SIV Vpx Is Essential for Macrophage Infection but Not for Development of AIDS

Analysis of rhesus macaques infected with a vpx deletion mutant virus of simian immunodeficiency virus mac239 (SIVΔvpx) demonstrates that Vpx is essential for efficient monocyte/macrophage infection in vivo but is not necessary for development of AIDS. To compare myeloid-lineage cell infection in monkeys infected with SIVΔvpx compared to SIVmac239, we analyzed lymphoid and gastrointestinal tissues from SIVΔvpx-infected rhesus (n = 5), SIVmac239-infected rhesus with SIV encephalitis (7 SIV239E), those without encephalitis (4 SIV239noE), and other SIV mutant viruses with low viral loads (4 SIVΔnef, 2 SIVΔ3). SIV+ macrophages and the percentage of total SIV+ cells that were macrophages in spleen and lymph nodes were significantly lower in rhesus infected with SIVΔvpx (2.2%) compared to those infected with SIV239E (22.7%), SIV239noE (8.2%), and SIV mutant viruses (10.1%). In colon, SIVΔvpx monkeys had fewer SIV+ cells, no SIV+ macrophages, and lower percentage of SIV+ cells that were macrophages than the other 3 groups. Only 2 SIVΔvpx monkeys exhibited detectable virus in the colon. We demonstrate that Vpx is essential for efficient macrophage infection in vivo and that simian AIDS and death can occur in the absence of detectable macrophage infection

Equine limb wound management with pinch grafting

Large excisional wounds located on the distal limbs of horses often undergo complications during the healing process. Exuberant granulation tissue is a potential complication to any excisional wound healing by second intention in the distal limb of a horse. Furthermore, large wounds in this location often cannot contract completely and require skin grafting. Therefore, when managing distal limb wounds in horses, meticulous care must be taken to prevent complications from occurring. Skin grafting is a common procedure used in management of non- healing wounds in horses. Pinch grafting, in particular, is a simple, straightforward and successful method of grafting that can be done on a standing horse. These practical characteristics allow for any equine practitioner to perform this procedure in either a hospital or field setting

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

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

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

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