VZV strain SuduVax has been used by a Korean pharmaceutical company to produce live attenuated vaccine for chickenpox since 1994. Although its efficacy and safety have been proven in the marketplace, molecular characteristics of the vaccine strain have not been available. In this study sequencing and analyses of the nucleotide sequence of the Korean varicella vaccine strain SuduVax were undertaken.
In the original paper on the first complete sequencing of VZV strain Dumas , 71 ORFs were proposed. However, the information obtained from the NCBI GenBank database for Dumas (NC_001348) identifies 73 ORF if three ORFs located in TRS are counted as separate ORFs. Sequencing of two Oka-derived vaccine strains, VarilRix (DQ008354) and VariVax (DQ008355), identified 72 ORFs . A Blast search using these three strains as queries produced 74 possible ORFs for VZV. We were presently able to locate ORF45 (position 81,523- 82,593) to Dumas and ORF33.5 to VarilRix (position 60,257 - 61,165) and VariVax (60,254 - 61,162). Extended from of ORF0 due to read-through mutation was identified in SuduVax as well as in Oka vaccine strains (see below). Using these reference strains Dumas and VarilRix as queries, we were able to identify and locate 74 ORFs in the genome of the strain SuduVax as well as in other 23 VZV strains analyzed in this study.
Phylogenetic analysis using the full nucleotide sequences of 24 VZV strains identified five distinct clades, consistent with previous findings [9, 10]. Phylogenetic trees constructed with concatenated amino acid sequences and coding nucleotide sequences also revealed five clades with the same members. The tree built using non-coding nucleotide sequences appeared similar to the other trees, except that the strains 8 and M2DR did not form a clear clade as in other trees. SuduVax co-clustered with Oka strains and this clade consisted exclusively of isolates from Japan and Korea in clade 2. SuduVax shares the minimum complement of single nucleotide polymorphism at 27 positions  with other members of the clade 2. Various genotyping methods using limited genetic information of VZV strains have been proved to represent genotyping using full genome information [11–15]. Any genotyping method unequivocally placed SuduVax to the same genogroup with Oka strains as in phylogenetic trees based on full or near-full genetic information (data not shown).
It is not presently certain, because of the lack of full genome sequences from other Asian isolates, whether this clade 2 could be extended to include isolates from other Asian countries or whether it is confined to isolates from Japan and Korea only. However, available data based on partial nucleotide sequences or restriction fragment length polymorphism suggest that all Korean isolates and Chinese isolates form a clade with Japanese isolates [16, 17]. Thus, it is possible that the clade 2 could be extended to include China, which is geographically close to Japan and Korea.
Coding sequences occupy approximately 91% of the VZV genome and reflect most of the sequence information of the whole genome. Thus, it was expected that the phylogenetic trees based on the coding sequences are very similar to the trees based on the full nucleotide sequences. We found that the coding sequence trees and amino acid trees were similar to the full nucleotide trees. Noncoding sequences were found to be interspersed between coding sequences or ORFs, accounting for approximately 9% of the VZV genome. The phylogenetic trees based on VZV noncoding sequences are not different from those based on full or coding nucleotide sequences or amino acid sequences. One notable difference is the location of pOka within clade 2. In full or coding sequence trees, pOka was separated from four vaccine strains to form two independent subclades within clade 2. On the contrary, pOka did not form a subclade separated from vaccine strains in noncoding sequence trees. pOka is a clinical strain. Thus, coding sequences or amino acid sequences of VZV genome may provide information distinguishing vaccine strains from clinical strains, while noncoding sequences does not.
Phylogenetic analyses using the nucleotide sequences of individual ORFs suggested 12 ORFs may be important in distinguishing vaccine strains from clinical strains. Yamanish identified 23 ORFs that are different between pOka and Oka vaccine , including 12 ORFs identified in this study. Moreover, our preliminary studies of single nucleotide polymorphism among the full genomic DNA sequences of the 24 VZV strains revealed 12 ORFs that may be characteristic for vaccine strains and these 12 ORFs coincide with the above-mentioned 12 ORFs [manuscript in preparation].
ORF0, also known as ORFS/L, is thought to be essential for VZV growth and encodes a membrane protein with 129 amino acid residues, which is possibly involved in vesicular trafficking and altering cell adhesion molecules in infected cells [18, 19]. ORF0 in SuduVax was determined to possess an extended C-terminal sequence due to a read-through mutation of its original stop codon TGA to CGA coding for Arg. The nearest downstream stop codon TGA was found to overlap with ORF1 and the extended ORF0 is expected to code for a new protein with 221 amino acid residues. Interestingly, this read-through mutation was also found in the three Oka-derived vaccine strains, while the stop codons were found to be unaltered in all of the clinical strains including the parent Oka strain. In cells infected with vOka, the extended form of ORF0 protein with 221 amino acid residues and its spliced form with 155 amino acid residues are expressed . Since other vaccine strains, including SuduVax, share 100% identical nucleotide sequences within and downstream of ORF0 up to the new stop codon, both forms of the extended ORF0 proteins are expected to be expressed in permissive cells infected with SuduVax. Thus, read-through mutation in ORF0 might be an important feature distinguishing vaccine strains from clinical strains.
Besides the read-through mutation in ORF0, SuduVax share same mutational events in ORFs 17, 29, 56 and 60 with Oka strains. ORF17 codes for an mRNA-specific RNase  and ORF29 encodes single strand DNA binding protein via its zinc-finger domain . The function of ORF56 has not been well characterized, but its gene product is reported to co-localize with regulatory protein ICP22 and nuclear protein UL3 in small, dense nuclear bodies (NCBI, http://www.ncbi.nlm.nih.gov/pubmed?Db=gene&Cmd=retrieve&dopt=full_report&list_uids=1487683. The gene product of ORF60 is glycoprotein L, which acts as a chaperon for glycoprotein H . Three bp deletions were found in ORFs 17 and 56, and an insertion of 3-bp was found in ORF60. While most of the clinical strains contain two tandem copies of 15 bp (AACATTTCAGGGTCA) elements in ORF29, while the SuduVax and Oka strains contain only one copy of this 15 bp element. Of these four deletion and insertion events, two events (ORFs 29, 60) are shared with the clinical strains 8 and M2DR, and one event (ORF29) is also found in the strain CA123. Since these deletion and insertion events are also found in some of the clinical strains including pOka, they by themselves may not be important in attenuation, although it is still possible that they, in combination with other events such as read-through mutation in ORF0, may play some roles in attenuation of vaccine strains.