VARICELLA-ZOSTER VIRUS

THE VARICELLA VACCINE

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VARICELLA-ZOSTER VIRUS The Virus Jeffrey I. Cohen, MD

Chickenpox has been described since antiquity. Chickenpox was first proved to be an infectious disease in 1875, when Steiner5Itransmitted the virus by inoculating volunteers with vesicle fluid. In 1892, von B ~ k a yreported ~~ that chickenpox occurred in individuals who were in close contact with herpes zoster patients, suggesting that the two diseases were caused by the same agent. In 1925, Kundratitz30proved this hypothesis by demonstrating that inoculation of children with herpes zoster vesicle fluid resulted in chickenpox. Subsequent serologic studies confirmed that the antibody responses to both diseases are identical.38 In 1943, GarlandI5suggested that zoster might be due to reactivation of varicella acquired earlier in life. In 1952, Weller and Stoddard60isolated varicella-zoster virus (VZV) from vesicle fluid of varicella patients. Subsequent studies showed that the viruses of chickenpox and zoster have identical histologic and growth characteristics in cell culture and identical morphology and antigens.'j' Restriction endonuclease patterns of isolates from a patient with varicella and subsequent zoster showed that the viral genomes are identical.55 In 1975, Takahashi and colleagues56produced a live VZV vaccine by passing the virus in cell culture. The complete sequence of the VZV genome was determined in 1986,l' the first genetically engineered VZV mutant was constructed in 1987,32 and a versatile method for site-directed mutagenesis of the virus was performed using DNA cosmids to produce infectious virus in 1993.'j

From the National Institutes of Health, Bethesda, Maryland

INFECTIOUS DISEASE CLINICS OF NORTH AMERICA VOLUME 10 * NUMBER 3 * SEPTEMBER 1996

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STRUCTURE OF THE

VIRION

VZV is classified as an Alphaherpesvirus in the same subfamily as herpes simplex virus (HSV) types 1 and 2. Like other herpesviruses, the VZV virion (Fig. 1)consists of a double-stranded DNA core surrounded by the nuc1eocapsid.l The latter is composed of 162 capsomeres arranged

Figure 1. Electron micrograph of varicella-zoster virus virions. A, Virion (phosphotungstic acid, original magnification). (From Straus SE, Ostrove JM, lnchauspe G, et al: Varicellazoster virus infections. Biology, natural history, treatment, and prevention. Ann Intern Med 108221-227, 1988; with permission.) 5, Nucleocapsids in a human melanoma cell in culture (original magnification, x 25,000).

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with icosahedral symmetry. The nucleocapsid consists of the major 155kd nucleocapsid protein (encoded by ORF40) as well as several other proteins. The nucleocapsid has a diameter of 90 to 95 nm. The nucleocapsid is surrounded by a proteinaceous tegument. This amorphous structure consists of several viral proteins including the immediate-early proteins (the products of ORFs 4, 62, and 63) and a late protein (the product of ORF10). The tegument is enclosed in a lipid envelope that is composed of host cell membranes and viral glycoproteins. These glycoproteins, originally termed gp1 to VI, have recently been renamed to correspond to their HSV homologues. They are gB (gpI1); gC (gpv); gE (gpI); gH (gpII1); gI ( g p W and gL ( g p W The virion has a diameter of 150 to 200 nm.

STRUCTURE OF THE GENOME

The prototype VZV genome is a double-stranded DNA molecule comprised of 125,000 base pairs of DNA (Fig. 2)." The genome consists of a unique long region (UL) flanked by terminal and internal repeat long sequences and a unique short region (US) flanked by terminal and internal repeat short sequences. Virions contain linear genomes predominantly in one of two isomers, differing in the orientation of the

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Figure 2. The structure of the varicella-zoster virus (VZV) genome witH selected genes expressed during viral replication. The VZV genome contains 125 kilobase pairs of DNA (top line) arranged into unique long (UL), unique short (US), terminal repeat long (TRL), terminal repeat short (TRS), internal repeat long (IRL) and internal repeat short (IRS) DNA segments (second line). During replication of the viral genome about 70 genes are expressed. Selected putative immediate-early (third line), early (fourth line), and late genes (fifth line) are depicted.

