Global identification of three major genotypes of varicella-zoster virus: longitudinal clustering and strategies for genotyping

By analysis of a single, variable, and short DNA sequence of 447 bp located within open reading frame 22 (ORF22), we discriminated three major varicella-zoster virus (VZV) genotypes. VZV isolates from all six inhabited continents that showed nearly complete homology to ORF22 of the European reference strain Dumas were assigned to the European (E) genotype. All Japanese isolates, defined as the Japanese (J) genotype, were identical in the respective genomic region and proved the most divergent from the E strains, carrying four distinct variations. The remaining isolates carried a combination of E- and J-specific variations in the target sequence and thus were collectively termed the mosaic (M) genotype. Three hundred twenty-six isolates collected in 27 countries were genotyped. A distinctive longitudinal distribution of VZV genotypes supports this approach. Among 111 isolates collected from European patients, 96.4% were genotype E. Consistent with this observation, approximately 80% of the VZV strains from the United States were also genotype E. Similarly, genotype E viruses were dominant in the Asian part of Russia and in eastern Australia. M genotype viruses were strongly dominant in tropical regions of Africa, Indochina, and Central America, and they were common in western Australia. However, genotype M viruses were also identified as a minority in several countries worldwide. Two major intertypic variations of genotype M strains were identified, suggesting that the M genotype can be further differentiated into subgenotypes. These data highlight the direction for future VZV genotyping efforts. This approach provides the first simple genotyping method for VZV strains in clinical samples.     

Complete-genome phylogenetic approach to varicella-zoster virus evolution: genetic divergence and evidence for recombination

Recent studies of varicella-zoster virus (VZV) DNA sequence variation, involving large numbers of globally distributed clinical isolates, suggest that this virus has diverged into at least three distinct genotypes designated European (E), Japanese (J), and mosaic (M). In the present study, we determined and analyzed the complete genomic sequences of two M VZV strains and compared them to the sequences of three E strains and two J strains retrieved from GenBank (including the Oka vaccine preparation, V-Oka). Except for a few polymorphic tandem repeat regions, the whole genome, representing approximately 125,000 nucleotides, is highly conserved, presenting a genetic similarity between the E and J genotypes of approximately 99.85%. These analyses revealed that VZV strains distinctly segregate into at least four genotypes (E, J, M1, and M2) in phylogenetic trees supported by high bootstrap values. Separate analyses of informative sites revealed that the tree topology was dependent on the region of the VZV genome used to determine the phylogeny; collectively, these results indicate the observed strain variation is likely to have resulted, at least in part, from interstrain recombination. Recombination analyses suggest that strains belonging to the M1 and M2 genotypes are mosaic recombinant strains that originated from ancestral isolates belonging to the E and J genotypes through recombination on multiple occasions. Furthermore, evidence of more recent recombination events between M1 and M2 strains is present in six segments of the VZV genome. As such, interstrain recombination in dually infected cells seems to figure prominently in the evolutionary history of VZV, a feature it has in common with other herpesviruses. In addition, we report here six novel genomic targets located in open reading frames 51 to 58 suitable for genotyping of clinical VZV isolates.     

Mutational analysis of the repeated open reading frames, ORFs 63 and 70 and ORFs 64 and 69, of varicella-zoster virus

Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuclear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleotides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and PCR-based mutagenesis to make single and dual deletions of these ORFs. VZV was recovered within 8 to 10 days when cosmids with single deletions were transfected into melanoma cells along with the three intact VZV cosmids. In contrast, VZV was not detected in transfections carried out with a dual deletion cosmid. Infectious virus was recovered when ORF63 was cloned into a nonnative AvrII site in this cosmid, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This requirement may be related to our observation that ORF63 interacts directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually or simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruses generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very large, multinucleated syncytia. Finally, all of the deletion mutants that yielded recombinants retained infectivity for human T cells in vitro and replicated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesis.     

