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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 −80°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 μM of each primer (Table (Table1;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 μl. 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 μl 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 μM oligonucleotide primers (Table (Table1).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 (Table2),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 SNP at position 107307 (T) was specific for both the E1 and E2 strains. According to additional unspecific SNPs, the E1 strains could be divided into 10 different genotype variants. ORF 62 of the 18 M1 strains, obtained exclusively from patients with varicella, proved to be very homogenous. As a specific M1 marker, the nucleotide at position 106569 (T) became apparent in all M1 strains (Table (Table2).2). In contrast to the M1 reference strain, VZV CA 123, all M1 strains revealed one nonsynonymous mutation at position 108015. In agreement with the sequence of E2 reference strain VZV 11, the nucleotide at position 105923 (T) was characterized as an E2-specific marker (Table (Table3),3), which was detected in 23 of 24 strains (95.8%) from varicella and in 11 of 13 strains (84.6%) from zoster. A second E2-specific marker of reference strain VZV 11 at nucleotide 107026 (G) was not shown to be specific for the strains from both varicella and zoster. In E2 strains, the analysis revealed exceedingly heterogeneous findings, resulting in 17 strain variants. As shown by the phylogenetic tree (Fig. (Fig.1),1), variant XVI (no. 1571-06) differed most significantly from the remaining E2 strains because of the differences in nucleotides 105429 and 105923. Variant XV (no. 052-07), containing one mutation at position 107252, specific to vOka, was isolated from one patient who had developed thoracic zoster 11 months after bone marrow transplantation at age 16 years. The patient suffered from a myelodysplastic syndrome and had developed varicella at age 3 years.
IE62 is likely to exert an important regulatory function in VZV replication since it is capable of transactivating viral genes and increasing viral DNA infectivity (4). Recent findings support the possibility that viral replication and cell-to-cell spread may be diminished in vOka due to the altered potency of IE62 for inducing VZV gene transcription (2, 13). This study revealed that VZV strains of genotypes E1 and E2, identified among 75% to 90% of European isolates (3, 10, 11), show a high variability of ORF 62. The SNP at position 107307, found in all strains of the genotypes E1 and E2, is indicative of a close phylogenetic relationship. Since most E variants were found in varicella, the results suggest that circulating viruses have a higher tendency to develop irregular SNPs. One E2 strain isolated from an immunocompromised patient with zoster contained one nonsynonymous vaccine mutation. This was surprising insofar as this patient had never received a varicella vaccination. The occurrence of this vaccine SNP could be the result of spontaneous mutation or might have emerged as a result of recombination. The described polymorphism has been found in rashes due to the VZV vaccine (6). Since the strain was cultured for only two passages, it is unlikely that the vaccine mutation was due to the cell culture passages (7, 12). In contrast to the E strains, the M1 strains in this study exhibited a uniform ORF 62 pattern. In Germany, M1 strains have been found only in varicella patients to date. It was concluded that these strains might have been imported from African and/or Asian countries during the last few years and must have spread more effectively in the population than the European strains (10). The uniform pattern of M1 strains found in this study gives further evidence that these strains were introduced via few sources from African and/or Asian countries.
This work was supported by grants of the Deutsche Forschungsgemeinschaft (DFG, Germany).
Published ahead of print on 26 August 2009.