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US region. Much less frequently, the UL region may be inverted. Each end of the viral genome contains a single unpaired nucleotide that can base pair to form a circular molecule during replication of the genome. The genome contains five repeat elements, termed RZ to R5, which are constant in length for a given strain of virus. The varying lengths of these repeat elements, as analyzed by restriction endonuclease analysis, are useful for distinguishing between different strains of VZV. For example, VZV strain Oka has four copies of the 42-base pair repeat (W), whereas strains Scott and Webster have about seven copies of the repeat.28The genome of VZV Oka differs from most wild-type strains of VZV by the presence of an additional BgII restriction endonuclease site and loss of a PstI site.3l Amplification of the appropriate-portion of the VZV genome using the polymerase chain reaction followed by digestion with BglI and PstI has been used clinically to determine whether strains are wild-type or Oka in origin. The viral genome encodes at least 69 unique genes. All but five of these genes have HSV-1 homologues. Although some VZV genes have similar functions as their HSV-1 counterparts and can complement them during replication (e.g., VZV OW61 and HSV-1 ICP035),other VZV genes do not complement their HSV-1 homologues (e.g./ VZV ORF4 and HSV-1 1CP27).36,41 At present, only one VZV gene (ORF42/45) is thought to be spliced.

REPLICATION OF THE VIRUS

VZV is thought to attach to cells by binding to heparan sulfate proteoglycans followed by binding to a mannose-6-phosphate receptor.h3 After attachment, viral glycoproteins fuse with cell membranes and the virion penetrates the cell. By analogy with HSV-1, VZV glycoproteins gB and gC may be important for attachment, whereas gB and gH may be critical for p e n e t r a t i ~ nRecent . ~ ~ studies suggest that VZV gH may be important for fusion with the cell membrane.44After penetration, the virion is uncoated and the nucleocapsid is transported to the nucleus. 25 may also be transported Tegument proteins (ORFs 4, 10, 62, and 63)24, to the nucleus where they initiate transcription of viral genomes. Initially, the viral immediate-early genes are expressed, which activate expression of the viral early genes. The latter encode proteins important for replication of viral DNA (e.g., thymidine kinase, polymerase, major DNA-binding protein). Finally, the late viral proteins are expressed. These are structural proteins that comprise the viral nucleocapsid, tegument, and envelope (Table 1).Viral DNA is packaged into nucleocapsids, is transported out of the nucleus, and acquires a glycoprotein envelope (either from the nuclear membrane or cytoplasmic vacuoles), and virions are released from the cell.9,l6

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Table 1. STRUCTURAL PROTEINS OF THE VZV VIRION

Class Glycoproteins

Gene ORF14 ORF31 ORF37

Tegument proteins Nucleocapsid proteins

ORF60 ORF67 ORF68 ORF4 ORFlO ORF62 ORF63 ORF33 ORF33.5 ORF40

Protein

Function

Attachment of virion to cell surface heparan sulfate Penetration of VZV into the cell? gB (gpll) Associates with gL, important for entry gH (gPw and exit of VZV from the cell gL (!JPVI) Associates with gH gl (gPlv) Associates with gE gE (gPU Associates with gl IE protein Transactivator late protein Transactivator IE protein Transactivator IE protein Transrepressor(?) Possible nucleocapsid protein Possible nucleocapsid protein Major nucleocapsid protein g c (SP V)