A full-genome phylogenetic analysis of varicella-zoster virus reveals a novel origin of replication-based genotyping scheme and evidence of recombination between major circulating clades

Varicella-zoster virus (VZV) is a remarkably stable virus that until recently was thought to exhibit near-universal genetic homogeneity among circulating wild-type strains. In recent years, the expanding knowledge of VZV genetics has led to a number of groups proposing sequence-based typing schemes, but no study has yet examined the relationships between VZV genotypes at a full-genome level. A central hypothesis of this study is that VZV has coevolved with humankind. In this study, 11 additional full VZV genomic sequences are presented, bringing the current number of complete genomic sequences publicly available to 18. The full-genome alignment contained strains representing four distinct clades, but the possibility exists that a fifth clade comprised of African and Asian-like isolates was not represented. A consolidated VZV genotyping scheme employing the origin-associated region between reiteration region R4 and open reading frames (ORFs) 63 and 70 is described, one which accurately categorizes strains into one of four clades related to the geographic origin of the isolates. The full-genome alignment also provided evidence for recombination having occurred between the major circulating VZV clades. One Canadian clinical isolate was primarily Asian-like in origin, with most of the genome showing strong sequence identity to the Japanese-like clade B, with the exceptions being two putative recombination regions, located in ORFs 14 to 17 and ORFs 22 to 26, which showed clear similarity to the European/North American clade A. The very low rate of single-nucleotide polymorphisms scattered across the genome made full-genome sequencing the only definitive method for identifying specific VZV recombination events.     

The Attenuated Genotype of Varicella-Zoster Virus Includes an ORF0 Transitional Stop Codon Mutation

Varicella-zoster virus (VZV) is the first of the human herpesviruses to be attenuated and subsequently approved as a live vaccine to prevent varicella and herpes zoster. Both the attenuated VZV vaccine, called vaccine Oka or vOka, and the parental strain pOka have been completely sequenced. Yet the specific determinants of attenuation are uncertain. The open reading frame (ORF) with the most single nucleotide polymorphisms (SNPs), ORF62, encodes the regulatory protein IE62, but IE62 studies have failed to define a specific SNP associated with attenuation. We have completed next-generation sequencing of the VZV Ellen genome, a strain known to be highly attenuated by its very limited replication in human skin xenografts in the SCID mouse model of VZV pathogenesis. A comparative analysis of the Ellen sequence with all other complete VZV sequences was extremely informative. In particular, an unexpected finding was a stop codon mutation in Ellen ORF0 (herpes simplex virus UL56 homolog) identical to one found in vOka, combined with the absence of polymorphisms in most Ellen ORFs that were known to be mutated in vOka. The mutated ORF0 protein was also imaged in both two dimensions and three dimensions by confocal microscopy. The probability of two VZV strains not connected by a recent common ancestor having an identical ORF0 SNP by chance would be 1 × 10(-8), in other words, extremely unlikely. Taken together, these bioinformatics analyses strongly suggest that the stop codon ORF0 SNP is one of the determinants of the attenuation genotype of live VZV vaccines.     

Sequencing of 21 varicella-zoster virus genomes reveals two novel genotypes and evidence of recombination

Genotyping of 21 varicella-zoster virus (VZV) strains using a scattered single nucleotide polymorphism (SNP) method revealed ambiguous SNPs and two nontypeable isolates. For a further genetic characterization, the genomes of all strains were sequenced using the 454 technology. Almost-complete genome sequences were assembled, and most remaining gaps were closed with Sanger sequencing. Phylogenetic analysis of 42 genomes revealed five established and two novel VZV genotypes, provisionally termed VIII and IX. Genotypes VIII and IX are distinct from the previously reported provisional genotypes VI and VII as judged from the SNP pattern. The alignments showed evidence of ancient recombination events in the phylogeny of clade 4 and recent recombinations within single strains: 3/2005 (clade 1), 11 and 405/2007 (clade 3), 8 and DR (clade 4), CA123 and 413/2000 (clade 5), and strains of the novel genotypes VIII and IX. Bayesian tree inference of the thymidine kinase and the polymerase genes of the VZV clades and other varicelloviruses revealed that VZV radiation began some 110,000 years ago, which correlates with the out-of-Africa dispersal of modern humans. The split of ancestral clades 2/4 and 1/3/5/VIII/IX shows the greatest node height.     

