Application of long PCR method of identification of variations in nucleotide sequences among varicella-zoster virus isolates

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Abstract
Restriction fragment length polymorphism (RFLP) analysis of whole viral DNA of varicella-zoster virus (VZV) requires the time-consuming and laborious preparation of a large amount of purified viral DNA. RFLP analysis of small DNA fragments amplified by PCR was developed as an alternative method. However, its use has been limited because of the small number of variations in VZV. To overcome these drawbacks and to identify variations in VZV, we developed an RFLP analysis method combined with the long PCR method which has recently been developed for the amplification of DNA fragments between 5 and 35 kb in length. We amplified three DNA regions ranging from 6.8 to 11.4 kb and demonstrated that RFLP analyses of these regions allowed for the classification of 40 VZV isolates in Japan into 17 groups. One-fourth of the isolates contained a nucleotide difference of C versus T, which abolished the StyI site at position 76530; this alteration was linked to the reported PstI site polymorphism at position 69349 (nucleotide positions are based on those of strain Dumas). Nucleotide sequence variation in the examined regions among VZV isolates in Japan was estimated at roughly less than 0.05%, confirming the previously proposed idea that VZV is genetically stable and not highly diversified. Our method will be useful for studies of the molecular epidemiology of VZV.