VIRAL PROTEINS Immediate-Early Proteins

At present, only two VZV genes have been definitively shown to encode immediate-early proteins (1)ORF62 and (2) ORF63.12,48Based on their homology with corresponding HSV immediate-early genes, VZV ORF4 and 61 are considered putative immediate-early genes. VZV ORF62 encodes a 175-kd phosphoprotein that acts as a potent transcriptional activator and is present in the tegument of the virion.25 OW62 protein activates expression of VZV putative immediate-early, early, and late genes in transient-expression assays and the protein can repress or activate its own promoter depending on the cell type used. VZV ORF62 protein contains a DNA-binding domain and a transcriptional-activation domain4,42, 43, 57 VZV ORF63 encodes a 45-kd phosphoprotein that is present in the viral tegument. There are conflicting reports in the literature as to whether ORF63 protein transrepresses VZV ORF62 protein in transientexpression assays.22, 29 ORF63 protein is expressed in latently infected ganglia in an animal model and in human skin during herpes zoster.I2 VZV ORF4 encodes a 55-kd phosphoprotein and O W 6 1 encodes a 62- to 65-kd phosphopr~tein.~~ ORF436,41 and ORF6135proteins transactivate putative immediate-early, early, and late viral genes in transientexpression assays. Early Proteins

By analogy with HSV, several proteins are required for replication of VZV DNA. The VZV DNA polymerase consists of a large and small

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subunit (encoded by ORF28 and ORF16, respectively). The large subunit has been expressed in baculovirus and the purified protein has enzymatic activity.I3VZV encodes two DNA binding proteins: (1) a singlestranded DNA-binding protein (encoded by ORF29)26and (2) the oribinding protein (encoded by ORF51) that binds to the origin of viral DNA replication in the US region of the g e n ~ m e The . ~ ORF28 and ORF29 genes have overlapping promoters that are activated by the combination of ORF62 protein and the upstream stimulating factor (USF) transcription fact0r.3~ Other early proteins also contribute to viral replication, but are probably not essential. The viral thymidine kinase is encoded by ORF36, the ribonucleotide reductase by ORFs 18 and 19, and th midylate synthetase by ORF13. The VZV thymidine kinase phosphory ates deoxycytidine, thymidine, and acyc10vir.~~ Phosphorylation of acyclovir is required for activation of the drug. Certain mutations in the thymidine kinase gene render VZV resistant to the antiviral activity of acyclovir.2 Drugs that inhibit the VZV ribonucleotide reductase inhibit the growth of virus in vitro and enhance the activity of acycl~vir.'~ VZV encodes two protein kinases, the products of ORF 47 and 66. The VZV ORF47 protein kinase can phosphorylate itself and the ORF62 encodes a serine-threonine protein k i n a ~ e . ~ ~ protein in ~ i t r o 40 . ~ORF66 ~,

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Late Proteins VZV encodes at least six glycoproteins. Infection with VZV induces both antibodies and T-cell responses to VZV glycoproteins gB, gC, gE, gI, and gH. Glycoprotein gB is a 140-kd glycoprotein consisting of 60and 70-kd proteins held together by disulfide bonds. Glycoprotein gB is a target for complement-independent antibody neutralization.16Glycoprotein gC varies in size from 95 to 105 kd, depending on the number of repeating units in the strain of VZV. Antibody to gC neutralizes virus in the absence of c ~ m p l e m e n t . ~ ~ Glycoprotein gE, a 98-kd glycoprotein, is the most abundant glycoprotein on the surface of infected cells and forms a complex with gI on the surface of cells. Glycoproteins gE and gI are targets for complementdependent antibody neutralization, and gE is a target for antibodydependent cellular cytotoxicity.*6* Glycoprotein gH is a 118-kd glycoprotein that is a target for complement-independent neutralizing antibodies. Glycoprotein gH forms a complex with gL, a 20-kd glycoprotein, on the surface of infected cells.I4 Monoclonal antibody to gH blocks entry of VZV into uninfected cells; therefore, gH may be important for the spread of virus between cells.'6,23,44 VZV ORFlO encodes a 50-kd protein that activates transcription and is present in the tegument of the virion.25,37 ORFlO protein activates expression of VZV ORF62 protein in transient-expression assays. Although VZV ORFlO is the homologue of HSV VP16, ORFlO has its transcriptional activation domain at the amino terminus, whereas VP16