Varicella-zoster virus (VZV) open reading frame 61 protein transactivates VZV gene promoters and enhances the infectivity of VZV DNA.

The varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein is the homolog of herpes simplex virus type 1 (HSV-1) ICP0. Both genes are located in similar parts of the genome, their predicted products share a cysteine-rich motif, and cell lines expressing VZV ORF61 are able to complement an HSV-1 ICP0 deletion mutant (H. Moriuchi, M. Moriuchi, H. A. Smith, S. E. Straus, and J. I. Cohen, J. Virol. 66:7303-7308, 1992). In transient expression assays, HSV-1 ICP0 is a transactivator alone and transactivates in synergy with another viral transactivator, ICP4. However, VZV ORF61 represses the activation by VZV-encoded proteins ORF62 (the homolog of ICP4) and ORF4. To further characterize the function of VZV ORF61 and its role(s) in regulation of viral gene expression, we performed transient expression assays using target promoters from VZV, HSV-1, and unrelated viruses. In the absence of other viral activators, VZV ORF61 transactivated most promoters tested. In addition, a cell line stably expressing VZV ORF61 complemented the HSV-1 mutant in 1814, which lacks the transactivating function of VP16. The cell line expressing VZV ORF61 enhanced the infectivity of HSV-1 virion DNA. Moreover, transient expression of VZV ORF61 also enhanced the infectivity of VZV DNA. These results indicate that VZV ORF61 can stimulate expression of HSV-1 and VZV genes at an early stage in the viral replicative cycle and that ORF61 has an important role in VZV gene regulation.     

Lineage and Genogroup-Defining Single Nucleotide Polymorphisms of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic human pathogen for which cattle are an important reservoir host. Using both previously published and new sequencing data, a 48-locus single nucleotide polymorphism (SNP)-based typing panel was developed that redundantly identified 11 genogroups that span six of the eight lineages recently described for E. coli O157:H7 (J. L. Bono, T. P. Smith, J. E. Keen, G. P. Harhay, T. G. McDaneld, R. E. Mandrell, W. K. Jung, T. E. Besser, P. Gerner-Smidt, M. Bielaszewska, H. Karch, M. L. Clawson, Mol. Biol. Evol. 29:2047–2062, 2012) and additionally defined subgroups within four of those lineages. This assay was applied to 530 isolates from human and bovine sources. The SNP-based lineage groups were concordant with previously identified E. coli O157:H7 genotypes identified by other methods and were strongly associated with carriage of specific Stx genes. Two SNP lineages (Ia and Vb) were disproportionately represented among cattle isolates, and three others (IIa, Ib, and IIb) were disproportionately represented among human clinical isolates. This 48-plex SNP assay efficiently and economically identifies biologically relevant lineages within E. coli O157:H7.     

SNP/RD typing of Mycobacterium tuberculosis Beijing strains reveals local and worldwide disseminated clonal complexes

The Beijing strain is one of the most successful genotypes of Mycobacterium tuberculosis worldwide and appears to be highly homogenous according to existing genotyping methods. To type Beijing strains reliably we developed a robust typing scheme using single nucleotide polymorphisms (SNPs) and regions of difference (RDs) derived from whole-genome sequencing data of eight Beijing strains. SNP/RD typing of 259 M. tuberculosis isolates originating from 45 countries worldwide discriminated 27 clonal complexes within the Beijing genotype family. A total of 16 Beijing clonal complexes contained more than one isolate of known origin, of which two clonal complexes were strongly associated with South African origin. The remaining 14 clonal complexes encompassed isolates from different countries. Even highly resolved clonal complexes comprised isolates from distinct geographical sites. Our results suggest that Beijing strains spread globally on multiple occasions and that the tuberculosis epidemic caused by the Beijing genotype is at least partially driven by modern migration patterns. The SNPs and RDs presented in this study will facilitate future molecular epidemiological and phylogenetic studies on Beijing strains.     

