Human herpesvirus 6 (HHV-6) causes severe thymocyte depletion in SCID-hu Thy/Liv mice

Abstract

Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive agent that may act as a cofactor in the progression of AIDS. Here, we describe the first small animal model of HHV-6 infection. HHV-6 subgroup A, strain GS, efficiently infected the human thymic tissue implanted in SCID-hu Thy/Liv mice, leading to the destruction of the graft. Viral DNA was detected in Thy/Liv implants by quantitative polymerase chain reaction (PCR) as early as 4 d after inoculation and peaked at day 14. The productive nature of the infection was confirmed by electron microscopy and immunohistochemical staining. Atypical thymocytes with prominent nuclear inclusions were detected by histopathology. HHV-6 replication was associated with severe, progressive thymocyte depletion involving all major cellular subsets. However, intrathymic T progenitor cells (ITTPs) appeared to be more severely depleted than the other subpopulations, and a preferred tropism of HHV-6 for ITTPs was demonstrated by quantitative PCR on purified thymocyte subsets. These findings suggest that thymocyte depletion by HHV-6 may be due to infection and destruction of these immature T cell precursors. Similar results were obtained with strain PL-1, a primary isolate belonging to subgroup B. The severity of the lesions observed in this animal model underscores the possibility that HHV-6 may indeed be immunosuppressive in humans.

Figure 1

Replication of HHV-6A_GS in Thy/Liv implants. Implants were harvested 4, 7, 11, 14, and 27 d after direct intrathymic injection with HHV-6A_GS or mock injection of UV-inactivated viral stock (n = 9) or culture medium (n = 12) (Mock). Data obtained at day 4, 7, and 11 were pooled. The amount of HHV-6 DNA and cell yield per implant are shown as means ± SEM. The numbers in parentheses indicate the number of mice analyzed at each of the indicated time points. Results include three independent experiments with six different cohorts of SCID-hu Thy/Liv mice. *P < 0.01, ^‡ P < 0.001 vs. mock; ^§ P < 0.05 vs. days 4–11, and P < 0.001 vs. day 27.

Figure 2

Transmission electron microscopy of a Thy/Liv implant harvested 11 d after inoculation with HHV-6A_GS. Naked viral nucleocapsids are visible in the nucleus, and two mature virions are visible in a cytoplasmic vacuole (insert). Original magnifications: ×10,000; inset, ×40,000.

Figure 3

Histopathology of HHV-6A_GS–infected Thy/Liv implants. (A) Severe thymocyte depletion associated with proliferation of granulation tissue in an implant harvested 14 d after inoculation with HHV-6A_GS (H&E; original magnification: ×40). (B) Typical appearance of a mock-infected Thy/Liv implant (H&E; original magnification: ×40). (C) Cytopathic effects induced by HHV-6A_GS. Atypical giant cells with prominent nuclear viral inclusions (Cowdry type A) (H&E; original magnification: ×1,000). (D) Immunohistochemical staining with 9A5D12, an mAb specific for the p41 early protein of HHV-6. Infected cells are scattered throughout the implant and include several large atypical cells (original magnification: ×100). (E) Double staining with CD3 (DAB) and 9A5D12 (Vector Red). All the cells that are positive for HHV-6 also coexpress CD3 (original magnification: ×1,000). (F) Double staining with an mAb to the macrophage marker CD68 (DAB) and to the p41 early protein of HHV-6 (9A5D12, Vector Red) (original magnification: ×1,000).