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has its activation domain at the carboxy terminus. The major nucleocapsid protein of VZV is encoded by 0RF40.58 GROWTH IN CELL CULTURE Cell Types Infected

VZV grows efficiently in a limited number of primate cells in culture. Human diploid fibroblasts, embryonic lung fibroblasts, foreskin fibroblasts, and primary keratinocytes as well as melanoma and schwannoma cells have all been used to propagate virus. VZV also has ~~ been grown in guinea pig embryo and monkey k i d n e y c e l l ~ .VZV grows less efficiently in human neuroblastoma, astrocyte, Schwann neuron, T cell, and Epstein-Barr virus-transformed B cells. CHARACTERISTICS OF INFECTION

VZV is highly cell-associated in culture and grows by spread from infected to uninfected cells. Unlike other Alphaherpesvirus, infection of cells with VZV in vitro results in production of little or no cell-free infectious virus. Electron microscopic studies indicate that viral particles are released into the media; however, the particle-to-infectivity ratio is quite high at lo6 to l.49 In contrast, fluid aspirated from vesicles has a much lower particle-to-infectivity ratio and the virus is more stable. Thus, VZV is not processed and released from cultured cells as it is from cells in vivo. VZV is grown in cell culture by passage of trypsinized infected cells onto uninfected cell monolayers. When cell-free virus is needed, infected cells are sonicated and after centrifugation the resulting supernatant contains from lo2 to lo5 infectious particles per mL.I7,56 Infection of cells in culture results in expression of viral proteins within 6 hours after inoculation. Virus spreads to adjacent cells within 12 hours after infection. Infected cells are refractile and subsequently round up and form multinucleated syncytia. Detachment of infected cells from the monolayer results in formation of plaques (Fig. 3). LATENCY

After primary infection, VZV enters the dorsal root and trigeminal ganglia where it remains latent for the lifetime of the individual. VZV DNA and RNA are present in these ganglia and may be located in neurons or in the surrounding satellite cells.'O Recent studies suggest that several VZV genes may be expressed during latency in human trigeminal ganglia. RNA transcripts corresponding to VZV ORFs 21, 29, and 62 have been detected in human ganglia? 33 whereas ORF63 has been detected in an animal model of

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Figure 3. Cytopathic effects of varicella-zoster virus in cell culture. Infection of human melanoma cells in culture results in a plaque with surrounding syncytia of fused cells.

VZV latency.I2 In contrast to HSV, homologues of the HSV latencyassociated transcripts have not been detected in VZV either during productive infection or during latency. Reactivation of VZV, resulting in zoster, usually occurs at a dermatome near a previous site of varicella infection. If immunocompromised individuals inoculated with the Oka vaccine virus develop zoster, it is often located at the site of vaccine inoculation.

INHIBITORS OF REPLICATION

Acyclovir is the most common agent used to treat VZV infections. Acyclovir is converted to acyclovir-monophosphate by the VZV thymidine kinase and subsequently converted to the active triphosphate form by cellular kinases. Acyclovir directly inhibits the viral DNA polymerase and is incorporated into viral DNA resulting in chain termination. Acyclovir inhibits replication of VZV in vitro at concentrations of 1 p,g/ mL. Famciclovir and sorivudine are ,also phosphorylated by the viral thymidine kinase; famciclovir has a longer intracellular half-life than acyclovir, whereas sorivudine inhibits VZV replication at concentrations of only 0.0003 p,g/mL. Foscarnet does not require phosphorylation by the VZV thymidine kinase; thus, viruses with mutations in the thymidine kinase gene are still susceptible to inhibition by f ~ s c a r n e t .Foscarnet ~~ inhibits DNA replication by directly inhibiting the viral DNA polymerase; thus, mutations in the DNA polymerase can result in resistance to foscarnet.

