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.

Vaccine-type mutations identified in Varicella zoster virus passaged in cell culture.

Varicella-zoster virus (VZV) is a causative agent for chickenpox and shingles. Comparative genomic sequence analysis of clinical and vaccine strains suggested potential sites responsible for attenuation. In this study, low and high passages of two VZV clinical strains cultured in human fibroblast cells were compared for genomic DNA sequences and growth characteristics. Mutations were detected at 187 and 162 sites in the strain YC01 and YC02, respectively. More than 86% of mutations were found in open reading frames, and ORF62 exhibited highest frequency of mutations. T to C and A to G transitions accounted for more 90% of all possible substitutions. Forty mutations were common to two strains, including 27 in ORF62. Mutations found in attenuated vaccine strains were also detected at 7 positions. Both high and low passage strains were infectious and grew similarly in human fibroblast cells. In guinea pig cells, however, high passage strain remained infectious while low passage strain lost infectivity. This study may provide new insight into the attenuating mutations associated with in vitro passaging of VZV.