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. 2006 Jul;116(7):1963-73.
doi: 10.1172/JCI27249. Epub 2006 Jun 22.

KSHV targets multiple leukocyte lineages during long-term productive infection in NOD/SCID mice

Christopher H Parsons  1 , Laura A Adang, Jon Overdevest, Christine M O'Connor, J Robert Taylor Jr, David Camerini, Dean H Kedes
Affiliations

KSHV targets multiple leukocyte lineages during long-term productive infection in NOD/SCID mice

Christopher H Parsons et al. J Clin Invest. 2006 Jul.

Abstract

To develop an animal model of Kaposi sarcoma-associated herpesvirus (KSHV) infection uniquely suited to evaluate longitudinal patterns of viral gene expression, cell tropism, and immune responses, we injected NOD/SCID mice intravenously with purified virus and measured latent and lytic viral transcripts in distal organs over the subsequent 4 months. We observed sequential escalation of first latent and then lytic KSHV gene expression coupled with electron micrographic evidence of virion production within the murine spleen. Using novel technology that integrates flow cytometry with immunofluorescence microscopy, we found that the virus establishes infection in murine B cells, macrophages, NK cells, and, to a lesser extent, dendritic cells. To investigate the potential for human KSHV-specific immune responses within this immunocompromised host, we implanted NOD/SCID mice with functional human hematopoietic tissue grafts (NOD/SCID-hu mice) and observed that a subset of animals produced human KSHV-specific antibodies. Furthermore, treatment of these chimeric mice with ganciclovir at the time of inoculation led to prolonged but reversible suppression of KSHV DNA and RNA levels, suggesting that KSHV can establish latent infection in vivo despite ongoing suppression of lytic replication.