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GENETICS Owing to the difficulty in preparing cell-free virus and plaquepurifying VZV in vitro, relatively few VZV mutants have been described. The Oka vaccine strain of VZV was isolated from a healthy child and passaged sequentially in human embryonic lung cells and guinea pig embryo cells.56Although the virus is attenuated, the genetic basis for its attenuation is unknown. Comparison of the growth of the vaccine strain of Oka with its wild-type parent indicates that the ratio between the infectivity of the vaccine strain in guinea pig embryo cells and in human embryo fibroblasts is higher than the corresponding ratio for wild-type virus.**Furthermore, the vaccine strain grows slightly less well at 39°C than wild-type virus. At present, the genotypic changes responsible for these effects are unknown. Two groups have inserted foreign genes into the VZV thymidine kinase gene. The gp350 glycoprotein from Epstein-Barr virus32and the hepatitis B surface antigen47have been inserted into the Oka VZV genome and both of these proteins are expressed in infected cells. Recently, a more versatile system has been developed to produce mutations in the VZV genome. Transfection of cultured cells with four cosmid DNAs that together contain the entire Oka VZV genome results in production of infectious VZV in culture. Site-directed mutation in a given cosmid results in VZV that has a specific mutation in a given gene. Using this procedure, the products of ORFs lo7; 13 (thymidylate synthetase)6;14 (glycoprotein V)5; 19 (ribonucleotide reductase)”; and 47 (a protein kinase)20have been shown to be dispensable for replication in cell culture. In some cases (e.g., ORF19) the phenotype is similar to that seen in HSV when the corresponding gene is deleted; in other cases (e.g., ORF10) the VZV mutant is able to grow in cell culture, whereas the corresponding HSV mutant (VP16) is unable to grow without a complementing cell line. This system has also been used to insert foreign genes (e.g., HSV glycoprotein D) into the VZV genome.21 CONCLUSIONS AND FUTURE AREAS FOR RESEARCH VZV is the etiologic agent of chickenpox and herpes zoster. The viral genome encodes at least 69 unique gene products, all but five of which have homologues with HSV-1. Although the complete nucleotide sequence of the VZV genome has been determined and the functions of many of the viral genes have been identified, there are many questions that remain unanswered. The inability to produce high titers of cell-free virus has limited the ability formally to assign genes to individual kinetic classes in the replicative cycle and to study viral DNA replication. We know little about the mechanism by which VZV establishes latency in the central nervous system and the function of viral genes in this process. Even less is known about the pathways responsible for reactiva-

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tion of virus from latency. Finally, despite the development of an effective live virus vaccine for prevention of chickenpox, we do not understand the basis for attenuation of the vaccine strain. Recent developments in the molecular genetics of VZV as well as more sensitive techniques (e.g., in situ polymerase chain reaction) to study viral genomes and their expression will begin to shed new light on some of these issues.

