Relative replication capacity of phenotypic SIV variants during primary infections differs with route of inoculation

Abstract

Background: Previous studies of human and simian immunodeficiency virus (HIV and SIV) have demonstrated that adaptive mutations selected during the course of infection alter viral replicative fitness, persistence, and pathogenicity. What is unclear from those studies is the impact of transmission on the replication and pathogenicity of the founding virus population. Using the SIV-macaque model, we examined whether the route of infection would affect the establishment and replication of two SIVmne variants of distinct in vitro and in vivo biological characteristics. For these studies, we performed dual-virus inoculations of pig-tailed macaques via intrarectal or intravenous routes with SIVmneCl8, a minimally pathogenic virus, and SIVmne027, a highly pathogenic variant that replicates more robustly in CD4+ T cells.

Results: The data demonstrate that SIVmne027 is the dominant virus regardless of the route of infection, indicating that the capacity to replicate efficiently in CD4+ T cells is important for fitness. Interestingly, in comparison to intravenous co-infection, intrarectal inoculation enabled greater relative replication of the less pathogenic virus, SIVmneCl8. Moreover, a higher level of SIVmneCl8 replication during primary infection of the intrarectally inoculated macaques was associated with lower overall plasma viral load and slower decline in CD4+ T cells, even though SIVmne027 eventually became the dominant virus.

Conclusions: These results suggest that the capacity to replicate in CD4+ T cells is a significant determinant of SIV fitness and pathogenicity. Furthermore, the data also suggest that mucosal transmission may support early replication of phenotypically diverse variants, while slowing the rate of CD4+ T cell decline during the initial stages of infection.

Figure 1

Quantitative real-time RT-PCR to measure relative levels of SIVmneCl8 and SIVmne027. The real-time RT-PCR assay was developed to detect two regions of the viral genome, a conserved gag sequence and an env V1 sequence specific to SIVmneCl8. The relative amount of SIVmneCl8 was determined from the amount of viral RNA detected by the SIVmneCl8 env V1 specific primer/probe set compared to the total amount of viral RNA detected by the primer/probe set recognizing the conserved gag sequence. Single-virus infections of activated pig-tail PBL with SIVmneCl8 (a) or SIVmne027 (b) shows that only SIVmneCl8 is recognized by the env V1 primer/probe, even when the overall viral RNA level is greater than 1 × 10⁹ viral RNA copies/ml of SIVmne027.

Figure 2

Dual-virus competition assays in primary pig-tailed macaque cells. Activated PBLs (a), DC/T cell co-cultures (b), and macrophages (c) were infected with equal doses of SIVmneCl8 and SIVmne027. Total virus was determined by measuring consensus sequence gag RNA transcripts (--). SIVmneCl8 viral RNA levels were measures and are reported as percentages of total viral RNA (····). Three representative experiments (black circle/open circle, black triangle/open triangle, and black square/open square) are shown for each graph and viral RNA values represent the average reading for each time point. Each infection used cells from a different macaque blood donor.

Figure 3

Plasma viral loads from in vivo co-infections. Macaques were infected with equal doses of SIVmneCl8 and SIVmne027 using either an intravenous route of inoculation (a), animals 29046 (gray circle/open circle), 29047 (gray triangle/open triangle) and 29048 (gray square/open square) or an intrarectal route of inoculation (b), animals 28488 (black circle/open circle), 28489 (black triangle/open triangle) and 28490 (black square/open square). Total virus in plasma was determined by measuring consensus sequence gag RNA transcripts (--) by quantitative real-time PCR. SIVmneCl8 specific viral RNA levels were also measured by quantitative real-time PCR and are reported as percentages of total viral RNA (····). Viral RNA values represent the average reading for each time point.

Figure 4

Comparison of IV vs IR viral loads and CD4⁺ T cell counts from SIVmneCl8/SIVmne027 co-inoculated macaques. Total plasma viral RNA transcripts (a), SIVmneCl8 env transcripts (b), and CD4⁺ T cell levels (c) from intravenously infected macaques (gray circles, triangles, and squares) and intrarectally infected macaques (black circles, triangles, and squares) are shown. Viral RNA measurements were determined as in Fig. 3. Values represent average readings for each time point ± the standard error. Levels of significance are denoted as follows: (**) p < 0.05, (*) p = 0.05. In panel (a) the p-value at peak plasma viral load is 0.0007. The p-value for differences in viral set-point at 20 and 24 weeks post-inoculation are 0.0544 and 0.0509, respectively. For panel (b) p-values at 2, 4, and 8 weeks post-inoculation are 0.049, 0.006, and 0.005, respectively. For panel (c) the p-value for period 0-8 weeks post-inoculation is 0.0013, and the p-value for period 8-24 weeks post-inoculation is 0.0006.

Figure 5

Comparison of central memory CD4⁺ T cells in intrarectally-infected and intravenously-infected pig-tailed macaques. Individual data points from intrarectally-infected animals (thin dotted lines with open symbols) with the predicted mean value (thick dotted line; Group 1) were compared with data from intravenously-infected animals (thin solid lines with closed symbols) with the predicted mean value (thick solid line; Group 2). Significant differences were found at all time points from week 1 through week 20. P-values for all time points (0, 1, 2, 4, 6, 8, 12, 16, 20, 24) are given here: 0.0528, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, 0.0003, 0.0009, 0.0585 and 0.9761 respectively.