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To locate the source of systemic viremia, this study employed a novel method to trace HIV\u20101 infection in vivo. We created JRCSF\u03be<jats:italic>nef<\/jats:italic>, a pool of infectious HIV\u20101 (strain JR\u2010CSF) with highly mutated <jats:italic>nef<\/jats:italic> gene regions by random mutagenesis PCR and infected this mutated virus pool into both Jurkat\u2010CCR5 cells and hematopoietic stem cell\u2010transplanted humanized mice. Infection resulted in systemic plasma viremia in humanized mice and viral RNA sequencing helped us to identify multiple lymphoid organs such as spleen, lymph nodes, and bone marrow but not peripheral blood cells as the source of systemic viremia. 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