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To assess the earliest events regarding transmission and dissemination of SIV following nontraumatic oral inoculation in macaques.Juvenile and neonate rhesus macaques were orally inoculated with SIVmac251 and necropsied at 1, 2, 4, 7, or 14 days post-inoculation. Sites of transmission and the extent of viral spread were assessed by using molecular techniques and in situ hybridization to identify SIV nucleic acid in lymphoid and nonlymphoid tissues.This study demonstrates that 1 day post-exposure, SIV nucleic acid was detected in the alimentary canal only in tissues proximal to the stomach, including the oral and esophageal mucosa as well as the tonsils. Following infection, virus was observed to spread rapidly to regional and peripheral lymph nodes by 1 and 2 days post-inoculation (dpi). Hundreds of copies of SIV-DNA were detected 4 dpi, increasing to > 10 000 copies/1 × 106 cells by 7 dpi. Identification of SIV positive T cells and macrophages implicates these cell types in viral spread, although dissemination of free virus is also likely.Here the oral and esophageal mucosa, as well as tonsils, are demonstrated to be potential sites for viral infection upon nontraumatic oral exposure to SIV in macaques. The rapid dissemination following oral transmission observed in this study is reflective of SIV transmission across other mucosal surfaces. The rapidity with which SIV, and probably HIV, spreads throughout the lymphatics indicates a major obstacle for a vaccine amnestic immune response to eliminate infected cells prior to dissemination.