Clinical and pathological features
As part of prior ongoing studies, rhesus macaque H445 was inoculated intravenously with the molecular clone, SIVsmE543-3 [20
]. H445 developed a transient antibody and cytotoxic T lymphocyte (CTL) response, had high plasma and tissue viral load, and rapidly progressed to AIDS with euthanasia at week 16 post inoculation (p.i.) [21
]. Previous studies have suggested that virus passaged in macaques have evolved to become more virulent [22
] Therefore, we sought to evaluate the pathogenicity of SIV isolates from this rapid progressor. Uncloned virus was isolated from H445 mesenteric lymph node and inoculated intravenously into a cohort of six rhesus macaques [19
All six animals became infected and had clinical and pathological symptoms characteristic of SIV-related disease [19
]. Although six animals were inoculated with virus isolated from a RP, only H635 progressed rapidly [27
] and was subsequently euthanized at week 9 p.i. [19
]. The survival time of the other five conventional progressor animals ranged from 52 to 116 weeks p.i. [19
High levels of plasma viral RNA were detected in all animals during primary viremia. These levels generally decreased by one to two logs by week 4 p.i. and were maintained at these levels until death (). The rapid progressor macaque, H635 was the one exception, showing increasing viral load until death. Detection of viral RNA in the CSF was observed by week 1 p.i. Only CSF viral RNA levels for H631, H635, and H636, increased terminally ().
Fig. 1 Clinical and pathological features. (A) Plasma and (B) CSF viral RNA levels. Samples were obtained at sequential time points post inoculation. Y axis represents viral RNA load (copies/ml) and the X axis represents weeks post inoculation. Macaques H631 (more ...)
CD4+ T cell levels gradually declined in all animals with a mean of 198 cells/µl at the time of euthanasia. The only exception was H635 where the number of CD4+ T cells recovered back to normal levels at death. Moderate to robust responses to SIV-Gag by week eight and to SIV-Env (gp160) by week 32 were observed in all animals except for H635 (data not shown).
Encephalitis Observed in Two Conventional Progressors
Histological examination of brain obtained during either necropsy or autopsy of all six animals revealed that half of the cohort developed SIV meningoencephalitis [19
]. SIVE in these three animals was characterized by the presence of MNGC. Perivascular lymphocytic cuffing was observed in the brains of the two conventional progressors, H631 and H636, a pathologic feature not observed in the RP brain sections. Immunohistochemistry using anti-CD20 and anti-CD3 antibodies, demonstrated that the infiltrating cells consisted of B and T cells, respectively, and that these latter cells were predominantly CD8+
T cells (). In contrast, only occasional T cells and no B cells were observed in the brain of H635. Additionally, infiltrating macrophages and MNGC were identified by HAM56, a macrophage marker, and these cells co-localized in lesions with SIV expression, as shown by in situ hybridization (ISH) (). Triple label confocal microscopy using ISH for SIV RNA and immunofluorescence for HAM56 and anti-CD3 demonstrated that the SIV-expressing cells in the brain were macrophages ().
Molecular evolution of SIV
To determine whether specific SIV variants were associated with the development of SIVE in these three animals, we examined the Env sequence of viral RNA amplified directly from CSF and plasma of H631, H636, and H635 and compared it to the inoculum, SIVsmH445. The SIVsmH445 virus inoculum stock showed few changes in the gp120 region of Env as compared to the parental SIVsmE543-3 [19
]. Of the few mutations observed, the majority were characteristic RP-specific mutations [19
] that we have described previously in RP macaques. However, clones with these mutations were a minority population. Analyses of sequences obtained directly from CSF (data not shown) and plasma [19
] of H635 at the time of death showed that those RP-specific mutations predominated in both compartments, consistent with specific selection of this minority viral variant from the inoculum. In contrast, sequencing of CSF and plasma from H631 and H636 did not reveal RP-specific mutations in any of the clones analyzed at any time point. Consequently, we have used the parental SIVsmE543-3 sequence for comparisons.
Sequential samples obtained during primary viremia and up to and including death at week 116 (H631) were analyzed. Animal H636 died unexpectedly; consequently, only samples from the last time point at week 76 p.i. were available. Sequencing analysis of the gp120 region revealed that virus during primary viremia in both compartments for both animals had few and random mutations compared to that of SIVsmE543-3. However, by week 32, mutations in the V1/V2 regions of gp120 could be observed () with extensive changes seen by week 76. By week 32, in H631 CSF and plasma clones (), except for two substitutions (S193A and N215D) in CSF, there were no consistent mutations observed. Interestingly, these two substitutions were maintained through death. The latter mutation resulted in a loss of a potential N-linked glycosylation site (PNGS). This suggests that these mutations may have conferred a selective advantage for viral fitness to the virus in H631 CSF.
Fig. 2 Sequence analysis of the V1/V2 region of gp120 clones directly from CSF and plasma viral RNA in H631 (A) and H636 (B) obtained from weeks 32 and 76 post inoculation. In addition, samples obtained at week 116 (death) for H631 are shown in (A). Amino acid (more ...)
In contrast, there appeared to be fewer total mutations in H636 clones () than in H631, and there were no consistent changes that were maintained from week 32 to week 76 as seen in H631. Overall, these data suggest that virus in H636 was possibly under different selective pressures at these two time points.