Genetic structure of natural populations of the nitrogen-fixing bacterium Rhizobium meliloti.

The genetic structure of populations of the symbiotic nitrogen-fixing soil bacterium Rhizobium meliloti was examined by analysis of electrophoretically demonstrable allelic variation in 14 metabolic, presumably chromosomal, enzyme genes. A total of 232 strains were examined, most of which were isolated from southwest Asia, where there is an unsurpassed number of indigenous host species for R. meliloti. The collection consisted of 115 isolates recovered from annual species of Medicago in Syria, Turkey, and Jordan; 85 isolates cultured from two perennial species of Medicago (M. sativa [alfalfa] and M. falcata) in northern Pakistan and Nepal; and 32 isolates collected at various localities in North and South America, Europe, South Africa, New Zealand, and Australia, largely from M. sativa. Fifty distinctive multilocus genotypes (electrophoretic types [ETs]) were identified, and cluster analysis revealed two primary phylogenetic divisions separated at a genetic distance of 0.83. By the criterion of genetic differentiation conventionally applied in defining species limits among members of the family Enterobacteriaceae and certain other bacteria, the two primary divisions of R. meliloti represent distinct evolutionary species. Division A included 35 ETs represented by 209 strains from the eastern Mediterranean basin, northern Pakistan, Nepal, and various other localities worldwide. This division contained the nine commercial alfalfa inoculant strains examined. Division B included 15 ETs represented by 23 isolates, 21 of which were isolated from annual medic species growing in previously uninoculated soils in the eastern Mediterranean basin. The two remaining strains in division B, both representing the same ET, were isolated in the United States and Australia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic structure of natural populations of the nitrogen-fixing bacterium Rhizobium meliloti.

The genetic structure of populations of the symbiotic nitrogen-fixing soil bacterium Rhizobium meliloti was examined by analysis of electrophoretically demonstrable allelic variation in 14 metabolic, presumably chromosomal, enzyme genes. A total of 232 strains were examined, most of which were isolated from southwest Asia, where there is an unsurpassed number of indigenous host species for R. meliloti. The collection consisted of 115 isolates recovered from annual species of Medicago in Syria, Turkey, and Jordan; 85 isolates cultured from two perennial species of Medicago (M. sativa [alfalfa] and M. falcata) in northern Pakistan and Nepal; and 32 isolates collected at various localities in North and South America, Europe, South Africa, New Zealand, and Australia, largely from M. sativa. Fifty distinctive multilocus genotypes (electrophoretic types [ETs]) were identified, and cluster analysis revealed two primary phylogenetic divisions separated at a genetic distance of 0.83. By the criterion of genetic differentiation conventionally applied in defining species limits among members of the family Enterobacteriaceae and certain other bacteria, the two primary divisions of R. meliloti represent distinct evolutionary species. Division A included 35 ETs represented by 209 strains from the eastern Mediterranean basin, northern Pakistan, Nepal, and various other localities worldwide. This division contained the nine commercial alfalfa inoculant strains examined. Division B included 15 ETs represented by 23 isolates, 21 of which were isolated from annual medic species growing in previously uninoculated soils in the eastern Mediterranean basin. The two remaining strains in division B, both representing the same ET, were isolated in the United States and Australia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence and functional analysis of the meta-cleavage pathway involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102.

Pseudomonas sp. strain KKS102 is able to degrade biphenyl and polychlorinated biphenyls via the meta-cleavage pathway. We sequenced the upstream region of the bphA1A2A3BCD (open reading frame 1 [ORF1]) A4 and found four ORFs in this region. As the deduced amino acid sequences of the first, second, and third ORFs are homologous to the meta-cleavage enzymes from Pseudomonas sp. strain CF600 (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), these ORFs have been named bphE, bphG, and bphF, respectively. The fourth ORF (ORF4) showed homology with ORF3 from Pseudomonas pseudoalcaligenes KF707 (K. Taira, J. Hirose, S. Hayashida, and K. Furukawa, J. Biol. Chem. 267:4844-4853, 1992), whose function is unknown. The functions of meta-cleavage enzymes (BphE, BphG, and BphF) were analyzed by using crude extracts of Escherichia coli which expressed the encoding genes. The results showed that bphE, bphG, and bphF encode 2-hydroxypenta-2,4-dienoate hydratase, acetaldehyde dehydrogenase (acylating), and 4-hydroxy-2-oxovalerate aldolase, respectively. The biphenyl and polychlorinated biphenyl degradation pathway of KKS102 is encoded by 12 genes in the order bphEGF (ORF4)A1A2A3BCD (ORF1)A4. The functions of ORF1 and ORF4 are unknown. The features of this bph gene cluster are discussed.     

