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Experimental rhesus lymphocryptovirus infection in immunosuppressed macaques: an animal model for Epstein-Barr virus pathogenesis in the immunosuppressed host

Pierre Rivailler, Angela Carville, Amitinder Kaur, Pasupuleti Rao, Carol Quink, Jeffery L. Kutok, Susan Westmoreland, Sherry Klumpp, Meredith Simon, Jon C. Aster and Fred Wang

Article Figures & Data

Figures

  • Figure 1.

    CD4+ T-cell counts and SHIV viral loads in 2 representative animals in group II. CD4+ T-cell counts (circles) and SHIV viral loads (triangles) from Mm255-96 (filled symbols) and Mm343-96 (open symbols) are shown.

  • Figure 2.

    Serum antibody responses to the rhesus LCV sVCA. (A) Animals in group I: Mm141-97 (♦), Mm144-97 (•), Mm211-98 (▪), Mm309-98(▴). (B) Animals in group II: Mm343-96 (♦), Mm165-97 (•), Mm255-96 (▪), Mm166-97 (▴). (C) Animals in group III: Mm260-98 (♦), Mm256-96 (•), Mm192-98 (▪), Mm321-98 (▴).

  • Figure 3.

    Rhesus LCV DNA viral loads in peripheral blood mononuclear cells after experimental infection. (A) Animals in group I: Mm144-97 (▪), Mm141-97 (▴) (B) Animals in group II: Mm165-97 (•), Mm255-96 (▪), Mm166-97 (▴).

  • Figure 4.

    Tissue invasion of rhesus LCV-positive cells in Mm321-98. (A) Hematoxylin and eosin–stained section of a lymph node biopsy performed on day 36 after intravenous inoculation of autologous rhesus LCV-immortalized B cells. Original magnification × 100. (B) EBERs in situ hybridization (original magnification × 200) and (C) EBNA-2 immunohistochemistry on sections of the same lymph node biopsy (original magnification × 200). (D) Hematoxylin and eosin–stained section from the spleen obtained at necropsy on day 57 (original magnification × 100). (E) EBERs in situ hybridization (original magnification × 20) and (F) EBNA-2 immunohistochemistry on sections from the spleen (original magnification × 100).

  • Figure 5.

    Rhesus LCV-positive B-cell lymphoma from Mm256-96. (A-B) Hematoxylin and eosin–stained section of submandibular tumor mass from necropsy on day 155 after intravenous inoculation of autologous rhesus LCV-immortalized B cells. Original magnification × 40 (A) and × 200 (B). (C-G) Immunohistochemical staining of tumor section for CD20 (C), CD3 (D), bcl-2 (E), Ki-67 (F), and EBNA-2 (G). Original magnification × 200. (H) In situ hybridization of tumor section for EBER expression. Original magnification × 200. (I-J) Fluorescent in situ hybridization with a rhesus LCV DNA probe on touch preps prepared from the (I) tumor section or (J) EBV-negative BJAB B lymphoma cells. Original magnification × 400.

  • Figure 6.

    Southern blot analysis for viral clonality. Genomic DNA from the rhesus LCV-producing B-cell line LCL8664, Mm256-96 tumor tissue, a cell line derived from the Mm256-96 tumor tissue, and the autologous, rhesus LCV–infected cell line used for intravenous inoculation were separated on a 0.6% agarose gel, transferred to a nylon membrane, and hybridized with a rhesus LCV DNA probe for the terminal repeats. Molecular weight markers in kilobases are shown to the right.

  • Figure 7.

    RT-PCR analysis for rhesus LCV latent infection gene expression in Mm256-96 tumor tissue. RT-PCR was performed on RNA extracted from the Mm256-96 tumor tissue with the use of primers specific for the mRNAs of rhesus LCV latent membrane protein 1 (LMP1), latent membrane protein 2A (LMP2A), and mRNAs spliced from the terminal exon of EBNA-LP to the coding region for EBNA-1,-2, -3A, -3B, and -3C. Control reactions consisting of all reagents except reverse transcriptase (-RT) or with water in place of RNA (W) are included. Molecular weight markers are shown in the leftmost column, with the brightest band representing 500 bp and minor bands at increments of 100 bp.

Tables

  • Table 1.

    Summary of rhesus LCV infections

    Animal no.SHIV infectionCD4+ T-cell nadir, cells/μLRhesus LCV infectionLCV seroconversionDeceasedDays after LCV infectionComments
    Group I
        211-98 None ND Oral Yes Yes 415 Asymptomatic, killed for +PPD
        309-98 None ND Oral Yes No 827 Alive and well
        141-97 None ND Oral Yes No 1177 Alive and well
        144-97 None ND Oral Yes No 1177 Alive and well
    Group II
        255-96 89.6P 10 Oral No Yes 122 PCP, calcinosis
        166-97 89.6P 3 Oral No yes 138 Lymphocytic pneumonia
        343-96 89.6P 35 Oral NA Yes 415 PCP, failure to thrive
        165-97 89.6P 11 Oral No Yes 511 Failure to thrive
    Group IIIa
        192-98 89.6P 67 B cells Yes No 700 Alive, asymptomatic
        260-98 89.6P 121 B cells Yes Yes 799 Failure to thrive
    Group IIIb
        321-98 89.6P 10 B cells No Yes 57 Tissue invasion, Staphylococcus sepsis
        256-96 89.6P 4 B cells No Yes 155 LCV+ tumor
    • ND indicates not done; PPD, purified protein derivative (of tuberculin); PCP, Pneumocystis carinii pneumonia; and NA, not applicable.