Information has been received from a published article. The live attenuated Oka varicella vaccine (vOka), derived from clade 2 wild-type (wt) virus pOka, was used for routine childhood immunization in several countries, including author's country, which had caused dramatic declines in the incidence of varicella. vOka could cause varicella, establish latency, and reactivate to cause herpes zoster (HZ). Three loci in varicella-zoster virus (VZV) open reading frame 62 (ORF62) (106262, 107252, and 108111) were used to distinguish vOka from wt VZV. A fourth position (105705) was also fixed for the vOka allele in nearly all vaccine batches. These 4 positions and two vOka mutations (106710 and 107599) reportedly absent from VARIVAX were analyzed on VARIVAX-derived ORF62 TOPO TA clones. The wt allele was detected at positions 105705 and 107252 on 3% and 2% of clones, respectively, but was absent at positions 106262 and 108111. Position 106710 was fixed for the wt allele, whereas the vOka allele was present on 18.4% of clones at position 107599. Author also evaluated the 4 vOka markers in an isolate obtained from a case of vaccine-caused HZ. The isolate carried the vOka allele at positions 105705, 106262, and 108111. However, at position 107252, the wt allele was present. Thus, all of the ORF62 vOka markers previously regarded as fixed occur as the wt allele in a small percentage of vOka strains. Characterization of all four vOka markers in ORF62 and of the clade 2 subtype marker in ORF38 is now necessary to confirm vOka adverse events. Materials and Methods Case report. In March 2011, approximately 23 months after receiving 1 dose of VARIVAX (vaccine lot number not indicated) (approximately in April 2009), an otherwise healthy 3-year-old female child was hospitalized. She presented with a rash consisting of 50 to 249 (exact number not specified) skin lesions on her left arm, affecting dermatomes C5, C6, and T1. the patient's guardian reported no history of varicella or HZ. The patient had been on acyclovir for 2 days prior to specimen collection. A swab was taken from a vesicle 2 days after HZ onset. On-site testing identified the presence of VZV DNA and absence of herpes simplex virus DNA by PCR. The patient was found to be VZV IgM negative and IgG positive. Patient outcome was not reported. DNA extraction. For the HZ case, total nucleic acid was extracted from 200 uL of vesicular fluid using the MagNA Pure LC automated NA purification system (Roche) according to the manufacturer's instructions and was eluted in 100 uL of elution buffer. For evaluation of the ORF62 SNPs on VARIVAX-derived clones, DNA was extracted from a batch of VARIVAX (lot LL46F, released in 2008) using a QIAamp DNA Minikit (Qlagen Ltd.) according to the manufacturer's instructions. Prior to extraction, the lyophilized vOka preparation was resuspended in 500 uL of the provided hydration buffer. DNA was extracted from 2 200-uL aliquots and 1 100-uL (mixed with 100 uL of PBS) aliquot. The 3 200-uL aliquots of eluted DNA were merged to form a 600-uL DNA pool which was used for subsequent experiments. PCRs and sequencing. Detection of VZV and discrimination between wt and vOka were performed at position 106262 using a Forster resonance energy transfer (FRET)-based real-time PCR assay as previously described (29) and at position 107252 using an in-house FRET assay. All FRET PCRs were performed using the LightCycler (Roche). A TaqMan-based realtime allelic discrimination assay, performed using the MX3000p platform (Stratagene), was used to analyze a third SNP (108111) in ORF62 that was reported to distinguish between vOka and wt strains (40, 48). Position 105705 was characterized by sequencing, which was also used to confirm results of the real-time PCR assays. Primers were designed to sequence SNPs 105705 (5'-CAAAGCGTGTTCTCTGTCGTCTG-3'), 106262 (5'-CTATGTGCCGCCTCGTCCA-3'), 106710 (5'-ATGATCAGAAGCCTCACATCCTCCG-3'), 107252 and 107599 (5'-GGTGTCTCCCTAATCTTGTCG-3'), and 108111 (5'-TGCTGCCTGTAGTTTCACTTCCC-3'). Sequencing reactions were performed using the BigDye Terminator cycle sequencing kit version 1.1 and analyzed on an ABI prism 3100 Genetic Analyzer (Applied Biosystems) in accordance with the manufacturer's instructions. SNPs in ORF1, -21, -22, -38, -50 and -54 that differentiate the 7 wt clades of VZV were analyzed as follows. The Bgll and Pstl sites in ORF54 and -38, respectively, were characterized using an in-house FRETbased real-time PCR assay. SNPs in ORF1, -21, -22, and -50 were analyzed by PCR and sequencing as previously described (1, 30). PCR amplification of the entire ORF62 (4,087 bp) was performed using a forward (5'-CCCGCACAGACAGACAGACACT-3') and reverse (5'-CTGCGAGAGCGTTTGGAAAACT-3') primer set. The PCR master mix included 12.5 uL Extensor Hi-Fidelity PCR master mix including buffer (AB-0794; Thermo Scientific), 4 uL of forward and reverse primers (each at 6 pmol/uL), and 4.5 ul of template DNA. PCRs were performed in a GeneAmp 9700 PCR system (Applied Biosystems) with initiation for 2 min at 95 degrees C followed by 10 cycles (94 degrees C for 10 s, 55 degrees C for 30 s, and 68 degrees C for 4 min) and 20 cycles (94 degrees C for 10 s, 55 degrees C for 30 s, and 68 degrees C for 4 min, increased by 10 s per cycle) of amplification, ending with a step at 68 degrees C for 7 min. The PCR product was visualized by