References -4

1. Almeida JD, Howatson AF, Williams MG: Morphology of varicella (chickenpox) virus. Virology 16:353-355, 1962 2. Boivin G, Edelman CK, Pedneault L, et a1 Phenotypic and genotypic characterization of acyclovir-resistant varicella-zoster viruses isolated from persons with AIDS. J Infect Dis 170:68-75, 1994 3. Chen D, Olivo P D Expression of the varicella-zoster virus origin-binding protein and analysis of its site-specific DNA-binding properties. J Virol 68:3841-3849, 1994 4. Cohen JI, Heffel D, Seidel K: The transcriptional activation domain of varicella-zoster virus open reading frame 62 protein is not conserved with its herpes simplex virus homolog. J Virol 674246-4251, 1993 5. Cohen JI, Seidel KE: Absence of varicella-zoster virus glycoprotein V does not alter growth of VZV in vitro or sensitivity to heparin. J Gen Virol 75:3087-3093, 1994 6. Cohen JI, Seidel KE: Generation of varicella-zoster virus (VZV) and viral mutants from cosmid DNAs: VZV thymidylate synthetase is not essential for replication in vitro. Proc Natl Acad Sci U S A 90:7376-7380, 1993 7. Cohen JI, Seidel KE: Varicella-zoster virus (VZV) open reading frame 10 protein, the homolog of the essential herpes simplex virus protein VP16, is dispensable for VZV replication in vitro. J Virol 68:7850-7858, 1994 8. Cohrs RJ, Barbour MB, Mahalingam R, et a1 Varicella-zoster virus (VZV) transcription during latency in human ganglia: Prevalence of VZV gene 21 transcripts in latently infected human ganglia. J Virol 6996742678, 1995 9. Cook ML, Stevens JG: Replication of varicella-zoster virus in cell culture: An ultrastructural study. J Ultrastructure Res 32334-350, 1970 10. Croen KD, Ostrove JM, Dragovic LJ, et al: Patterns of gene expression and sites of latency in human nerve ganglia are different for varicella-zoster and herpes simplex viruses. Proc Natl Acad Sci U S A 85:9773-9777, 1988 11. Davison AJ, Scott J: The complete DNA sequence of varicella-zoster virus. J Gen Virol 6 71759-1 816, 1986 12. Debrus S, Sadzot-Delvaux C, Nikkels AF, et a1 Varicella-zoster virus gene 63 encodes an immediate-early protein that is abundantly expressed during latency. J Virol 69:3240-3245, 1995 13. Ertl PF, Thomas MS, Powell KL: High level expression of DNA polymerases from herpesviruses. J Gen Virol 721729-1734, 1991 14. Forghani B, Lin N, Grose C: Neutralization epitope of the varicella-zoster virus gH:gL glycoprotein complex. Virology 199:458-462, 1994 15. Garland J: Varicella following exposure to herpes zoster. N Engl J Med 228:336-337, 1943 16. Grose C: Glycoproteins encoded by varicella-zoster virus: Biosynthesis, phosphorylation, and intracellular trafficking. Annu Rev Microbiol 44:59-80, 1990 17. Grose C, Perrotta DM, Brunell PA, Smith GC: Cell-free varicella-zoster virus in cultured human melanoma cells. J Gen Virol 43:15-27, 1979 18. Hayakawa Y, Torigoe S, Shiraki K, et al: Biologic and biophysical markers of a live varicella vaccine strain (Oka): Identification of clinical isolates from vaccine recipients. J Infect Dis 149956-963, 1984

