Significance Identifying the source and dynamics of persistent HIV-1 during combinational antiretroviral therapy (cART) is crucial for understanding the barriers to curing HIV infection. Through genetic characterization of HIV-1 DNA in infected cells from peripheral blood and gut-associated lymphoid tissue from patients after long-term suppressive cART, our study reveals that the primary barrier to a cure is a remarkably stable pool of infected memory CD4 + T cells. Through in-depth phylogenetic analyses, we determined that the HIV-1 reservoir in these cells from eight patients is kept stable during long-term cART and, with little evidence of viral replication, this population could be maintained by homeostatic cell proliferation or other processes.

Neither the number of HIV-1 proviruses within individual infected cells in HIV-1–infected patients nor their genetic relatedness within individual infected cells and between cells and plasma virus are well defined. To address these issues we developed a technique to quantify and genetically characterize HIV-1 DNA from single infected cells in vivo. Analysis of peripheral blood CD4 + T cells from nine patients revealed that the majority of infected cells contain only one copy of HIV-1 DNA, implying a limited potential for recombination in virus produced by these cells. The genetic similarity between HIV populations in CD4 + T cells and plasma implies ongoing exchange between these compartments both early and late after infection.

Genetic recombination contributes to the diversity of human immunodeficiency virus (HIV-1). Productive HIV-1 recombination is, however, dependent on both the number of HIV-1 genomes per infected cell and the genetic relationship between these viral genomes. A detailed analysis of the number of proviruses and their genetic relationship in infected cells isolated from peripheral blood and tissue compartments is therefore important for understanding HIV-1 recombination, genetic diversity and the dynamics of HIV-1 infection. To address these issues, we used a previously developed single-cell sequencing technique to quantify and genetically characterize individual HIV-1 DNA molecules from single cells in lymph node tissue and peripheral blood. Analysis of memory and naïve CD4+ T cells from paired lymph node and peripheral blood samples from five untreated chronically infected patients revealed that the majority of these HIV-1-infected cells (>90%) contain only one copy of HIV-1 DNA, implying a limited potential for productive recombination in virus produced by these cells in these two compartments. Phylogenetic analysis revealed genetic similarity of HIV-1 DNA in memory and naïve CD4+ T-cells from lymph node, peripheral blood and HIV-1 RNA from plasma, implying exchange of virus and/or infected cells between these compartments in untreated chronic infection.

The progression of a natural bacterial infection is a dynamic process influenced by the physiological characteristics of the target organ. Recent developments in live animal imaging allow for the study of the dynamic microbe-host interplay in real-time as the infection progresses within an organ of a live host. Here we used multiphoton microscopy-based live animal imaging, combined with advanced surgical procedures, to investigate the role of uropathogenic Escherichia coli (UPEC) attachment organelles P and Type 1 fimbriae in renal bacterial infection. A GFP+ expressing variant of UPEC strain CFT073 and genetically well-defined isogenic mutants were microinfused into rat glomerulus or proximal tubules. Within 2 h bacteria colonized along the flat squamous epithelium of the Bowman's capsule despite being exposed to the primary filtrate. When facing the challenge of the filtrate flow in the proximal tubule, the P and Type 1 fimbriae appeared to act in synergy to promote colonization. P fimbriae enhanced early colonization of the tubular epithelium, while Type 1 fimbriae mediated colonization of the center of the tubule via a mechanism believed to involve inter-bacterial binding and biofilm formation. The heterogeneous bacterial community within the tubule subsequently affected renal filtration leading to total obstruction of the nephron within 8 h. Our results reveal the importance of physiological factors such as filtration in determining bacterial colonization patterns, and demonstrate that the spatial resolution of an infectious niche can be as small as the center, or periphery, of a tubule lumen. Furthermore, our data show how secondary physiological injuries such as obstruction contribute to the full pathophysiology of pyelonephritis.