Research ArticleHEALTH AND MEDICINE

Defining early SIV replication and dissemination dynamics following vaginal transmission

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Science Advances  29 May 2019:
Vol. 5, no. 5, eaav7116
DOI: 10.1126/sciadv.aav7116
  • Fig. 1 Plasma viral load (pVL) data.

    Longitudinal pVL determined by qRT-PCR from animals during ramp up and necropsy with day of necropsy indicated by different colors (red, d14; orange, d8; pink, d7; blue, d6; green, d5; cyan, d3). Open symbols represent animals with a plasma viremia of less than 30 copies/ml (limit of detection) at necropsy.

  • Fig. 2 Viral nucleic acid distribution during primary infection.

    Viral RNA (vRNA) (A) and viral DNA (vDNA) (B) quantities were assessed by qRT-PCR or qPCR from nucleic acids obtained from ~1-cm3 pieces of tissue. Individual tissue viral loads were averaged and plotted for each tissue indicated. Background shading indicates different types of tissues [green, FGT; yellow, lymphatic system; purple, gastrointestinal (GI) tract; blue, bone marrow and peripheral blood mononuclear cells (PBMCs)]. Individual symbols are color-coded based on days after infection until necropsy (red, d14; orange, d8; pink, d7; blue, d6; green, d5; cyan, d3) with animals grouped based on phases of infection (negative, local, transitional, and systemic). Missing symbols indicate no data available. Open symbols indicate no detectable vRNA or vDNA. For simplicity, the limit of detection for each tissue type is not identified in this figure (see fig. S4 for limit of detection per sample).

  • Fig. 3 Proportion and quantification of individual variants.

    (A to C) Schematic representation of the three distinct possible mechanisms for multilineage infection; (A) multiple breaches in mucosal barrier allowing for a single variant each, (B) single breach where all detected lineages enter, and (C) a few breaches each containing more than one lineage. Nucleic acid–positive tissues are displayed as the height of the bar corresponding to the log scale on the left axis. The relative proportion of each viral variant is represented by color coding within each bar representing the linear proportion of variants A to wild type (WT). Individual foci of infection are indicated schematically within the FGT. In addition to PCR-positive tissue, RNAscope positive sites are indicated (+). Animal D290 (D) has detectable virus within the FGT and within some draining and distal LNs, GI tract, bone marrow, and plasma. Animal E048 (E) has detectable virus with multiple variants both locally and within some draining and distal LNs, spleen, GI tract, and plasma. Individual sections from the cervix of ELV were stained for vRNA, and highlighted regions were captured for DNA extraction and sequencing (F and G). The proportion of each variant is shown in the pie chart adjacent to the region isolated, with the number of sequences indicated within the pie chart. Cervix 1 tissues were analyzed in two separate sections containing one to three distinct variants. Cervix 2 contained WT virus found in two distinct aggregates (H). Scale bars, 100 μm.

  • Fig. 4 Proportion of individual variants using NGS.

    The proportion of each variant was determined in a subset of SIV-positive tissues, cells, and plasma using NGS and is plotted for animal D290 necropsied at day 8 (A) and animal EZN necropsied at day 14 (B). Rectangular symbols are used for each variant (A to WT; legend on the right). The lower limit of detection is indicated with a black line if above 0.01%. All other samples had a limit of detection less than 0.01%. Left of dashed line, tissue samples; right of dashed line, plasma samples.

  • Fig. 5 A tissue infection threshold for viral dissemination to plasma.

    (A) The sum of vRNA and vDNA for all pieces of the FGT and for LNs/spleen is plotted for animals with different levels of viral dissemination. Viremic animals are indicated in red, delayed animals are in blue, transitional animals are in green, and negative animals are in orange. Open symbols indicate undetected in plasma. Gray shading indicates the approximate threshold level of tissue infection observed in animals that had detectable plasma virus. (B) The proportion of the total viral load for each individual lineage was plotted with the same symbol as the total but shaded. Where individual lineages were not found in plasma, this is displayed as an open symbol. Gray-filled symbols represent variant D. Similar threshold levels of infection appear to apply for individual lineages, since only a minority of lineages with concentrations below the threshold are observed in plasma. The limit of detection is indicated by a dashed line.

