Variability of Immediate-Early Gene 62 in German Varicella-Zoster Virus Wild-Type Strains

Abstract

Varicella-zoster virus strains of European genotypes have developed a high variability of open reading frame(ORF) 62 during their occurrence over many years in Germany. M1 strains in Germany display a uniform ORF 62 pattern, suggesting that these strains were introduced from Africa and/or Asia via few sources during the last years.During the surveillance of circulating varicella-zoster virus (VZV) strains, the analysis of the immediate-early gene 62 region has an important function, since 15 of the described 42 vaccine mutations of the Oka vaccine strain (vOka) were found in open reading frame (ORF) 62 alone. However, to date it is not clear that these mutations play an important role in the attenuation of vOka (1,5). In this study, ORF 62 of VZV wild-type strains was analyzed by DNA sequencing. The primary objective was to search for both genotypic markers and vaccine-related mutations.The study included 86 VZV isolates from 59 patients with varicella and 27 patients with zoster. Patients were randomly selected between 2003 and 2007, as described previously (8, 10). No patient had received a varicella vaccination. All patients or their parents gave the consent for the specimen collection. VZV isolates were cultured for two to four passages (9), genotyped (10), and stored at Ϫ80°C. For sequencing of ORF 62, DNA was isolated by means of a QIAamp blood kit (Qiagen, Hilden, Germany) and amplified using High-Fidelity enzyme mix (Fermentas, St.-Leon-Rot, Germany). The amplification mixture contained 10 M of each primer (Table 1; ORF 62, VZV-4F and VZV-26R; fragment A, VZV-25F and VZV-10R; fragment B, VZV-9F and VZV-17R) plus approximately 50 ng template DNA per 50 l. Reaction mixtures were cycled 35 times at 95°C for 40 s, at 55°C (ORF 62, fragment A) or 50°C (fragment B) for 45 s, and at 68°C for 1 min/kbp. An amount of 200 ng gel-purified DNA per l was used for the sequencing reaction, carried out by means of the cycle sequencing method using a DYEnamic ET Terminator cycle sequencing kit (Amersham Biosciences Europe, Freiburg, Germany) and 10 M oligonucleotide primers (Table 1). The thermal conditions of amplification followed 25 cycles of 95°C for 20 s, 50°C for 15 s, and 60°C for 1 s. DNA fragments were analyzed as described previously (10). Results were compared with published sequences of the reference strains Dumas (genotype E1; GenBank accession no. NC_001348 and X04370), VZV 11 (genotype E2; accession no. DQ479955), VZV CA 123 (genotype M1; accession no. DQ457052), VZV 8 (genotype M2; accession no. DQ479960), pOka (genotype J; accession no. AB097933), and vOka (genotype J; accession no. AB097932).The genotype E1 strains (Table 2), 17 from patients with varicella and 14 from patients with zoster, contained three genotype-specific nucleotides in positions 107165 (C), 108747 (A), and 108951 (G). Whereas the single nucleotide polymorphism (SNP) at nucleotide 108951 was observed in all E1 strains, the SNPs in positions 107165 and 108747 were present in 16 varicella strains (94.1%) and all zoster strains. The ...