electrophoresis on a 1% gel at 85 V for 1.5 h. Cloning of VARIVAX ORF62. Gel purification and cloning of the VARIVAX-derived ORF62 PCR product were performed using a TOPO XLPCR cloning kit with One Shot TOP10 chemically competent Escherichia coli (KA4750-10; Invitrogen) and suicide gene selection according to the manufacturer's instructions. Over three hundred copies were picked, and colony PCR using M13 vector primers was performed to amplify a 4,335-bp fragment (ORF62 insert plus M13 primer and flanking vector regions 132 bp from reverse primer site to ORF62 insert and 118 bp from forward priming site to ORF62 insert) to confirm the presence of the ORF62 insert. Positions 105705, 106262, 106710, 107599, and 108111 were characterized on each clone by sequencing as described above. Position 107252 was characterized by restriction fragment length polymorphism (RFLP) analysis using Nael restriction endonuclease as described below. ORF62 from pOka was also cloned as described above except that only 2 colonies were screened by PCR. RFLP analysis. The NEBcutter tool (New England BioLabs) was used to verify previously described restriction endonuclease cleavage sites that could be used to determine the presence of the wt allele at positions 106262 and 107252 in the ORF62 clones. NEBcutter also generates a virtual gel image to illustrate how the restriction fragment length polymorphisms (RFLPs) should look depending on which allele is present. The vOka alleles at position 106262 and 107252 have been reported previously to create Smal and Nael restriction endonuclease sites, respectively (15, 29), creating unique vOka and wt RFLPs. In our ORF62 clone, the presence of an additional 8 Smal sites produced RFLPs that could not be interpreted by gel electrophoresis. Three additional Nael sites were identified in ORF62 at nucleotides (nt) 105511 to 105516, not 105768 to 105773, and nt 108780 to 108785. We replaced the vOka and wt alleles at all 16 vOka loci in ORF 62 and found that these additional sites were not affected, nor were any new sites created, by any of the vOka mutations. Using Nael, RFLPs that clearly differentiated between the presence of the vOka allele and the wt allele were produced. Nael digestion in the presence of the wt allele resulted in a total of 4 fragments due to the absence of the restriction site at position 107252. The sizes of the minor fragments varied slightly depending on the orientation of the ORF62 fragment inserted into the vector (3012 bp, 535 to 552 bp or 514 to 531 bp, and 257 bp). The presence of the vOka allele at position 107252 caused cleavage of the Nael site, resulting in 5 fragments (1530 bp, 1482 bp, 535 to 552 bp or 531 to 514 bp, and 257 bp). Restriction digestion of the PCR product was performed in a 10-ul reaction volume consisting of 2 ul of PCR product, 5 U of Nael restriction endonuclease (New England BioLabs), 1 ul of 10 x EBuffer 4, and 6.5 ul molecular-grade water. Reaction mixtures were incubated at 37 degrees C for 3 h, and fragments were separated by gel electrophoresis on 1% agarose at 80 V for 3 h. The 3 additional Nael sites served as an internal control for the enzymatic reaction. Position 107252 was also sequenced in all clones to confirm the RFLP results. Statistical calculations. Authors estimated the probability of occurrence of the wt allele as the number of variant (i.e., wt) alleles that they found divided by the total number of sequences that we analyzed. Authors assumed a binomial distribution to obtain a 95% confidence interval for the probability, using the calculator at the website. Reference strain. All nucleotide positions referred to were those in strain Dumas (GenBank accession no. X04370; RefSeq NC_001348). Results Analysis of HZ case virus. The SNP profile for the VZV isolate obtained from the HZ case was shown. DNA was analyzed at positions 105705, 106262, 107252, and 108111 in ORF 62 in order to determine if the virus was vOka derived or wt. At positions 105705, 106262, and 108111 the virus was found to have the vOka allele, confirming it as vOka derived. However, at position 107252 the virus carried the wt allele. Analysis of SNPs in ORF1, -21, -22, -38, -50, and -54 collectively identified the virus as a clade 2 strain with the ORF38/ORF54 SNP profile of the vOka. Analysis of SNPs in VARIVAX-derived ORF62 clones. The frequencies of the wt allele at four loci in ORF62 (105705, 106262, 107252, and 108111) in 304 ORF62 clones derived from a batch of VARIVAX were determined. At positions 106262, and 108111, no clones with the wt allele were detected. At positions 105705 and 107252, the wt allele was found on 3% (9/304) and 2% (6/304) of clones, respectively. Among the 6 clones with the wt allele at position 107252, 3 also carried the wt allele at position 105705. The clones were also analyzed at positions 106710 and 107599, both of which are reported to lack the novel vOka allele present in VARILRIX and/or vOka/Biken. At position 107599, the vOka allele was found on 18.4% (56/304) of clones, but only the wt allele was detected at position 106710. The authors considered the patient's herpes zoster as Vaccine-Associated. "The frequencies of the wt allele at four loci in ORF62 (105705, 106262, 107252, and 108111) in 304 ORF62 clones derived from a batch of VARIVAX were determined." Additional information has been requested.