P24 staining of HAD brain tissue
The tissue stains of the meninges for patient CX showed a background of HIV p24 positivity with intense staining localized to perivascular macrophages. The section shown in is typical of HIV p24 large vessel staining of a HAD brain section. P24 staining of the meninges for the other patients was very low, presumably due to the late stage of disease; however, as previously reported (Lamers et al., 2010
), frontal lobe tissues for all patients stained positive for p24 with varying degrees of intensity.
HIV p24 stain of meninges: localization to perivascular and parenchyma macrophages in patient CX
Phylogenetic analysis of HIV-1 in post-mortem brain and peripheral tissues
The maximum clade credibility (MCC) tree was obtained for each patient under a Bayesian framework. In patient CX (), the MCC tree showed two well-supported clades of brain sequences (basal ganglia, occipital lobe/grey, occipital lobe white matter, periventricular, choroid plexus, temporal lobe) that shared a common origin at the root of the tree, separated by very long branches. The V3 loop was predicted to utilize the CCR5 co-receptor with a charge of 3+ or less. Results from the Hudson test indicated there was no population structure evident among brain tissues (p>0.05) except for the temporal lobe (p<0.05). Interestingly, the peripheral sequences (colon and lymph node) emerged from one of the major brain clades. Another back migration from the periphery occurred to the basal ganglia. The meninges were again dispersed throughout the brain clade. Patient CX displayed signs of dementia over several years and was the only patient in the study that was not on HAART therapy. Although only two peripheral tissues were sampled in this patient, the virus in these tissues appeared to evolve directly from the brain.
For patient GA, the MCC tree () was also consistent with an early viral infection model. Similar to the sequence of patient CX, all of GA’s sequences were predicted to utilize the CCR5 co-receptor, with a V3 loop charge of 3+ or less. A well-supported peripheral and brain clade shared a common ancestor at the root of the tree. Results from the Hudson test showed that the peripheral sequences (lymph and spleen) did not have structure between them (p>0.05). The temporal and frontal lobe/white matter were structured with respect to each other (p<0.05). Also similar to patient CX, the meninges were interspersed within the brain clade only. Again one well-supported clade consisting of peripheral sequences from both lymph and spleen emerged from a larger brain clade. This result indicated re-seeding of the peripheral tissues from the brain.
The MCC tree for patient DY () provided support for late viral invasion into brain tissues. In addition, all sequences from patient DY had a charge of +6 or greater, indicating infection of the brain with CXCR4 viruses, a phenotype typically associated with T-cell tropic viruses. One primary well-supported brain clade emerged from the peripheral tissues (liver, lymph, spleen). Again, results from the Hudson test indicated no population structure among the peripheral tissues (p>0.05). Sequences from the meninges were interspersed within both brain and peripheral tissue. Four additional introductions occurred from periphery to the brain. Sequences from specific regions of the brain lacked population structure (basal ganglia, frontal lobe/white matter, frontal lobe/grey matter, p>0.05), with the exception of the temporal lobe (p<0.05). Furthermore, a few sequences from peripheral tissues emerged from the brain clade, suggesting a back-migration event(s).
The MCC tree for patient AZ () provided another example of late viral invasion into the brain. The peripheral sequences (liver, lymph, and spleen) were basal to the brain clade and were not clustered by tissue of origin. A separate clade of peripheral sequences (liver and spleen) was separated by a long branch, all peripheral sequences potentially using the CXCR4 co-receptor with a V3 loop charge of +5 (all other AZ sequences had a v3 loop charge of 3+ or less). The Hudson test confirmed a lack of population subdivision among the three peripheral tissues (p>0.05). A well-supported monophyletic brain clade comprised of frontal lobe/white matter sequences emerged late in the tree. Again, the meninges are not monophyletic, but in this case were interspersed within the peripheral tissue and appear to be recently infected by peripheral tissues.
The MCC tree for patient BW () was consistent with the classic pattern of compartmentalization: a well-supported peripheral clade and a brain clade shared a common ancestor at the root of the tree. The brain clade was comprised of several monophyletic clades of sequences from different regions (frontal lobe/white matter, frontal lobe/grey matter, basal ganglia, and temporal lobe). The Hudson test indicated strong evidence for population subdivision (p<0.001). The only exception was the meninges sequences, which were interspersed throughout the brain clades, indicating that the meninges were infected either by virus from different regions of the brain or by different peripheral isolates over the course of infection. The V3 loop charge for BW’s sequence were predominantly +4, a charge associated with both CCR5 and CXCR4 phenotypes.
The phylogenetic trees for patients CX and GA displayed more evolutionary distance between major clades than the trees from patients AZ, DY and BW (note the distance bar at the bottom of the phylogenies).
Viral gene flow among post-mortem tissues
The Slatkin-Maddison test was used to investigate the hypothesis of gene flow among brain, peripheral, and meninges sequences. The average number of observed migrations among the three tissues was calculated for the maximum clade credibility tree (). In four patients (BW, DY, GA, and CX) significant migration was detected from the brain to the meninges (p<0.0001). Migration was also detected from the meninges to the periphery in DY, and from the meninges to the brain in patient CX. Significant migration was found from the periphery to the brain in three patients (AZ, DY, and CX). A similar analysis with the program LAMARC, which allows estimating the direction and rate of gene flow among different sub-population with maximum likelihood, gave identical results (data not shown). In we summarize the conclusions from the combined phylogenetic and migration analyses. Gene flow patterns among different lymphoid tissues, as well as different brain tissues, were also examined. In all cases, however, the number of observed migrations among strains infecting different tissues lied within the 95% confidence interval of a distribution of 10,000 trees where tissue assignments on the tips were randomly scrambled (data not show). Therefore, no statistically significant inferences could be made about preferential/asymmetric gene flow in this case.
Migration of the virus among tissues.
Summary of Phylogenetic Results.