Infected mice lacking T cells develop cognitive impairment and have elevated number of monocytes/macrophages in the brain.

<p><b>A.</b> Control and EcoHIV-infected nude mice with or without ART were tested 24 h after fear conditioning training for contextual fear response and 24 h later for cued fear response. *P<0.05, **P<0.01, EcoHIV vs. EcoHIV+ART; #P<0.05, EcoHIV vs. PBS. <b>B.-D.</b> Flow cytometry analysis of macrophage or monocytes in brains of nude mice with EcoHIV, EcoHIV with cART or PBS showing representative dot plots. Cell populations were gated based on isotype control antibodies. <b>B.</b> Staining for CD45 and CD11b and negative for Ly-6G/C. <b>C.</b> Staining for CD45, CD11b and Ly-6C and negative for Ly-6G. <b>D.</b> As determined by flow cytometry using 5 mice per group, the number of cells in each population per total mouse brain are shown. <b>E.</b> Total and integrated vDNA was measured by QPCR and nested QPCR, respectively, in infiltrating leukocytes isolated from the brain of EcoHIV-infected mice with and without cART. *P<0.05, **P<0.01, ***P<0.001.</p

Features of EcoHIV-infected mice compared to HIV-infected people.

<p>Features of EcoHIV-infected mice compared to HIV-infected people.</p

LASER ART (L-ART) fails to reverse HIV-NCI in chronically-infected mice.

<p><b>A.</b> Two days prior to EcoHIV infection, mice were injected once with L-ART and their virus burdens in spleen and PC were evaluated seven days after infection. <b>B.</b> Design of the experiment. L-ART was administered twice, at 28 days and 56 days p.i. <b>C.</b> Results of RAWM conducted on chronically EcoHIV-infected L-ART-treated and untreated mice starting 82 days p.i.; L-ART treated, uninfected mice served as controls; left panel: errors; middle panel: latency; right panel: visible platform control. T represents the learning trial and RT the retention trial (*Mock vs. EcoHIV, <i>p</i><0.002; #Mock vs. EcoHIV+L-ART, <i>p</i><0.03). <b>D.</b> HIV burdens of treated and untreated EcoHIV-infected mice tested in spleen cells, peritoneal macrophages, and brain 94 days p.i.</p

Both EcoHIV and MLV infect mouse brain but only EcoHIV-infected mice develop cognitive impairment.

<p><b>A.</b> Design of the experiment. <b>B.</b> Groups of mice were gavaged with cART or vehicle before and during infection with indicated viruses or vehicle. At day 21 after infection, mice were tested for errors (left panel) and latency (middle panel) in finding the hidden platform, the right panel shows latency to reach the visible platform. RT represents the retention trial. <b>C.</b> Two days after completion of RAWM, the same mice were used to test contextual response to fear conditioning training. The percentage of freezing time was calculated for contextual fear memory deficit in each group of mice. <b>D.</b> Mice were euthanized on day 40 after infection and levels of total vDNA in spleen and brain measured by QPCR (left panel) and integrated viral DNA measured by nested QPCR in PM (right panel). All data are mean ± standard errors. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 EcoHIV vs. EcoHIV+cART; ^#<i>P</i><0.05, ^##P<0.01, ^###<i>P</i><0.001 EcoHIV vs. MLV.</p

Mouse macrophages are susceptible to EcoHIV but not MLV.

<p><b>A.</b> Expression of CAT-1 was determined in peritoneal cells (PC) and thymus (TH) of C57BL/6 mice by QPCR. <b>B.</b> Ten days after EcoHIV or MLV infection of mice, the indicated tissues were harvested for measurement of viral nucleic acids by QPCR. <b>C.</b> Eight weeks after mouse infection by EcoHIV or MLV, F4/80⁺ macrophages were purified from PC and integrated viral DNA in unfractionated or F4/80⁺ PC was measured by nested-QPCR. <b>D.</b> C57BL/6 euthymic and athymic mice were infected by EcoHIV and PC harvested from groups at the times indicated to measure spliced EcoHIV RNA. Mean and standard errors are shown (*<i>P</i><0.05). <b>E.</b> At day 86 after EcoHIV infection, PC were isolated from 129X1 euthymic and athymic mice and forms of the viral DNA and RNA were measured by QPCR, each symbol represents a single mouse. <b>F.</b> Flow cytometric analysis of percentage of F4/80⁺ PM expressing EcoHIV-encoded EGFP in nude and wild-type mice at 25 days after infection. <b>G.</b> Expression of EcoHIV-encoded EGFP in F4/80⁺ PM at 25 days after infection in mice was detected by confocal imaging.</p