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19. Heineman TC, Cohen JI: Deletion of the varicella-zoster virus large subunit of ribonucleotide reductase impairs the growth of virus in vitro. J Virol 68:3317-3323, 1994 20. Heineman TC, Cohen JI: The varicella-zoster (VZV) open reading frame 47 (ORF47) protein kinase is dispensable for viral replication and is not required for phosphorylation of OW63 protein, the VZV homolog of HSV ICP22. J Virol 69:7367-7370, 1995 21. Heineman TC, Connelly BL, Bourne N, et al: Immunization with recombinant varicellazoster virus expressing herpes simplex virus type 2 glycoprotein D reduces the severity of genital herpes in guinea pigs. J Virol 69:8109-8113, 1995 22. Jackers P, Defechereux P, Baudoux L, et a1 Characterization of regulatory functions of varicella-zoster virus gene 63-encoded protein. J Virol 663899-3903, 1992 23. Keller PM, Davison AJ, Lowe RS, et al: Identification and sequence of the gene encoding gpIII, a major glycoprotein of varicella-zoster virus. Virology 157526-533, 1987 24. Kinchington PR, Bookey D, Turse SE: The transcriptional regulatory proteins encoded by varicella zoster virus open reading frames (ORFs) 4 and 63, but not ORF61 are associated with purified virus particles. J Virol 69:42744282, 1995 25. Kinchington PR, Hougland JK, Arvin AM et al: The varicella-zoster virus immediateearly protein IE62 is a major component of virus particles. J Virol 66:359-366, 1992 26. Kinchington PR, Inchauspe G, Subak-Sharpe JH, et al: Identification and characterization of a varicella-zoster virus DNA-binding protein by using antisera directed against a predicted synthetic oligonucleotide. J Virol 62502-809, 1988 27. Kinchington PR, Ling P, Pensiero M, et al: The glycoprotein products of varicellazoster virus gene 14 and their defective accumulation in a vaccine strain (Oka). J Virol 64:4540-4548, 1990 28. Kinchington PR, Remenick J, Ostrove JM, et a1 Putative glycoprotein gene of varicellazoster virus with variable copy numbers of a 42-base-pair repeat sequence has homology to herpes simplex virus glycoprotein C. J Virol 59:660-668, 1986 29. Kost RG, Kupinsky H, Straus SE: Varicella zoster virus gene 63: Transcript mapping and regulatory activity. Virology 209:218-224, 1995 30. Kundratitz K Uber die atiologie des zoster and uber seine beiziehungen zu varizellen. Wien Klin Wochenschr 38:502-503, 1925 31. LaRussa P, Lungu 0, Hardy I, et al: Restriction fragment length polymorphism of polymerase chain reaction products from vaccine and wild-type varicella-zoster virus isolates. J Virol 66:1016-1020, 1992 32. Lowe RS, Keller PM, Keech BJ, et al: Varicella-zoster virus as a live vector for the expression of foreign genes. Proc Natl Acad Sci U S A 84:389&3900, 1987 33. Meier JL, Holman RP, Croen KD, et al: Varicella-zoster virus transcription in human trigeminal ganglia. Virology 193:193-200, 1993 34. Meier JL, Luo X, Sawadogo M, Straus SE: The cellular transcription factor USF cooperates with varicella-zoster virus immediate-early protein 62 to symmetrically activate a bidirectional viral promoter. Mol Cell Biol 146896-6906, 1994 35. Moriuchi H, Moriuchi M, Smith HA, et al: Varicella-zoster open reading frame 61 protein is functionally homologous to herpes simplex virus type 1 ICPO. J Virol 66:7303-7308, 1992 36. Moriuchi H, Moriuchi M, Smith HA, et a1 Varicella-zoster open reading frame 4 protein is functionally distinct from and does not complement its herpes simplex virus type 1 homolog, ICP27. J Virol 68:1987-1992, 1994 37. Moriuchi H, Moriuchi M, Straus SE, Cohen JI: Varicella-zoster virus open reading frame 10 protein, the herpes simplex virus VP16 homolog, transactivates herpesvirus immediate-early gene promoters. J Virol 672739-2746, 1993 38. Netter A, Urbain A: Les relations du zona et de la varicelle. Etude serologique de 100 cas de zona. C R SOCBiol (Paris) 94:98-102, 1926 39. Ng TI, Grose C: Serine protein kinase associated with varicella-zoster virus ORF 47. Virology 191:9-18, 1992 40. Ng TI, Keenan L, Kinchington PR, Grose C: Phosphorylation of varicella-zoster virus open reading frame (OW) 62 regulatory product by viral ORF47-associated protein kinase. J Virol 68:1350-1359, 1994 41. Perera LP, Kaushal S, Kinchington PR, et al: Varicella-zoster open reading frame 4

