Use of a Replication-Defective Vector to Track Cells Initially Infected by SIV in Vivo : Infected Mononuclear Cells Rapidly Appear in the Draining Lymph Node after Intradermal Inoculation of Rhesus Monkeys

Abstract

A better understanding of the mechanisms of HIV dissemination, a key step in pathogenesis, would be possible if the cellular pathways of viral dissemination could be followed in simian immunodeficiency virus (SIV)- inoculated monkeys or HIV-infected people. In an initial attempt to follow this process using a traceable virus infection, we inoculated rhesus monkeys intradermally (ID) or directly into lymph nodes with a replicationdefective SIV-based vector expressing the enhanced jellyfish green fluorescent protein (EGFP), V1EGFP. EGFP expression was detected in mononuclear cells isolated from the sites of inoculation (skin and lymph node) at 5 and 16 hr after inoculation and then cultured in vitro for 6 days to allow maximum EGFP expression. Similarly, EGFP-expressing, SIV-infected cells could be detected at 16 hr postinfection in the lymph nodes that drained the sites of ID inoculation. Since V1EGFP is a replication-defective vector, the EGFP-expressing cells are the initial target cells infected by the virions in the original inoculum. The results of flow cytometric analysis were confirmed by a nested PCR assay to detect SIV DNA and hence infection of cells and reverse transcription. These experiments indicate that 16 hr after ID inoculation newly infected cells either remain in the skin at the site of inoculation or have migrated to the draining lymph node. The results in this SIV vector model probably reflect the short time (less than 16 hr) required for HIV to move from a site of epithelial penetration to the lymphoid tissues via lymphatic vessels.