Nucleotide sequence of a Madagascar hepatopancreatic parvovirus (HPV) and comparison of genetic variation among geographic isolates

A segment of Madagascar hepatopancreatic parvovirus (HPV) genomic sequence (5742 nucleotides) was determined through PCR and direct sequencing. This nucleotide sequence was compared to isolates from Australia, Thailand, Korea, and Tanzania, and the mean distance was determined to be 17%. The Madagascar HPV is closest to the Tanzania isolate (12%), followed by isolates from Korea (15%), Australia (17%) and Thailand (20%). Analysis of the genomic structure revealed that this HPV sequence is comprised of one partial Left open reading frame (ORF) (349 amino acids, aa) and complete Mid (578 aa) and Right (820 aa) ORFs. The amino acid sequences of the 3 ORFs were compared among isolates. The Right ORF was found to have the highest variation with a mean distance of 24%. This was followed by the Left and Mid ORF with distances of 13 and 7%, respectively. A phylogenetic analysis based on the amino acid sequence of the Right ORF divides 7 HPV isolates into 3 well-separated groups: Korea, Thailand, and Australia. The Madagascar HPV clustered with the Korea and Tanzania isolates. In Madagascar, HPV has been detected by histological examination since the 1990s. PCR analysis of a recent (2007) sampling showed a 100% prevalence. HPV was also detected in Mozambique with a 100% prevalence. High (95%) prevalence of HPV was found in wild Penaeus merguinesis collected from New Caledonia. These results indicate that HPV displays a high degree of genetic diversity and is distributed worldwide among populations of penaeid shrimp.     

Differentiation of varicella-zoster virus ORF47 protein kinase and IE62 protein binding domains and their contributions to replication in human skin xenografts in the SCID-hu mouse

To investigate the role of the ORF47 protein kinase of varicella-zoster virus (VZV), we constructed VZV recombinants with targeted mutations in conserved motifs of ORF47 and a truncated ORF47 and characterized these mutants for replication, phosphorylation, and protein-protein interactions in vitro and for infectivity in human skin xenografts in the SCID-hu mouse model in vivo. Previous experiments showed that ROka47S, a null mutant that makes no ORF47 protein, did not replicate in skin in vivo (J. F. Moffat, L. Zerboni, M. H. Sommer, T. C. Heineman, J. I. Cohen, H. Kaneshima, and A. M. Arvin, Proc. Natl. Acad. Sci. USA 95:11969-11974, 1998). The construction of VZV recombinants with targeted ORF47 mutations made it possible to assess the effects on VZV infection of human skin xenografts of selectively abolishing ORF47 protein kinase activity. ORF47 mutations that resulted in a C-terminal truncation or disrupted the DYS kinase motif eliminated ORF47 kinase activity and were associated with extensive nuclear retention of ORF47 and IE62 proteins in vitro. Disrupting ORF47 kinase function also resulted in a marked decrease in VZV replication and cutaneous lesion formation in skin xenografts in vivo. However, infectivity in vivo was not blocked completely as long as the capacity of ORF47 protein to bind IE62 protein was preserved, a function that we identified and mapped to the N-terminal domain of ORF47 protein. These experiments indicate that ORF47 kinase activity is of critical importance for VZV infection and cell-cell spread in human skin in vivo but suggest that it is the formation of complexes between ORF47 and IE62 proteins, both VZV tegument components, that constitutes the essential contribution of ORF47 protein to VZV replication in vivo.     

