Mouse Adenovirus Type 1 Infection in SCID Mice: an Experimental Model for Antiviral Therapy of Systemic Adenovirus Infections

Abstract

The importance of human adenovirus infections in immunocompromised patients urges for new and adequate antiadenovirus compounds. Since human adenoviruses are species specific, animal models for systemic adenovirus infections rely on a nonhuman adenovirus. We established mouse adenovirus type 1 (MAV-1) infection of BALB/c SCID mice as a model for the evaluation of antiadenovirus therapy. In vitro studies with mouse embryonic fibroblasts pointed to the acyclic nucleoside phosphonate cidofovir and the N-7-substituted acyclic derivative 2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine (S-2242) as markedly active compounds against MAV-1. SCID mice, infected intranasally with MAV-1, developed a fatal disseminated infection after approximately 19 days, characterized by hemorrhagic enteritis. Several techniques were optimized to monitor viral, immunological, and pathological aspects of MAV-1 infection. Real-time PCR quantification of viral DNA revealed that after replication in the lungs, virus disseminated to several organs, including the brain, liver, spleen, intestine, heart, and kidneys (resulting in viruria). Immunohistochemical staining showed that MAV-1 was localized in the endothelial cells of the affected organs. Using reverse transcription-PCR, tissue levels of proinflammatory cytokines (i.e., interleukin-1␤ and tumor necrosis factor alpha) were found to be markedly increased. The MAV-1/SCID model appears to be an appropriate model for in vivo evaluation of antiadenovirus agents. Treatment with cidofovir or S-2242 at a dose of 100 mg per kg of body weight resulted in a significant delay in MAV-1-related death, although these antivirals were unable to completely suppress virus replication despite continued drug treatment. These findings suggest that complete virus clearance during antiviral therapy for disseminated adenovirus infection may require an efficient adaptive immune response from the host.

Recovery of Humoral Immunity Is Critical for Successful Antiviral Therapy in Disseminated Mouse Adenovirus Type 1 Infection

“…The C3H/3T3 mouse embryonic fibroblast cell line was cultured as described previously (27). For the virus neutralization assays, the concentration of heat-inactivated newborn calf serum was reduced to 2%.…”

“…For the analysis of MAV-1 titers in tissues of infected mice, cardiac perfusion with PBS and dissection were performed and organs were recovered, homogenized, and subjected to DNA extraction according to the tissue protocol of the QIAamp DNA mini kit (Qiagen, Hilden, Germany). MAV-1 DNA was quantified by real-time PCR analysis using an ABI Prism 7000 apparatus (Applied Biosystems, Foster City, CA) and a previously published protocol (27). To correct for experimental variations, the primerprobe set of the 18S genomic endogenous control kit (Eurogentec, Seraing, Belgium) was included in the same PCR mixture as the target primer-probe set.…”

“…Virus stocks were prepared in C3H/3T3 cells as described before (27). The titer of the virus stocks, determined by a standard plaque assay, was ϳ8 ϫ 10 5 PFU per ml.…”

“…From the following organs, parts were fixated in 4% formaldehyde for 24 h and then embedded in paraffin: spleen, kidney, adrenal glands, liver, small intestine, lung, heart, and brain. Tissue sections, 4 m thick, were stained with hematoxylin and eosin for routine histologic examination and immunohistochemically with a rabbit polyclonal antibody to the MAV-1 E3 class 1 protein by using a standard avidin-biotin immunoperoxidase staining procedure, as previously described (27).…”

“…We previously demonstrated that continued antiviral treatment with cidofovir causes a marked delay in MAV-1-induced disease but cannot prevent a fatal outcome in severe combined immunodeficient (SCID) mice (27). In other studies, mice with genetic deficiencies in specific immunological functions were used to investigate the roles of distinct leukocyte subsets in MAV-1 infection (31,32).…”