Varicella-zoster virus neurotropism in SCID mouse-human dorsal root ganglia xenografts.

Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus and the causative agent of varicella and herpes zoster. VZV reactivation from latency in sensory nerve ganglia is a direct consequence of VZV neurotropism. Investigation of VZV neuropathogenesis by infection of human dorsal root ganglion xenografts in immunocompromised (SCID) mice has provided a novel system in which to examine VZV neurotropism. Experimental infection with recombinant VZV mutants with targeted deletions or mutations of specific genes or regulatory elements provides an opportunity to assess gene candidates that may mediate neurotropism and neurovirulence. The SCID mouse-human DRG xenograft model may aid in the development of clinical strategies in the management of herpes zoster as well as in the development of "second generation" neuroattenuated vaccines.

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.

Granulysin blocks replication of varicella-zoster virus and triggers apoptosis of infected cells.

Granulysin, a lytic protein present in cytolytic granules of human natural killer and cytotoxic T cells, entered cells infected with varicella-zoster virus (VZV). Exposure to granulysin accelerated death of infected cells as assessed by apoptosis markers. The functional domain of granulysin that mediated its antiviral effects was amino acid 23-51; this domain also mediates the additional antitumor cell effects of granulysin. Because granulysin is a product of natural killer cells and T lymphocytes, it is possible that its antiviral activity may act as a mediator of innate and adaptive immune mechanisms.

Varicella-zoster virus infection of a human CD4-positive T-cell line.

Varicella-zoster virus (VZV) is a human alpha-herpesvirus that causes varicella (chickenpox) at primary infection and may reactivate as herpes zoster. VZV is a T-lymphotropic virus in vivo. To investigate the T-cell tropism of VZV, we constructed a recombinant virus expressing green fluorescent protein (VZV-GFP) under the CMV IE promoter. Coculture of VZV-GFP-infected fibroblasts with II-23 cells, a CD4-positive human T-cell hybridoma, resulted in transfer of virus to II-23 cells. II-23 cells are susceptible to VZV-GFP infection as demonstrated by expression of immediate/early (IE62), early (ORF4), and late (gE) genes. Recovery of infectious virus was limited, with only 1 to 3 in 10(6) cells releasing infectious virus by plaque assay, indicating that transfer of virus results in a limited productive infection. In vitro infection of II-23 cells will be useful for further analysis of VZV tropism for T-lymphocytes.

The ORF47 and ORF66 putative protein kinases of varicella-zoster virus determine tropism for human T cells and skin in the SCID-hu mouse.

The varicella-zoster virus (VZV) genes ORF47 and ORF66 are predicted to encode serine/threonine protein kinases, which are homologs of herpes simplex virus 1 (HSV-1) UL13, and US3. When mutants were constructed by inserting stop codons into ORF47 and ORF66, the recombinants ROka47S and ROka66S, as well as intact ROka replicated in tissue culture. In contrast, inoculation of human thymus/liver or skin implants in SCID-hu mice showed that ORF47 protein was required for viral growth in human T cells and skin. Eliminating ORF66 expression inhibited VZV infectivity for T cells partially but did not impair replication in skin compared with ROka. Infectivity for T cells and skin was restored when ROka47S virus was complemented by insertion of ORF47 into a distant, noncoding site. The ORF47 gene product is the first VZV protein identified as necessary for T cell tropism. It also is essential for skin infectivity in vivo, as is glycoprotein C. Expression of ORF66 did not compensate for the absence of the ORF47 protein. The requirement for ORF47 expression in T cells and skin indicates that this gene product, which is dispensable in vitro, has a critical role within differentiated cells that are essential targets for VZV pathogenesis in vivo.

Analysis of the persistence of humoral and cellular immunity in children and adults immunized with varicella vaccine.

The persistence of humoral and cellular immunity to varicella-zoster virus (VZV) was evaluated in 60 children and 18 adults immunized with live attenuated VZV vaccine. At a mean of 5 years after vaccination, 93% of children and 94% of adults had IgG antibodies to VZV as determined by ELISA. VZV antibody concentrations were significantly higher at 5 years than at 1 year after immunization in children and adults. Cell-mediated immunity to VZV was detected in 87% of children and 94% of adults at 5 years. The mean stimulation index was significantly higher at 5 years than at 1 year among children and adults. Cytokine responses to VZV, including interleukin-2, interferon-gamma, and interleukin-10 were equivalent between children and adults at 5 years. In summary, varicella immunization induced long-term humoral and cellular immunity, and initial differences between cell-mediated responses in children and adults diminished over time.

Attenuation of the vaccine Oka strain of varicella-zoster virus and role of glycoprotein C in alphaherpesvirus virulence demonstrated in the SCID-hu mouse.