EcoHIV in macrophage reservoirs is transmissible <i>in vivo</i> and can be activated by TNF-α but not SAHA.

<p><b>A.</b> Two weeks after transfer of PC from EcoHIV-infected male mice to females, females were euthanized and total vDNA and a Y chromosome transcript were measured in CD4⁺ spleen cells by QPCR. <b>B.</b> Eight weeks after EcoHIV infection, PM were isolated and stimulated by TNF-α or SAHA+prostratin for 48 h. Integrated vDNA and spliced <i>vif</i> and <i>tat</i> RNA were determined by nested QPCR and QPCR, respectively. <b>C.</b> Five weeks after EcoHIV infection PM were isolated and cultured either with no additions, 1 μM ABC and 1 μM RAL, TNF-α, or ABC, RAL, and TNF-α for 2 days prior to collection for QPCR detection of spliced <i>vif</i> RNA.</p

EcoHIV infects mice and induces antiviral immune responses but not immunodeficiency in mice.

<p><b>A.-B.</b> Kinetics of production of total EcoHIV DNA (left panels), integrated EcoHIV DNA (middle panels), and genomic EcoHIV RNA (right panels) were measured by QPCR in <b>A.</b> spleen and <b>B.</b> PC at the times indicated after infection. Each point represents a single mouse. <b>C.</b> EcoHIV <i>gag</i> RNA burden at each time point in copies per ml of whole blood was measured at the times after infection indicated. The dashed line indicates limit of detection of 10 copies. <b>D.</b> The frequency of interferon-γ expression by CD4⁺ or CD8⁺ spleen cells at the time indicated after EcoHIV or mock infection was measured by flow cytometry. <b>E.</b> The number of CD4⁺ and CD8⁺ T cells was measured by flow cytometry at the indicated times and the CD4⁺:CD8⁺ ratio is shown. Symbols represent individual mice, the horizontal lines represent the mean. <b>F.</b> Longitudinal anti-NDK Gag antibodies in 3 EcoHIV-infected mice were measured by ELISA at the times indicated after infection. Each panel represents titers in a single mouse, mean +/- standard errors are shown.</p

EcoHIV establishes latent reservoirs in resting CD4⁺ cells that are inducible by epigenetic modulators.

<p><b>A.</b> CD4⁺ T cells isolated from spleen at day 25 post-infection were cocultured with uninfected cells in the presence or absence of ABC as indicated prior to vDNA amplification, each symbol represents culture from a single mouse. <b>B.</b> Six weeks after EcoHIV infection, CD4⁺ splenic T cells were harvested and purified from donor male 129X1/SvJ mice and injected into the recipient female 129X1 nude mice; their PM were collected after one week. Y chromosome and EcoHIV <i>gag</i> RNA levels in PM were determined by QPCR. Each symbol represents a single mouse. <b>C.-D.</b> Twelve weeks after infection, resting CD4⁺ T cells were purified from the spleen and <b>C.</b> The levels of integrated EcoHIV DNA and <b>D.</b> Genomic RNA were measured by QPCR. <b>E.</b> Eight weeks after EcoHIV infection of mice, resting CD4⁺ T cells were isolated and exposed to the agents indicated in culture for 2 days prior to collection and measurement of EcoHIV mRNA QPCR. <b>F.</b> Six weeks after infection by EcoHIV-Luc, mice were treated as shown, euthanized and splenic CD4⁺ cells isolated for measurement of virus RNA expression (left panel) or protein expression (right panel). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p