Double-label immunohistochemistry and <i>in situ</i> hybridization of colon.

<p><b>(A).</b> Representative images of SIV ISH (blue) with double-label immunohistochemistry for macrophage marker Ham56 (DAB, brown) in rhesus macaques in groups SIV239E (left), SIV239noE (middle), and SIVΔ<i>vpx</i> (right). SIV+ Ham56+ macrophages were frequent in SIV239E monkeys (left), but absent in the SIVΔ<i>vpx</i> group (right). <b>(B).</b> SIVΔ<i>vpx</i>-infected monkeys had significantly less virus in the colon (mean 10.5 SIV+ cells) compared to monkeys infected with SIV239 (SIV239E mean 105.3 SIV+ cells, SIV239noE mean 33.5 SIV+ cells), but no difference with animals in the SIVΔ<i>nef</i>/SIVΔ3 group. <b>(C).</b> SIVΔ<i>vpx</i> monkeys (mean 0 cells) had significantly fewer SIV+ Ham56+ macrophages compared to SIV239E (mean 66.7 cells), SIV239noE (mean 9.8 cells), and SIVΔ<i>nef</i>/SIVΔ3 groups (mean 3.3 cells) groups. <b>(D).</b> There was a significantly lower percentage of SIV+ cells that were macrophages in SIVΔ<i>vpx</i>-infected rhesus (mean 0%) compared to both the SIV239E (mean 63.3%) and SIV239noE (mean 22.4%) groups and a trend in the SIVΔ<i>nef</i>/SIVΔ3 group (mean 25.6%).</p

Survival and viral load data.

<p>(<b>A)</b> Survival days post-inoculation (dpi) for rhesus macaques inoculated with SIVΔ<i>vpx</i> compared to animals inoculated with SIVmac239 (SIV239E with encephalitis or SIV239noE without encephalitis) or other mutant viruses (SIVΔ<i>nef</i> or SIVΔ3. SIVΔ<i>vpx</i>-infected macaques survived significantly longer with a slower disease progression compared to SIV239E or SIV239noE animals, but did not differ in survival length compared to SIVΔ<i>nef</i> or SIVΔ3. <b>(B)</b> Plasma viral RNA from SIVΔ<i>vpx</i>-infected rhesus macaques expressed as RNA copy equivalents per ml plasma from chronic disease or near-terminal collections (range 329–1140 dpi, median 730 dpi).</p

Amino acid sequences from chronic infection plasma viral RNA from SIVΔ<i>vpx</i>-infected monkeys.

<p><b>(A).</b> Vpx N-terminal sequences accumulated debilitating mutations in two animals (cases 4 and 5) (*). <b>(B).</b> Novel amino acid sequence changes of Vif were present in case 4 (with highest viral loads) with leucine to proline (L->P) and alanine to threonine (A->T). <b>(C).</b> Analysis of Vpr revealed changes in amino acid sequences detected at the C-terminal region in cases 3 and 4 (I94T, P95L, S99N or G). <b>(D).</b> Alignments of Tat demonstrated multiple non-conservative changes, such as I22T, S32L, L35P.</p

Summaryoflentiviral infection of macrophages in spleen, lymph node, anin macaques inoculated intravenously with SIVΔ<i>vpx</i> compared to SIVmac239 (with or without SIV encephalitis) and SIVΔ<i>nef</i> or Δ3 assessed in spleen, lymph node, and colon.

<p>*p<0.05 difference between SIVΔ<i>vpx</i> and both SIVmac239 and SIVΔ<i>nef</i>/Δ3cases.</p

Double-label immunohistochemistry and <i>in situ</i> hybridization of lymphoid tissues.

<p><b>(A)</b> Representative images of SIV <i>in situ</i> hybridization (ISH, blue) with double-label immunohistochemistry for monocyte/macrophage lineage cell marker Ham56 (DAB, brown) in rhesus macaques in groups SIV239E (left), SIV239noE (middle), and SIVΔ<i>vpx</i> (right). The top images of lymphoid follicles depict Ham-56+ cells morphologically consistent with follicular DCs. Images in the second row depict HAM56+ cells in the red pulp of the spleen (left and center) or paracortex in lymph node (right). Frequent SIV+ HAM56+ macrophages/DCs (Mφ) were observed in tissues from the SIV239E and SIV239noE groups, while only rare SIV+ Ham56+ macrophages/DCs were observed in lymph node from a SIVΔ<i>vpx</i>-infected rhesus (arrow). <b>(B).</b> The overall numbers of infected cells in the spleen and lymph node from the 4 groups of animals were not significantly different. <b>(C)</b> However, there were significantly fewer SIV+ Mφ in SIVΔ<i>vpx</i> monkeys (mean 0.5 SIV+ Mφ) compared to the SIV239E (mean 13.64 SIV+ Mφ), SIV239noE (mean 4.5 SIV+ Mφ) and SIVΔ<i>nef</i>/SIVΔ3 monkeys (mean 6.25 SIV+ Mφ). <b>(D)</b> SIV infected macrophages made up a much lower percentage of all SIV+ cells in SPL and LN of SIVΔ<i>vpx</i>-infected rhesus (mean 2.2%) compared to SIV239E (mean 22.7%), SIV239noE (mean 8.3%), and SIVΔ<i>nef</i>/SIVΔ3 monkeys (10.1%) (p<0.05).</p

Phenotype of SIV+ cells in spleen, peripheral lymph node, and colon of SIVΔ<i>vpx</i>-infected monkeys by double-label <i>in situ</i> hybridization and immunohistochemistry.

<p>Phenotype of SIV+ cells in spleen, peripheral lymph node, and colon of SIVΔ<i>vpx</i>-infected monkeys by double-label <i>in situ</i> hybridization and immunohistochemistry.</p

Quantification of tissue macrophages and lymphocytes in SIVΔ<i>vpx</i> compared to SIVmac239-infected rhesus.

<p><b>(A).</b> Rhesus, lymph node: representative image of SIV <i>in situ</i> hybridization (ISH, blue) with double-label immunohistochemistry for CD3+ T lymphocytes (DAB, brown); <b>(B).</b> Quantification of immunophenotyped SIVΔ<i>vpx</i>-infected cells demonstrates almost exclusive infection of CD3+ T lymphocytes and not Ham56+ macrophages in spleen and lymph nodes (p<0.05); <b>(C).</b> Numbers of Ham56+ tissue macrophages in the spleen or lymph node and colon were not significantly different in SIVΔ<i>vpx</i>-infected rhesus macaques compared to SIVmac239-infected rhesus; <b>(D).</b> Numbers of CD3+ lymphocytes were not significantly different in the colon in SIVΔ<i>vpx</i>-infected rhesus macaques compared to SIVmac239-infected rhesus.</p

Spectral imaging and colocalization of double-label immunohistochemistry slides of spleen and colon.

<p>Immunofluorescence with SIV nucleic acid (green), Ham56+ macrophages (red), and coexpression (yellow, arrows) demonstrate that infected macrophages are readily evident in SIVmac239-infected monkeys (<b>A, B, arrows</b>), particularly within the colon (<b>B</b>), but are absent in spleen and colon from SIVΔ<i>vpx</i>-infected monkeys (<b>C, D</b>). Snowflake symbols in <b>C</b> denote autofluorescence in red pulp macrophages. Original magnification 40× (<b>A</b>–<b>D</b>).</p