Genetic changes in plaque-purified varicella vaccine strain Suduvax during in vitro propagation in cell culture.

Infection by varicella-zoster virus (VZV) can be prevented by using live attenuated vaccines. VZV vaccine strains are known to evolve rapidly in vivo, however, their genetic and biological effects are not known. In this study, the plaque-purified vaccine strain Suduvax (PPS) was used to understand the genetic changes that occur during the process of propagation in in vitro cell culture. Full genome sequences of three different passages (p4, p30, and p60) of PPS were determined and compared for genetic changes. Mutations were found at 59 positions. The number of genetically polymorphic sites (GPS) and the average of minor allele frequency (MAF) at GPSs were not significantly altered after passaging in cell culture up to p60. The number of variant nucleotide positions (VNPs), wherein GPS was found in at least one passage of PPS, was 149. Overall, MAF changed by less than 5% at 52 VNPs, increased by more than 5% at 42 VNPs, and decreased by more than 5% at 55 VNPs in p60, compared with that seen in p4. More complicated patterns of changes in MAF were observed when genetic polymorphism at 149 VNPs was analyzed among the three passages. However, MAF decreased and mixed genotypes became unequivocally fixed to vaccine type in 23 vaccine-specific positions in higher passages of PPS. Plaque-purified Suduvax appeared to adapt to better replication during in vitro cell culture. Further studies with other vaccine strains and in vivo studies will help to understand the evolution of the VZV vaccine.

Increase in the genetic polymorphism of varicella-zoster virus after passaging in in vitro cell culture.

Primary infections with the varicella-zoster virus (VZV) result in varicella, while latent reactivation leads to herpes zoster. Both varicella and zoster can be prevented by live attenuated vaccines. There have been reports suggesting that both clinical VZV strains and those in vaccine preparations are genetically polymorphic, containing mixtures of both wild-type and vaccine-type sequences at certain vaccine-specific sites. In this study, the genetic polymorphism of the VZV genome was examined by analyzing the frequencies of minor alleles at each nucleotide position. Next-generation sequencing of the clinical VZV strain YC02 passaged in an in vitro cell culture was used to identify genetically polymorphic sites (GPS), where the minor allele frequency (MAF) exceeded 5%. The number of GPS increased by 7.3-fold at high passages (p100) when compared to low passages (p17), although the average MAF remained similar. GPS were found in 6 open reading frames (ORFs) in p17, 35, and 54 ORFs in p60 and p100, respectively. GPS were found more frequently in the dispensable gene group than the essential gene group, but the average MAF was greater in the essential gene group. The most common two major/minor base pairs were A/g and T/c. GPS were found in all three passages at 16 positions, all located in the reiterated (R) region. The population diversity as measured by Shannon entropy increased in p60 and p100. However, the entropy remained unchanged in the R regions.