  • Fig. 6 Using estimated growth rates to estimate the time to detectable viral load.

    (A) Viral load data for viremic animals following vaginal infection (n = 5) were plotted with 15 intravenously (iv) infected animals. Viremic animals and animals sampled at days 3 and 5 fall within the animal-to-animal variability of exponential growth. Animals sampled after day 6 are outside of this range and are classified as delayed. (B) For individual viral lineages within each animal, the proportion of the total viral load is plotted with and the time each sample would reach 30 copies/ml are shown assuming a constant growth rate for each lineage. The differences in time to detectable viremia between individual lineages within the same animal can be as high as 5 days.

  • Fig. 7 Early recruitment of CD4+T cells and antiviral factors.

    CD4+ T cell recruitment to FGT was monitored by quantitative IHC image analysis. CD4 recruitment was correlated with vRNA (A) and vDNA (B) within the FGT. Representative images of CD4+ T cell recruitment (brown) are shown during local (C), transitional (D), and systemic (E) infection with macrophages (red). RNAscope was used to localize and identify the cell morphology of vRNA+ cells in the FGT from animals displaying local (F), transitional (G), and systemic (H) infection. The cell morphology is consistent with CD4+ T cells being the dominant target cell following vaginal transmission. Scale bars, 100 μm. Quantitative image analysis was used to identify the increase in Mx1 (I) and APOBEC3G (J) expression during progressive infection, which correlated to CD4+ T cell recruitment. The relative expression levels of Mx2, APOBEC3G, Tetherin, and TRIM5α were quantified together in SIV-negative or SIV-infected CD4+ cells using fluorescent confocal microscopy and expressed as the median fluorescence intensity (MFI) per T cell. Neither transitional nor systemic animals exhibited any difference in the MFI between SIV-positive and SIV-negative cells (K).

  • Fig. 8 Accumulation of Mx1 in draining and distal LNs during progressive infection.

    Mx1 staining (brown) in FGT draining and distal LNs from an SIV-negative animal (A), local infection animal D226 (B), transitional infection animal E024 (C), and systemic infection animal D290 (D). Mx1 was increased in the draining LNs as early as during the local infection phase, which progressed throughout the rest of the phases of infection. Distal LNs showed evidence of up-regulated Mx1 commencing only in animals in the transitional phase, which increased further as animals became systemically infected. Scale bars, 100 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaav7116/DC1

    Fig. S1. No viral nucleic acid signal detected in two exposed animals.

    Fig. S2. Detection of rare vRNA- and vDNA-positive cells in draining LN before systemic dissemination.

    Fig. S3. Schematic representation of FGT tissue collection schema.

    Fig. S4. Viral nucleic acid distribution within individual tissue pieces.

    Fig. S5. Proportion and quantification of each variant in virus-positive tissues.

    Fig. S6. Proportional representation of sequences following NGS.

    Fig. S7. Proportion of individual variants using NGS.

    Fig. S8. Growth rates during FGT and systemic viral replication.

    Fig. S9. No significant difference in the local expression of many proinflammatory and AVFs.

    Fig. S10. AVFs measured in SIV-positive and SIV-negative cells.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. No viral nucleic acid signal detected in two exposed animals.
    • Fig. S2. Detection of rare vRNA- and vDNA-positive cells in draining LN before systemic dissemination.
    • Fig. S3. Schematic representation of FGT tissue collection schema.
    • Fig. S4. Viral nucleic acid distribution within individual tissue pieces.
    • Fig. S5. Proportion and quantification of each variant in virus-positive tissues.
    • Fig. S6. Proportional representation of sequences following NGS.
    • Fig. S7. Proportion of individual variants using NGS.
    • Fig. S8. Growth rates during FGT and systemic viral replication.
    • Fig. S9. No significant difference in the local expression of many proinflammatory and AVFs.
    • Fig. S10. AVFs measured in SIV-positive and SIV-negative cells.

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