Kedes, unpublished observations). To more precisely distinguish NOD/SCID splenocyte subpopulations and determine the specific cellular targets of KSHV contamination, we employed MIFC, a novel approach (see Methods) that integrates multispectral imaging of individual cells with the statistical power of flow cytometry. suppression of lytic replication. Introduction Over the past decade, studies have implicated human herpesviruses as the causative brokers for a number of illnesses arising in patients with compromised immune Benzenepentacarboxylic Acid systems related to HIV-1 contamination or receipt of immunosuppressive therapies (1C3). A herpesvirus, Kaposi sarcomaCassociated herpesvirus (KSHV or HHV8), is the etiologic agent of KS, the most common AIDS-associated malignancy worldwide, as well as other malignant lymphoproliferative processes, including primary effusion lymphoma and multicentric Castleman disease (4C6). Although initiation of highly active antiretroviral therapy for HIV-1 contamination has reduced the incidence of KSHV-related complications in this populace (7), prevention or regression of tumors is usually often incomplete, and therapeutic alternatives, including systemic chemotherapy, or reductions in antirejection therapies are less effective (8, 9). In vitro experiments and transgenic animal models have begun to elucidate the potential roles of individual KSHV proteins (2). However, a more thorough understanding of the relative contribution of these proteins to host-viral interactions and KSHV-related disease hinges on the creation of an in vivo model supporting whole-virus contamination, latent and lytic viral gene expression, and a larger array of target cell and tissue types. Such an in vivo approach would also provide a more rigorous model for the development of effective anti-KSHV therapeutics. To date, attempts to establish long-term de novo contamination within relevant target cells following the introduction of KSHV to either primate or chimeric hosts have been unsuccessful (10C12). In this study, we injected NOD/SCID mice with purified KSHV and exhibited latent and lytic viral gene expression within murine spleens and long-term contamination within specific leukocyte cell populations. Parallel experiments employing NOD/SCID mice implanted with functional human hematopoietic tissue grafts (NOD/SCID-hu model) revealed a human KSHVCspecific humoral immune response within a subset of infected animals, and administration of short-term preemptive antiviral therapy Rabbit polyclonal to EPHA4 to these chimeric mice resulted in long-term but reversible suppression of viral DNA and mRNA levels. Results Longitudinal increase in latent and lytic viral transcripts within NOD/SCID mice following intravenous injection of KSHV. To ascertain whether KSHV establishes long-term contamination within an immunocompromised murine host, we injected NOD/SCID mice intravenously with either KSHV or UV-inactivated KSHV (UV-KSHV) once weekly for 3 weeks and followed the levels of viral DNA and RNA longitudinally (below). Amplification of DNA sequences within the Benzenepentacarboxylic Acid KSHV open reading frame 73 (ORF73) encoding the latency-associated nuclear antigen (LANA) (13) from total DNA extracted from NOD/SCID spleen tissue 24 hours after injection revealed approximately comparative amounts of KSHV DNA within animals injected with UV-KSHV or KSHV (Physique ?(Figure1A).1A). At all subsequent time points, however, only the mice injected with untreated computer virus showed steadily increasing levels of KSHV DNA, reaching a viral genome copy to cell ratio of approximately 1:300 by 4 months after contamination. In contrast, the levels of viral DNA from mice injected with UV-KSHV fell below the threshold of detection after the 1-day time point, likely reflecting degradation of the inactivated input virus. Open in a separate window Physique 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, sound lines) or UV-KSHV (triangles, dashed lines) intravenously and Benzenepentacarboxylic Acid euthanized 1 day (DNA only) and 1, 2, and 4 months following the third injection. (A) Genomic KSHV DNA values were decided using qPCR to calculate the Ct, representing KSHV Ct normalized to mouse GAPDH Ct (mean of triplicate determinations for each) for each sample. (BCD) 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. 0.001 for mean size comparisons between small-sized (~115 m2; CD3+ and B220+) and intermediate-sized (~130 m2; Ly49+, CD11b+, and CD11c+) cells and between intermediate-sized and large-sized (~155 m2; CD117+) cells. 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. values were.