A spatio-temporal assessment of simian/human immunodeficiency virus (SHIV) evolution reveals a highly dynamic process within the host

<div><p>The process by which drug-resistant HIV-1 arises and spreads spatially within an infected individual is poorly understood. Studies have found variable results relating how HIV-1 in the blood differs from virus sampled in tissues, offering conflicting findings about whether HIV-1 throughout the body is homogeneously distributed. However, most of these studies sample only two compartments and few have data from multiple time points. To directly measure how drug resistance spreads within a host and to assess how spatial structure impacts its emergence, we examined serial sequences from four macaques infected with RT-SHIV_mne027, a simian immunodeficiency virus encoding HIV-1 reverse transcriptase (RT), and treated with RT inhibitors. Both viral DNA and RNA (vDNA and vRNA) were isolated from the blood (including plasma and peripheral blood mononuclear cells), lymph nodes, gut, and vagina at a median of four time points and RT was characterized via single-genome sequencing. The resulting sequences reveal a dynamic system in which vRNA rapidly acquires drug resistance concomitantly across compartments through multiple independent mutations. Fast migration results in the same viral genotypes present across compartments, but not so fast as to equilibrate their frequencies immediately. The blood and lymph nodes were found to be compartmentalized rarely, while both the blood and lymph node were more frequently different from mucosal tissues. This study suggests that even oft-sampled blood does not fully capture the viral dynamics in other parts of the body, especially the gut where vRNA turnover was faster than the plasma and vDNA retained fewer wild-type viruses than other sampled compartments. Our findings of transient compartmentalization across multiple tissues may help explain the varied results of previous compartmentalization studies in HIV-1.</p></div

Compartmentalization relationships between different samples reveal transient and stable patterns.

<p>Probability of a significant pairwise compartmentalization test between vRNA subsampled from each compartment for the four macaques (columns) over time (x-axis). Each row represents comparisons between the vRNA in each compartment marked at the right (from top to bottom: plasma, PBMC, LN, gut and vagina vRNA) to all other compartments. Because all pairwise relationships are shown for each compartment, lines are repeated (i.e., the lymph node versus plasma line is present in both the lymph node and plasma rows). The y-axis indicates the proportion of K_ST tests significant at the 5% significance level when subsampled to 10 sequences per compartment 1000 times. Coloration indicates that the comparison was done between the focal compartment for the row and plasma (green), PBMC (pink), LN (purple), gut (dark blue) or vagina (light blue). Rx1 is treatment FTC+TFV+EFV and Rx2 is the treatment TFV+L870812+DRV/r.</p

Plasma viremia and drug resistance in four macaques over the course of infection.

<p>Plasma RT-SHIV (black) and frequency of plasma vRNA with DRMs (red) are plotted over time for each animal, as determined by SGS. Red lines show the frequency of K103N (solid) or M184I/V (dashed) in the plasma vRNA. Gray shading denotes administration of drug(s) between weeks 12–20 and weeks 26–44. Black dotted line indicates the final time point for each animal. Rx1 is treatment FTC+TFV+EFV and Rx2 is the treatment TFV+L870812+DRV/r.</p

Compartmentalization test results reveal pairwise RT-SHIV RNA compartmentalization relationships between blood, lymph node, gut, and vagina.

<p>Each circle summarizes the results of three compartmentalization tests performed pairwise between the vRNA from compartments within a macaque at a given time, and the position of the segment represents the test performed (left: KST, top-right: Slatkin-Maddison (SM), bottom-right: AMOVA). The coloration indicates the probability of a significant test result at the 5% significance level when subsampling 10 sequences from each compartment averaged across each macaque and all time points with red indicating a high probability and white indicating a low probability of a significant test. The area of the segment indicates the number of comparisons contributing to that average (i.e., the number of time points with a sufficient number of sequences in each compartment within a macaque). Because we used 10 unique subsampled sequences to perform the SM test, the SM test could not be performed on some macaque time points, resulting in smaller sample sizes (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006358#sec011" target="_blank">Materials and Methods</a> for details).</p

Drug resistance increase over time across all compartments.

<p>Drug resistance was defined as the proportion of vRNA (dashed lines) or vDNA (solid lines) sequences having K103N and/or M184V/I within each compartment for animals T98133, A99039, and A99165. Gray shading indicates the FTC or EFV treatment periods for the macaques.</p

Change in compartmental viral composition over time varies by compartment and drug pressure.

<p>For each macaque, K_ST tests are plotted between all time points with adjacent samples (i.e., week 12 or 13 compared to week 15 or 16) for each compartment (vRNA top, vDNA bottom). Black dots indicate time points with samples of at least 3 sequences. Red lines indicate that a K_ST test comparing those two samples is significantly different (with a 5% FDR). Grey lines indicate a failure to reject the null hypothesis of being well-mixed at the 5% FDR level. Grey shading indicates monotherapy or combination therapy, as indicated below the x-axis. Rx1 is treatment FTC+TFV+EFV and Rx2 is the treatment TFV+L870812+DRV/r.</p

Drug resistance spreads dynamically across several compartments in macaque A99165 over the course of treatment.

<p>Colored polygons show the prevalence of different genotypes of RT-SHIV RNA (A) or DNA (B) over time in different compartments. Vertical distance at black vertical lines indicates the frequency of the sample with an exact set of mutations (i.e., genotype) at the time of sampling. Grey indicates WT virus, and striations on the background of a color indicate a drug-resistant genotype. Common mutations are labeled, and known DRMs are labeled in pink. For plasma/PBMC, plasma vRNA and PBMC vDNA are shown. Two sequences are considered the same genotype when all mutations found above frequency 1% in the macaque are shared. Sample sizes for at each sampling point are given above each vertical line. (C) Time-structured phylogenies reveal relationships between vRNA sequences (shown as leaf nodes). Branch lengths indicate sequence sampling time, and branches are colored to match (A) and (B). Sampling location is indicated for each sequence, and the identity of all mutations at frequency >1% is shown to the right. Colors indicating mutation type (synonymous, nonsynonymous, DRM, stop or nonsense/missense) are shown in the legend. Note, except for WT, the coloration is not preserved between figures (i.e., the yellow in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006358#ppat.1006358.g003" target="_blank">Fig 3</a> does not represent the same genotype as the yellow in Fig 4). Rx1 is treatment FTC+TFV+EFV.</p

Summary of results from a subset of HIV-1 compartmentalization studies.

<p>Summary of results from a subset of HIV-1 compartmentalization studies.</p