWs) without evidence of immunity to varicella-zoster virus (VZV) are recommended to undergo varicella vaccination. Immunogenicity of live attenuated varicella vaccine has rarely been investigated among HCWs in Taiwan.MethodsAnti-VZV immunoglobulin G (IgG) titer was checked for all HCWs at Changhua Christian Hospital from 2011 to 2017. One-dose and two-dose (separated by 4–8 weeks) vaccines were administered to HCWs with equivocal and negative anti-varicella IgG results, respectively. Follow-up anti-VZV IgG was determined at least 4 weeks after completion of vaccination. Factors associated with seroconversion to varicella vaccination were analyzed.ResultsAmong 2406 included HCWs, the anti-VZV IgG serostatus was tested positive, equivocal and negative in 1924 (79.9%), 117 (4.9%) and 365 (15.2%), respectively. The seroprevalence had decreased from 88.0% (235/267) in 2011 to 72.2% (270/374) in 2017 (p for trend <0.05). A total of 67.8% (327/482) HCWs completed scheduled vaccination and serological follow-up. The seroconversion rates for HCWs with baseline equivocal and negative anti-VZV IgG results were 100% (80/80) and 79.4% (196/247) after one- and two-dose vaccination, respectively. In multivariate analysis, obesity (adjusted odds ratio, 0.308; 95% confidence interval [CI], 0.11–0.94, p = 0.039) was the only factor statistically significantly associated with seroconversion to vaccination.ConclusionDecreasing trends of seroprevalence of VZV were observed among HCWs from 2011 to 2017. HCWs who were obese were less likely to respond to varicella vaccination.     

An evaluation of single nucleotide polymorphisms used to differentiate vaccine and wild type strains of varicella-zoster virus

Rashes following immunization with the vaccine strain (vOka) of varicella-zoster virus (VZV) may occur in up to 5% of children and 10% of adults. In 40% of cases, the causative virus is the vaccine strain and in 60% wild type virus is found. Several reports have identified three restriction site polymorphisms in ORF 62 and the loss of one in ORF 6, which differentiate vOka from wild type VZV, including the parental wild type strain from which vOka, is derived. Using polymerase chain reaction (PCR), restriction enzyme analysis, and sequencing, we analyzed the presence of these markers in the GlaxoSmithKline (GSK, UK) and Merck vaccine preparations as well as in 15 vaccine virus rashes and 15 wild type UK viruses. Our data suggest that a Sma1 positive and an Nae1 positive site in ORF 62 are present in the GSK and Merck vaccine preparations and all vaccine virus rashes. By contrast, a BssHII positive vaccine virus restriction site in ORF 62 and an Alu1 negative site in ORF 6 were mixed in the GSK and Merck vaccines and absent in some of the vaccine rashes. The BssHII site was also present in the European wild type C viruses in UK. The data suggest that unlike the Biken vaccine preparation, the Merck and GSK vaccine preparations are polymorphic for the BssHII and Alu1 restriction sites. These sites are also present variably in the vaccine viruses causing rashes following vaccination, and are therefore unreliable markers for differentiating vOka and wild type VZV strains.     

Improved identification and differentiation of varicella-zoster virus (VZV) wild-type strains and an attenuated varicella vaccine strain using a VZV open reading frame 62-based PCR

A new method was developed to identify and differentiate varicella-zoster virus (VZV) wild-type strains from the attenuated varicella Oka vaccine strain. The PCR technique was used to amplify a VZV open reading frame (ORF) 62 region. A single specific amplicon of 268 bp was obtained from 71 VZV clinical isolates and several laboratory strains. Subsequent digestion of the VZV ORF 62 amplicons with SmaI enabled accurate strain differentiation (three SmaI sites were present in amplicons of vaccine strain VZV, compared with two enzyme cleavage sites for all other VZV strains tested). This method accurately differentiated the Oka vaccine strain from wild-type VZV strains circulating in countries representing all six populated continents. Moreover, the assay more reliably distinguished wild-type Japanese strains from the vaccine strain than did previously described methods.