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Figures

Figure 1
Figure 1. Sequential increases in KSHV genomic DNA, latent (ORF73) and lytic (ORF50 and ORF65) transcripts within the spleens of KSHV-injected NOD/SCID mice.
Mice were administered 3 weekly doses of KSHV (squares, solid lines) or UV-KSHV (triangles, dashed lines) intravenously and euthanized 1 day (DNA only) and 1, 2, and 4 months following the third injection. (A) Genomic KSHV DNA values were determined using qPCR to calculate the ΔCt, representing KSHV Ct normalized to mouse GAPDH Ct (mean of triplicate determinations for each) for each sample. (B–D) Total splenic RNA was subjected to qRT-PCR using primers specific for ORFs 73 (B), 50 (C), and 65 (D) as well as mouse GAPDH. ΔδCt = (ΔCt(G)) – (ΔCt(K)), where ΔCt(K) and ΔCt(G) are the differences between the Ct of each sample (mean of duplicate determinations) without and with reverse transcriptase (RT) for KSHV (K) and GAPDH (G), respectively. Each symbol represents the mean and SE of ΔCt (A) or ΔδCt (B–D). Numbers of mice are indicated in parentheses. Graph-wide dotted lines mark the lower limit of sensitivity of each assay. Of note, UV-KSHV did not establish infection within either primary dermal microvascular endothelial cells (pDMVECs) or HeLa cells in vitro (not shown).
Figure 2
Figure 2. KSHV latent and lytic protein expression escalated over time following intravenous injection of NOD/SCID mice.
NOD/SCID spleens were collected 1 month (A and B) and 4 months (C and D) after intravenous injection with PBS (A), KSHV (B and C), and UV-KSHV (D). Spleen sections were incubated with DAPI (blue) and mAbs to either LANA (A–D, large panels) or K8.1 (C and D, insets) and the images merged to assess colocalization. Solely to emphasize the qualitative aspects of the LANA staining pattern, we chose C from a mouse spleen with one of the highest overall infection rates (3%). Staining characteristic of cytoplasmic or cell-surface localization of K8.1 was also evident within rare cells (approximately 0.01%) in sections from KSHV-injected mice (C, inset, arrows) while no staining over background staining was observed within sections from UV-KSHV–injected mice (D, inset). Original magnification, ×40 (large panels); ×20 (insets).
Figure 3
Figure 3. Thin section EM of NOD/SCID-hu spleens demonstrated virion production 3 months after infection with KSHV.
(A) Angular C capsid (arrow) within cytoplasmic vesicle. (B) A capsids. (C) Cytoplasmic virion. (D) Cytoplasmic virion likely sectioned asymmetrically, allowing good visualization of angular capsid. (E) Virion within cytoplasmic vesicle likely during egress toward plasma membrane (pm). (F) Released virion in extracellular space(es). (G–I) Identical to images D–F, respectively, but with an additional ×2.5 magnification to reveal subviral structural components. t, tegument; c, capsid; gp, glycoproteins; e, envelope; v, vesicle; d, viral DNA. Original magnification, ×28,000. Scale bars, 0.2 μm.
Figure 4
Figure 4. Identification of murine leukocyte subpopulations within NOD/SCID spleens.
Splenocyte suspensions from KSHV-infected NOD/SCID mice were incubated with antigen-specific monoclonal antibodies (large panels) or their respective isotype controls (small panels). (A–F) Approximately 10,000 total live cells were evaluated for the coexpression of B220, Ly49, CD11b, and CD11c for all 2-way combinations. (G and H) In separate experiments gating only on the lymphocyte population, coexpression was observed for Ly49 and IL-2Rβ (G), but not Ly49 and CD3 (H).
Figure 5
Figure 5. Distinct NOD/SCID spleen cell populations were latently infected with KSHV.
(A) For cells from mice injected with KSHV, intranuclear LANA staining was noted in the B220+, Ly49+, CD11b+, and CD11c+ but not CD3+ or CD117+ populations. (B) Analogous populations of cells from mice receiving UV-KSHV revealed no specific staining suggestive of LANA. (C) The proportion of LANA+ cells within the positive populations in A. (D) Four populations (see also text and Table 1 for details) accounted for nearly all LANA+ cells. (E and F) BF imaging and specific nuclear staining with DRAQ5 (NUC) allowed determinations of area (E) and N/C ratios (F) for individual cells. P < 0.001 for mean size comparisons between small-sized (~115 μm2; CD3+ and B220+) and intermediate-sized (~130 μm2; Ly49+, CD11b+, and CD11c+) cells and between intermediate-sized and large-sized (~155 μm2; CD117+) cells. P < 0.001 for N/C comparisons between populations with low (~0.31; Ly49+ and CD11b+) and high (~0.33-0.35; B220+, CD3+, CD11c+, and CD117+) N/C ratios. P values were calculated as outlined in Methods. (G) Relative proportions of different cell types are similar in mice 4 months after injection with either KSHV (white bars) or UV-KSHV (black bars). SM, surface marker.
Figure 6
Figure 6. KSHV-specific human antibody production in NOD/SCID-hu mice inoculated with KSHV.
(A) Compared with unimplanted mice (U), sera from NOD/SCID mice receiving human tissue implants (I) contained detectable amounts of total human IgG. (B–E). Serum (1:80) from an implanted and KSHV-injected mouse probed with either anti-human (B) or anti-mouse (C) secondary antibodies. Serum from a human KSHV–infected patient (1:640) (D) or rabbit polyclonal anti-LANA antiserum (1:40) (E) followed by anti-human or anti-rabbit secondary antibodies conjugated to Texas Red, respectively. Original magnification, ×60.
Figure 7
Figure 7. Increases in KSHV genomic DNA in NOD/SCID-hu mouse spleens were inhibited temporally and quantitatively by pretreatment with GCV.
Mice received daily (days –24 to +1) intraperitoneal administration of either GCV (diamonds, dotted line) or PBS (squares, solid line) during the 3-week KSHV infection period. A third group of mice received UV-KSHV (triangles, dashed line). (A) Mean ΔCt values of genomic KSHV DNA were calculated as in Figure 1. *Data from 1-month GCV-treated mice were not collected, indicated by the discontinuous line. (B–D). Mean ΔδCt values representing KSHV RNA determinations for ORFs 73 (B), 50 (C), and 65 (D) were calculated as in Figure 1. Each symbol represents the mean and SE of ΔCt (A) or ΔδCt (B–D). Numbers of mice and the lower limit of sensitivity of the assays are indicated as in Figure 1.
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References

    1. Okano M., Gross T.G. A review of Epstein-Barr virus infection in patients with immunodeficiency disorders. Am. J. Med. Sci. 2000;319:392–396. - PubMed
    1. Boshoff C., Chang Y. Kaposi’s sarcoma-associated herpesvirus: a new DNA tumor virus. Annu. Rev. Med. 2001;52:453–470. - PubMed
    1. Gandhi M.K., Khanna R. Human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments. Lancet Infect. Dis. 2004;4:725–738. - PubMed
    1. Cesarman E., Chang Y., Moore P.S., Said J.W., Knowles D.M. Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N. Engl. J. Med. 1995;332:1186–1191. - PubMed
    1. Cesarman E., Knowles D.M. The role of Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) in lymphoproliferative diseases. Semin. Cancer Biol. 1999;9:165–174. - PubMed

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