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. 2000 Sep;38(9):3156-60.
doi: 10.1128/JCM.38.9.3156-3160.2000.

Improved identification and differentiation of varicella-zoster virus (VZV) wild-type strains and an attenuated varicella vaccine strain using a VZV open reading frame 62-based PCR

V N Loparev  1 , T Argaw, P R Krause, M Takayama, D S Schmid
Affiliations

Improved identification and differentiation of varicella-zoster virus (VZV) wild-type strains and an attenuated varicella vaccine strain using a VZV open reading frame 62-based PCR

V N Loparev et al. J Clin Microbiol. 2000 Sep.

Abstract

A new method was developed to identify and differentiate varicella-zoster virus (VZV) wild-type strains from the attenuated varicella Oka vaccine strain. The PCR technique was used to amplify a VZV open reading frame (ORF) 62 region. A single specific amplicon of 268 bp was obtained from 71 VZV clinical isolates and several laboratory strains. Subsequent digestion of the VZV ORF 62 amplicons with SmaI enabled accurate strain differentiation (three SmaI sites were present in amplicons of vaccine strain VZV, compared with two enzyme cleavage sites for all other VZV strains tested). This method accurately differentiated the Oka vaccine strain from wild-type VZV strains circulating in countries representing all six populated continents. Moreover, the assay more reliably distinguished wild-type Japanese strains from the vaccine strain than did previously described methods.

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Figures

FIG. 1
FIG. 1
Representative results for experimental ORF 62 primer pairs. Amplification products were produced with gradient annealing temperature cycling (ranging from 55 to 75°C) with the following primer pairs: PHKR1-PHKR2 (A), PHKR1-PKVL4L (B), PKVL7U-PKVL2L (C), and PKVL6U-PKVL1L (D). Apart from the controlled variation in annealing temperature, all reactions were carried out under identical conditions. Lane 11 is the negative control for all four gels (template DNA prepared from uninfected HLF cells). Lanes M contain a molecular size marker set (100 to 1,500 bp in 100-bp multiples).
FIG. 2
FIG. 2
Comparative RFLP test results for wild-type and Oka vaccine strain VZV using amplicons generated with the PKVL6U-PKVL1L primer pair. Shown are results for the SmaI RFLP assay for VZV ORF 62 amplicons obtained with wild-type viruses (lanes 1 to 3 and 5 to 9 correspond to samples 44 to 46 and 48 to 52 in Table 1) and Oka vaccine strain (lane 4). Lane M, molecular size marker set (100 to 1,500 bp in 100-bp multiples).
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