Complete sequences of two highly divergent european isolates of TT virus

TT virus is a virus distantly related to the Circoviridae family. We report here the complete genome characterization of two European human isolates (T3PB and TUPB) using a new and simple protocol for sequencing GC-rich genomic regions. Sequence analysis confirmed the existence of two major ORFs, of a CAV-like VP2 motif in ORF2 and of potential stem-loop structures in non-coding regions. Phylogenetic analyses based on complete genomic sequences of human isolates suggested that three different lineages exist at least. The first lineage includes genotypes 1, 2, and 3, and two other lineages include viruses related to the Japanese SANBAN and to the North American TUS01 isolates respectively. Sequence comparison made it possible to assign strain T3PB to genotype 3, and strain TUPB to the TUS01 group. Consequently, this study reports the first full-length sequence of a genotype 3 isolate and demonstrates that viruses belonging to the TUS01 lineage are present in the Old Word.     

Genetic and pathogenic diversity of Neofusicoccum parvum in New Zealand vineyards

Genetic diversity of 50 isolates of Neofusicoccum parvum, the predominant species of the Botryosphaeriaceae recovered from grapevines displaying symptoms of dieback and decline in New Zealand, was compared to that of isolates from Australia, South Africa, and California. The eight universally primed polymerase chain reaction (UP-PCR) primers distinguished 56 genotypes, with only four clonal pairs found. Seven main groups were identified in a neighbour-joining (NJ) tree with isolates from different regions and vineyards of New Zealand, Australia, and California distributed in different groups, indicating a high level of intra and intervineyard genetic variation. All of the South African isolates were positioned in a separate UP-PCR group, indicating that these isolates were less related to the other N. parvum isolates. When compared to fungi that reproduce sexually the genetic diversity and Shannon diversity indices were low (0.076-0.249; 0.109-0.367, respectively), genetic identity levels were high (0.76-0.95), and genetic distance levels were low (0.04-0.27). The large number of genotypes and the low number of clones in the New Zealand N. parvum populations may be explained by parasexual recombination as anastomosis was observed between nonself pairings. Pathogenicity tests using isolates from different UP-PCR groups inoculated onto either green shoots or 1-y-old grapevines detected virulence diversity, indicating intra and intervineyard variation between isolates, however, no correlation was detected between UP-PCR group and virulence.     

Phosphorylation of varicella-zoster virus open reading frame (ORF) 62 regulatory product by viral ORF 47-associated protein kinase.

Varicella-zoster virus (VZV) encodes within its unique long region a gene product with protein kinase motifs. In a previous study, we demonstrated that immunoprecipitated VZV open reading frame (ORF) 47 protein was associated with a functional protein kinase activity, on the basis of its ability to both autophosphorylate and phosphorylate artificial substrates. To further define potential substrates of ORF 47-associated protein kinase, we analyzed individual viral phosphoproteins to determine whether any were modified by the viral protein kinase. These candidates included gene products of VZV ORFs 4, 61, 62, and 63, which are homologs of herpes simplex virus type 1 (HSV-1) immediate-early proteins. Each of the above VZV proteins was coimmunoprecipitated with ORF 47 kinase, and the immune complex was incubated in a protein kinase assay. Under these conditions, only the VZV immediate-early ORF 62 protein was phosphorylated by ORF 47-associated protein kinase. The specificity of this phosphorylation event was analyzed by a competition assay in which a recombinant ORF 47 protein lacking enzymatic activity was able to reduce the amount of phosphorylation of ORF 62 protein by VZV ORF 47-associated kinase. To provide an additional evaluation of specificity, the experiment was repeated with [32P]GTP instead of [32P]ATP, because the VZV ORF 47 kinase has the distinctive property of using GTP as a phosphate donor. Again the ORF 62 substrate was phosphorylated. In summary, the VZV ORF 47-associated protein kinase (the HSV-1 UL13 homolog) catalyzed the in vitro phosphorylation of the VZV ORF 62 protein, the homolog of the HSV-1 ICP4 regulatory protein.