The SCID-hu mouse implanted with human fetal tissue is a novel model for investigating human viral pathogenesis. Infection of human skin implants was used to investigate the basis for the clinical attenuation of the varicella-zoster virus (VZV) strain, V-Oka, from which the newly licensed vaccine is made. The pathogenicity of V-Oka was compared with that of its parent, P-Oka, another low-passage clinical isolate, strain Schenke (VZV-S), and VZV-Ellen, a standard laboratory strain. The role of glycoprotein C (gC) in infectivity for human skin was assessed by using gC-negative mutants of V-Oka and VZV-Ellen. Whereas all of these VZV strains replicated well in tissue culture, only low-passage clinical isolates were fully virulent in skin, as shown by infectious virus yields and analysis of implant tissues for VZV DNA and viral protein synthesis. The infectivity of V-Oka in skin was impaired compared to that of P-Oka, providing the first evidence of a virologic basis for the clinical attenuation of V-Oka. The infectivity of V-Oka was further diminished in the absence of gC expression. All strains except gC-Ellen retained some capacity to replicate in human skin, but cell-free virus was recovered only from implants infected with P-Oka or VZV-S. Although VZV is closely related to herpes simplex virus type 1 (HSV-1) genetically, experiments in the SCID-hu model revealed differences in tropism for human cells that correlated with differences in VZV and HSV-1 disease. VZV caused extensive infection of epidermal and dermal skin cells, while HSV-1 produced small, superficial lesions restricted to the epidermis. As in VZV, gC expression was a determinant for viral replication in skin. VZV infects human CD4+ and CD8+ T cells in thymus/liver implants, but HSV-1 was detected only in epithelial cells, with no evidence of lymphotropism. These SCID-hu mouse experiments show that the clinical attenuation of the varicella vaccine can be attributed to decreased replication of V-Oka in skin and that tissue culture passage alone reduces the ability of VZV to infect human skin in vivo. Furthermore, gC, which is dispensable for replication in tissue culture, plays a critical role in the virulence of the human alphaherpesviruses VZV and HSV-1 for human skin.

Reliability of a history of previous varicella infection in adults.

CONTEXT: Apparent second episodes of varicella are reported in immunocompetent hosts, but laboratory confirmation of prior immune status has rarely been possible. OBJECTIVE: To evaluate adult patients with varicella who claimed to have had previous varicella to determine whether they had true second episodes or primary cases with inaccurate clinical histories. DESIGN: Adult subjects with varicella who enrolled in an antiviral treatment trial were interviewed about a history of varicella. The clinical course of varicella was documented prospectively in all subjects. Serum samples that predated the acute illness were obtained from the US Navy's central serum storage facility for subjects who reported a previous episode of varicella. These stored samples were tested in parallel by enzyme-linked immunosorbent assay, latex agglutination, and Western blot for IgG antibodies to varicella-zoster virus (VZV). PARTICIPANTS: Twenty military personnel with varicella and a history of the disease. SETTING: A military hospital in San Diego, Calif. MAIN OUTCOME MEASURE: Presence or absence of antibodies to VZV. RESULTS: Twenty (10.8%) of 184 adults with serologically confirmed acute varicella reported a prior history of varicella. The clinical course of these 20 patients did not differ from those with no history of varicella. Serum samples that had been collected a mean of 12.4 months (median, 12 months; range, 3 days to 34 months) before the incident episode were available for 19 subjects. All 19 serum samples lacked IgG antibodies to VZV. CONCLUSION: A history of previous varicella infection in adults with varicella may not be reliable. True second episodes of varicella are probably rare in immunocompetent adults.

Early reconstitution of immunity and decreased severity of herpes zoster in bone marrow transplant recipients immunized with inactivated varicella vaccine.

Varicella-zoster virus (VZV) causes herpes zoster after bone marrow transplantation (BMT). The immunogenicity of heat-inactivated varicella vaccine and effects on VZV pathogenesis were evaluated in 75 BMT patients randomized to receive vaccine or no intervention. Among 14 patients given a single dose at 1 month after transplantation, the mean (+/-SE) stimulation index (SI) was 12.20 +/- 3.13 compared with 4.83 +/- 2.74 (P = .036) in 14 unvaccinated patients, but clinical disease was not altered. Among 24 patients vaccinated at 1, 2, and 3 months, mean SI was 8.43 +/- 3.89 versus 2.00 +/- 0.33 (P = .014) in 23 unvaccinated patients at 4 months and 8.56 +/- 2.81 versus 5.30 +/- 2.47 (P = .043) at 5 months. Disease severity associated with VZV reactivation was decreased dramatically in vaccinees given three doses; severity scores were 6.4 +/- 1.0 versus 11.8 +/- 1.1 (P = .007). This experience with varicella vaccine in BMT patients is the first evidence that active immunization can reduce morbidity due to herpesvirus reactivation in high-risk populations.