Effective monitoring for vOka adverse events requires genomic markers that reliably distinguish vOka from wt viruses. The VZV genome is highly conserved, however, and even the most distantly related wt clades have only about 0.1% sequence variation at the DNA level (34
). Complete sequencing of vOka and pOka revealed only 42 SNP that differ between the two viruses (16
). vOka has been shown to contain a mixture of strains, and only 4 of 42 vOka-associated SNPs were reported as fixed for the vOka allele in all vOka strains (16
). The remaining SNPs occur as mixtures of vOka and wt alleles in the vaccine preparations, and the SNP profiles vary both between manufacturers and between batches from the same source (22
The vaccine is known to be attenuated for replication in skin (35
), but the genetic basis for attenuation remains elusive. It may involve multiple mutations within both ORF62 and several other genes (reviewed in reference 39
). The presence of the wt alleles is also likely to contribute to the immunogenicity and pathogenicity of the vaccine.
The approach to discriminating vOka from wt strains has evolved with improved understanding of genomic variation in vOka and with apparent shifts in the biogeography of wt VZV clades (16
). At first, vOka-derived viruses were distinguished using two SNPs located in ORF38 and ORF54 that distinguished the wt clade 2 variant of vOka (clade 2 PstI−
) from all other wt strains (24
). This was an unacceptable approach in Asia, where strains of this subclade were in common circulation, but it was initially useful in the United States since none of these clades appeared to be present. After pOka and vOka had been completely sequenced, SNPs that could distinguish vOka from all wt strains were identified (16
). Based on conventional DNA sequencing methods, four SNPs appeared to be fixed in vOka, all of which were located in ORF62 (positions 105705, 106262, 107252, and 108111) (16
). This finding was supported by the observed SNP profiles of viruses isolated from laboratory-confirmed cases of vOka varicella and HZ, although four isolates with the wt SNP at position 105705 were detected (41
). Assays targeting any of the SNPs at position 106262, 107252, or 108111were therefore regarded as the most reliable approach for identifying vOka, particularly if a combination of methods targeting more than one SNP was used. Complicating matters, however, was the observation of a wt clade 5 strain containing the vOka allele at position 107252 isolated from varicella cases that had been transmitted from an 86-year-old HZ patient in a long-term care facility (32
). Since the index case patient would likely have experienced her primary infection in childhood, this finding suggested that wt strains bearing one of the vOka markers from a clade known to be circulating in North America were present in the early 20th century and are likely to still be circulating today. Thus, it has now become necessary to characterize both wt genotyping markers (1
) and vOka-specific markers to maximize the reliability of vOka-wt discrimination.
Recognizing the inherent limit in the sensitivity of DNA sequencing methods for the detection of mixed alleles, we sequenced a large number of TA clones derived from one lot of vOka to determine whether a small fraction of strains in vOka carried the wt allele at any of the fixed vOka loci. In our study, 2% of vOka clones carried the wt allele at position 107252 in ORF62, and 3% of clones carried the wt allele at position 105705. None of the 304 ORF62 clones evaluated in this study carried the wt alleles at either position 106262 or 108111. A recent study of shorter clones (47
), each containing one of the fixed vOka SNPs, found precisely the opposite: 6% of clones had the wt marker at positions 106262 and 108111, and none of the clones had the wt allele at either position 105705 or 107252. While it is unclear whether the discrepant findings reflect lot-to-lot variation, differences between the experimental approaches or the numbers and types of clones evaluated, or other factors, it is now apparent that vOka contains small numbers of strains that carry at least one wt allele at any of the four vaccine markers previously regarded as fixed. Moreover, we found that 3 of the 6 clones that carried the wt allele at position 107252 also carried the wt marker at position 105705. On the basis of the findings from our study and that by Thiele and colleagues (47
), reliance on a single “fixed” vOka marker would result in a 1-in-17 to 1-in-50 chance of mischaracterizing a vOka adverse event as a wt infection. A testing algorithm that evaluates all 4 of these SNPs would reduce the risk of mistaken identification by multiple orders of magnitude. In addition, since one of the vOka markers (position 107252) has been observed in a North American clade 5 virus, additional testing to evaluate the PstI marker at position 69349 in ORF38 will also be needed to determine whether an isolate has the SNP profile of vOka-like clade 2 strains.
We initially screened position 107252 using RFLPs generated by the NaeI restriction endonuclease. We demonstrated that this approach is a robust, rapid, and cost-effective screening tool for characterization of this locus through RFLP patterns generated from the entire OFR62 fragment. The consistency of the additional NaeI sites in all vOka genomes makes this method applicable to the analysis of all batches of vOka.
Our observation of a case of vOka HZ illustrates the need for a more complex approach to discriminating vOka from wt virus. If a method targeting only the 107252 locus had been employed, this virus would have been identified as wt. Evaluation of SNPs at multiple loci in ORF1, -21, -22, -38, -50, -54, and -62 unambiguously identified the strain as a clade 2 virus of the vOka/pOka subtype, (clade 2 PstI−), with 3 of the 4 vOka markers that were previously considered fixed.
The Varivax vaccine preparation has also been reported to contain two fixed wt alleles in ORF62 (positions 106710 and 107599) that are present as a mixture of wt and vOka alleles in vOka/Biken and/or Varilrix (16
). Since the clones used in our study included the entire ORF62 reading frame, we were also able to evaluate the frequency of these markers in Varivax and to detect any linkage between one or more vOka markers. We detected the vOka allele at position 107599 on 18% of clones, which was previously reported to be fixed for the wt in all batches (31
). This percentage is well within the detection limits of all currently employed methodologies and may therefore be indicative of interbatch variation. None of the 304 clones carried the vOka marker at position 106710, consistent with previous observations using DNA prepared directly from the vOka (31
). The apparent loss of this vaccine marker may reflect differences in the manufacturing processes used by the three companies that produce this vaccine. The Varivax and Varilrix vaccines underwent additional passages in human cells, which may have introduced selective pressure against the vaccine marker at position 106710.
Another concern for diagnostic approaches to discriminating between vOka and wt strains is the possibility of recombination between vOka and wt virus in coinfected persons. Recombination between wt VZV strains has been reported in vitro
and in vivo
), and phylogenetic analyses of the published complete genomic sequences for VZV have led to the conclusion that recombination has played an important role in the establishment of currently circulating VZV clades (34
). Our characterization of the HZ case virus was limited to the analysis of widely scattered SNPs across a large portion of the genome. While the data are not sufficient to make definitive conclusions about recombination, the SNP profile of this virus provided no suggestion that recombination between vOka and a wt virus had occurred.
It is now clear that all 42 of the vOka-associated SNPs are present as a mixture of vaccine and wt alleles, albeit in widely variable proportions. The basis for vOka attenuation is therefore likely to be more difficult to ascertain than might have been hoped. A number of the genetic differences between vOka and pOka therefore seem likely to contribute collectively to the reduced pathogenicity of the vaccine. We are currently working to isolate a series of viable clones from the vOka preparation, each with different vOka-associated SNP profiles, for characterization using in vitro and in vivo pathogenicity assays.
In view of our observations, it now seems necessary to evaluate all four of the high-frequency vOka markers in ORF62 (positions 105705, 106262, 107252, and 108111) in addition to the clade 2 subtype marker in ORF38 in order to confidently verify vOka adverse events. Additional genotypic analysis may prove necessary in the future, particularly if the occurrence of vOka-wt recombination is documented.