Phylogenetic analyses were then performed to study the relationship of these sequences (figure 3). Sequences obtained from H631 CSF and plasma viral RNA (figure 3A) were found to be clustered based on their time points at week 1 and 32. At week one, CSF and plasma sequences were still closely related to one another and to SIVsmE543-3, indicating that very little evolution occurred. At week 32, CSF and plasma sequences for the most part, formed their own clade suggesting that selective pressures in these compartments may have been different from that of week 1. Viral sequences isolated at week 76 and at death continued to diverge from week 32, but virus from CSF and virus from plasma remained clustered based on their respective compartment.
Similar to what was observed at week 1 in H631, CSF and plasma sequences in H636 were also very similar to one another and to SIVsmE543-3 (figure 3B). By week 32, while virus could be seen to have diverged from SIVsmE543-3, CSF and plasma sequences did not form their own clades. Half of the sequences from plasma at week 76 appear to be direct descendents of virus from plasma at week 32. In contrast, the other half, possibly under different selective pressures, had formed a different clade with virus from CSF.
To determine if the changes observed in the gp120 sequences were due to random mutations or due to positive selection from immune pressure, the rate of nonsynonymous (dN) to synonymous (dS) change was calculated for each clone at sequential timepoints and the average ratio is shown in . Positive selection is evidenced by a dN/dS ratio > 1. In H631, increasing positive selection could be seen in both CSF and plasma gp120 sequences throughout the course of infection, with positive selection occurring by week 76. Strong positive selection was evident by death indicating that immune pressures in these compartments drove the evolution of these viruses in H631. In the case of H636, increasing positive selection was also observed for plasma sequences; however, with sequences obtained from CSF, only slight positive selection was seen by week 32, but had weakened slightly by week 76.
Analysis of Nonsynonymous to Synonymous Changes in gp120 at Sequential Timepoints
Replicative abilities of viruses isolated from H631 and H636
To study the biological consequences of the observed mutations, we isolated virus from CSF and plasma from both animals at the last available time point. Additionally, for H631, virus was isolated from the brain. Virus isolates from H631 brain, CSF, and plasma (Fig. 4A), as well as from H636 CSF and plasma (Fig. 4B) were all able to infect donor PBMC. However, the replication efficiency varied among the viruses. Virus from both H631 plasma and H636 plasma were found to replicate much more efficiently than the corresponding virus from the central nervous system (CNS) of the same animal.
Based on our earlier observation of SIV-infected macrophages in the brain, we then wanted to evaluate viral replication in MDM (figure 4C–D). Again, all viruses from both animals were able to replicate in MDM. However, in contrast to what was observed in the PBMC infections, infectivity of MDM by virus isolated from CNS of both macaques was much higher than that of virus isolated from PBMC.
To ensure that there were no donor biases, as different macaques may have variable susceptibilities to infection [21
], this infectivity assay was repeated using an additional three different donor macaques. The same donor macaques were used in both the PBMC and MDM experiments. Data from the four different infection assays were then combined to obtain an average, and the experiment was then independently repeated a second time. These experiments (data not shown) confirmed the initial results and demonstrated a marked trend for virus isolated from plasma of either H631 or H636 to replicate in PBMC better than their counterpart virus isolated from the CNS. Similarly, replication of viruses isolated from CNS was observed to infect MDM better than virus isolated from plasma. Thus, our data suggests that there is a preference for cell tropism that is dependant on the origin of the virus compartment.
Phylogenetic analyses of isolated viruses
Based on the observed differences in biological phenotypes, we wanted to examine the gp160 sequences to evaluate the phylogenetic relationships of the isolated viruses. Additionally, bootstrapping support tests were done to examine the reliability of the topology or branching order of the tree. Bootstrap support values were based on 1000 replicates and only values greater than 70% at the major nodes are shown. With the exception of one envelope clone obtained from a plasma virus, all sequences from H631 brain, CSF, and plasma clustered within their respective groups (figure 5A). Although the viruses were quite distinct and formed their own clades, bootstrap values did not support compartmentalization of the viruses. However, phylogenetic analyses and bootstrap support tests indicated that H636 viruses from the CSF and plasma compartments were indeed compartmentalized (bootstrap values 916 and 736, respectively) (figure 5B). In contrast to H631 and H636, gp160 sequences amplified directly from CSF and plasma viral RNA of H635 showed that there was intermingling of virus between plasma and CSF and no tissue-specific localization of virus (figure 5C).
The rates of nonsynonymous to synonymous changes were also calculated for H631 and H636 isolated viruses. For all cases, the dN/dS ratio was greater than one, indicating that positive selection was responsible for the evolution of these viruses (data not shown).
Loss of PNGS
Sequencing analyses revealed that there were changes in the PNGS pattern (figure 2 and data not shown) resulting in a statistically significant loss of PNGS in gp160 of all H631 virus isolates and H636 CSF virus isolates compared to E543-3 (P< 0.0001 to P<0.0054) (figure 6). Furthermore, the number of PNGS in Env of H631 brain virus isolates was found to be lower than that of H631 CSF and plasma virus (P< 0.0001 and P<0.0005, respectively).