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encodes a transcriptional activator that is functionally distinct from that of herpes simplex virus homolog ICP27. J Virol 682468-2477, 1994 42. Perera LP, Mosca JD, Ruyechan WT, et al: A major transactivator of varicella-zoster virus, the immediate-early protein IE62, contains a potent N-terminal activation domain. J Virol 674744483, 1993 43. Perera LP, Mosca JD, Ruyechan WT, et al: Regulation of varicella-zoster virus gene expression in human T lymphocytes. J Virol 66:5298-5304, 1992 44. Rodriquez JE, Moninger T, Grose C: Entry and egress of varicella-zoster virus blocked by same anti-gH monoclonal antibody. Virology 196:840-844, 1993 45. Safrin S, Berger TG, Gilson I, et al: Foscamet therapy in five patients with AIDS and acyclovir-resistant varicella-zoster virus infection. Ann Intern Med 11519-21, 1991 46. Sawyer MH, Ostrove JM, Felser JM, Straus SE: Mapping of the varicella-zoster virus deoxypyrimidine kinase gene and preliminary identification of its transcript. Virology 149:1-9, 1986 47. Shiraki K, Hayakawa Y, Mori H, et al: Development of immunogenic recombinant Oka varicella vaccine expressing hepatitis B surface antigen. J Gen Wrol 72:139>1399, 1991 48. Shiraki K, Hyman RW: The immediate early proteins of varicella-zoster virus. Virology 156:423426, 1987 49. Shiraki K, Takahashi M: Virus particles and glycoproteins excreted from cultured cells infected with varicella-zoster virus. J Gen Virol 61:271-275, 1982 50. Spear PG: Entry of alphaherpesviruses into cells. Semin Virol 4167-180, 1993 51. Steiner I? Zur inokulation der varicellen. Wein Med Wochenschr 25:306, 1875 52. Stevenson D, Colman KL, Davison AJ: Characterization of the varicella-zoster virus gene 61 protein. J Gen Virol 73:521-530, 1992 53. Stevenson D, Colman KL, Davison AJ: Characterization of the putative protein kinases specified by varicella-zoster virus genes 47 and 66. J Gen Virol 75:317-326, 1994 54. Straus SE, Ostrove JM, Inchauspe G, et al: Varicella-zoster virus infections. Biology, natural history, treatment, and prevention. Ann Intem Med 108:221-227, 1988 55. Straus SE, Reinhold W, Smith HA, et al: Endonuclease analysis of viral DNA from varicella and subsequent zoster infections in the same patient. N Engl J Med 311:13621364, 1984 56. Takahashi M, Okuno Y, Otsuka T, et a1 Development of a live attenuated varicella vaccine. Biken J 18:25-33, 1975 57. Tyler JK, Everett RD: The DNA binding domain of the varicella-zoster virus gene 62 protein interacts with multiple sequences which are similar to the binding site of the related protein of herpes simplex virus type 1. Nucleic Acids Res 21:513-522, 1993 58. Vafai A, Wroblewska Z, Graf L: Antigenic cross-reaction between a varicella-zoster virus nucleocapsid protein encoded by gene 40 and a herpes simplex virus nucleocapsid protein. Virus Res 15163-174, 1990 59. von Bokay J: Das auftreten der schafblattern uter besonderen umstanden. Unger Arch Med 1:159, 1892 60. Weller TH, Stoddard MB: Intranuclear inclusion bodies in cultures of human tissue inoculated with varicella vesicle fluid. J Immunol 68:311-319, 1952 61. Weller TH, Witton HM, Bell EJ: The etiologic agents of varicella and herpes zoster: Isolation, propagation, and cultural characteristics in vitro. J Exp Med 108:843-868, 1958 62. Yao Z, Jackson W, Forghani 8, Grose C: Varicella-zoster virus glycoprotein gpI/gpIV receptor: expression, complex formation, and antigenicity within the vaccinia virus-T7 RNA polymerase transfection system. J Virol 67:305-314, 1993 63. Zhu 2, Gershon MD, Ambron R, et a1 Infection of cells by varicella-zoster virus: Inhibition of viral entry by mannose 6-phosphate and heparin. Proc Natl Acad Sci U S A 923546-3550, 1995

Address reprint requests to: Jeffrey 1. Cohen, MD Building 10, Room llN214 Laboratory of Clinical Investigation National Institutes of Health Bethesda, MD 20892