Infection of human T lymphocytes with varicella-zoster virus: an analysis with viral mutants and clinical isolates

Abstract

Varicella-zoster virus (VZV) disseminates in the body in peripheral blood mononuclear cells during chickenpox. Up to 1 in 10,000 mononuclear cells are infected during the viremic phase of the disease. We developed an in vitro system to infect human mononuclear cells with VZV by using umbilical cord blood. In this system, 3 to 4% of T cells were infected with VZV. VZV mutants unable to express certain genes, such as open reading frame 47 (ORF47) or ORF66, were impaired for growth in T cells, while other mutants showed little difference from parental virus. VZV unable to express ORF47 was even more impaired for spread from umbilical cord blood cells to melanoma cells in vitro. Early-passage clinical isolates of VZV infected T cells at a similar rate to the Oka vaccine strain; however, the clinical isolates were more efficient in spreading from infected T cells to melanoma cells. This in vitro system for infecting human T cells with VZV should be useful for identifying cellular and viral proteins that are important for virus replication in T cells and for the spread of virus from T cells to other cells.

FIG. 1

Immunofluorescence microscopy of umbilical cord mononuclear cells infected with ROka and ROka47S VZV. (A) Mononuclear cells were infected with ROka (top panels) and ROka47S (bottom panels) and then incubated with murine antibody to VZV gE followed by rhodamine-labeled anti-mouse antibody and then fluorescein isothiocyanate-labeled anti-human CD3 antibody. Panels (left to right) show cells staining for CD3 (green), VZV gE (red), and both CD3 and VZV gE (yellow). (B) Mononuclear cells infected with VZV ROka (top panel) and ROka47S (bottom panel) were incubated with murine antibody to VZV ORF62 protein followed by fluorescein isothiocyanate-labeled anti-mouse antibody.

FIG. 2

Flow cytometry of umbilical cord blood T cells infected with VZV ROka, ROka47S, ROka47SR, ROka66S, and ROka32D. The cells were incubated with anti-VZV gE antibody (x axis) and anti-human CD3 (y axis). The percentage of VZV-infected T (CD3) cells is shown in the upper right quadrant, while the percentage of virus-infected non-T cells is shown in the lower right quadrant. Results from three independent experiments (A, B, and C) are shown.

FIG. 3

Percentage of human T cells infected with VZV mutants determined by flow cytometry. Cells infected with VZV mutants were stained with antibodies as described in the legend to Fig. 2, and the median percentage of VZV-infected T cells was determined. Each median value was derived from at least five independent experiments with samples from different cord blood donors, except VZV ROka1S, which was determined from three experiments.

FIG. 4

The infectious-focus assay detects transfer of VZV mutants from umbilical cord mononuclear cells to melanoma cells. VZV-infected human mononuclear cells were incubated with melanoma cells, and 7 to 8 days later the melanoma cells were fixed and stained with crystal violet. Results from two separate experiments (A and B) are shown.

FIG. 5

Growth of Oka vaccine virus and early-passage varicella isolates in melanoma cells. Melanoma cells were inoculated with VZV-infected cells, aliquots were harvested on days 1, 2, 3, and 4 after infection, and the titer of virus was determined by plating on melanoma cells. Day 0 indicates the titer of virus in the inocula. The titer (log₁₀ of the mean number of PFU per dish) is indicated on the y axis.

FIG. 6

Percentage of human T cells infected with VZV ROka and early-passage clinical isolates of VZV. The infected cells were incubated with anti-VZV and anti-human CD3 antibodies (as described in the legend to Fig. 2), and the median percentage of VZV-infected T (CD3) cells was determined. Each median value was derived from at least five independent experiments with samples from different cord blood donors, except VZV Emily, which was determined from three experiments.

FIG. 7

The infectious-focus assay detects increased transfer of infectious virus from human mononuclear cells infected with early-passage clinical isolates (VZV Molly and Emily) compared with VZV ROka. Results from two separate experiments (A and B) are shown.