Viral Diversity and Diversification of Major Non-Structural Genes vif, vpr, vpu, tat exon 1 and rev exon 1 during Primary HIV-1 Subtype C Infection

To assess the level of intra-patient diversity and evolution of HIV-1C non-structural genes in primary infection, viral quasispecies obtained by single genome amplification (SGA) at multiple sampling timepoints up to 500 days post-seroconversion (p/s) were analyzed. The mean intra-patient diversity was 0.11% (95% CI; 0.02 to 0.20) for vif, 0.23% (95% CI; 0.08 to 0.38) for vpr, 0.35% (95% CI; −0.05 to 0.75) for vpu, 0.18% (95% CI; 0.01 to 0.35) for tat exon 1 and 0.30% (95% CI; 0.02 to 0.58) for rev exon 1 during the time period 0 to 90 days p/s. The intra-patient diversity increased gradually in all non-structural genes over the first year of HIV-1 infection, which was evident from the vif mean intra-patient diversity of 0.46% (95% CI; 0.28 to 0.64), vpr 0.44% (95% CI; 0.24 to 0.64), vpu 0.84% (95% CI; 0.55 to 1.13), tat exon 1 0.35% (95% CI; 0.14 to 0.56 ) and rev exon 1 0.42% (95% CI; 0.18 to 0.66) during the time period of 181 to 500 days p/s. There was a statistically significant increase in viral diversity for vif (p = 0.013) and vpu (p = 0.002). No associations between levels of viral diversity within the non-structural genes and HIV-1 RNA load during primary infection were found. The study details the dynamics of the non-structural viral genes during the early stages of HIV-1C infection

Timing Constraints of In Vivo Gag Mutations during Primary HIV-1 Subtype C Infection

Background: Aiming to answer the broad question “When does mutation occur?” this study examined the time of appearance, dominance, and completeness of in vivo Gag mutations in primary HIV-1 subtype C infection. Methods: A primary HIV-1C infection cohort comprised of 8 acutely and 34 recently infected subjects were followed frequently up to 500 days post-seroconversion (p/s). Gag mutations were analyzed by employing single-genome amplification and direct sequencing. Gag mutations were determined in relation to the estimated time of seroconversion. Time of appearance, dominance, and completeness was compared for different types of in vivo Gag mutations. Results: Reverse mutations to the wild type appeared at a median (IQR) of 62 (44;139) days p/s, while escape mutations from the wild type appeared at 234 (169;326) days p/s (p<0.001). Within the subset of mutations that became dominant, reverse and escape mutations appeared at 54 (30;78) days p/s and 104 (47;198) days p/s, respectively (p<0.001). Among the mutations that reached completeness, reverse and escape mutations appeared at 54 (30;78) days p/s and 90 (44;196) days p/s, respectively (p = 0.006). Time of dominance for reverse mutations to and escape mutations from the wild type was 58 (44;105) days p/s and 219 (90;326) days p/s, respectively (p<0.001). Time of completeness for reverse and escape mutations was 152 (100;176) days p/s and 243 (101;370) days p/s, respectively (p = 0.001). Fitting a Cox proportional hazards model with frailties confirmed a significantly earlier time of appearance (hazard ratio (HR): 2.6; 95% CI: 2.3–3.0), dominance (4.8 (3.4–6.8)), and completeness (3.6 (2.3–5.5)) of reverse mutations to the wild type Gag than escape mutations from the wild type. Some complex mutational pathways in Gag included sequential series of reversions and escapes. Conclusions: The study identified the timing of different types of in vivo Gag mutations in primary HIV-1 subtype C infection in relation to the estimated time of seroconversion. Overall, the in vivo reverse mutations to the wild type occurred significantly earlier than escape mutations from the wild type. This shorter time to incidence of reverse mutations remained in the subsets of in vivo Gag mutations that reached dominance or completeness

Transmission of single and multiple viral variants in primary HIV-1 subtype C infection.

To address whether sequences of viral gag and env quasispecies collected during the early post-acute period can be utilized to determine multiplicity of transmitted HIV's, recently developed approaches for analysis of viral evolution in acute HIV-1 infection [1,2] were applied. Specifically, phylogenetic reconstruction, inter- and intra-patient distribution of maximum and mean genetic distances, analysis of Poisson fitness, shape of highlighter plots, recombination analysis, and estimation of time to the most recent common ancestor (tMRCA) were utilized for resolving multiplicity of HIV-1 transmission in a set of viral quasispecies collected within 50 days post-seroconversion (p/s) in 25 HIV-infected individuals with estimated time of seroconversion. The decision on multiplicity of HIV infection was made based on the model's fit with, or failure to explain, the observed extent of viral sequence heterogeneity. The initial analysis was based on phylogeny, inter-patient distribution of maximum and mean distances, and Poisson fitness, and was able to resolve multiplicity of HIV transmission in 20 of 25 (80%) cases. Additional analysis involved distribution of individual viral distances, highlighter plots, recombination analysis, and estimation of tMRCA, and resolved 4 of the 5 remaining cases. Overall, transmission of a single viral variant was identified in 16 of 25 (64%) cases, and transmission of multiple variants was evident in 8 of 25 (32%) cases. In one case multiplicity of HIV-1 transmission could not be determined. In primary HIV-1 subtype C infection, samples collected within 50 days p/s and analyzed by a single-genome amplification/sequencing technique can provide reliable identification of transmission multiplicity in 24 of 25 (96%) cases. Observed transmission frequency of a single viral variant and multiple viral variants were within the ranges of 64% to 68%, and 32% to 36%, respectively

HIV-1 subtype C phylogenetic relationship and diversity of HIV non-structural genes is consistent with the multiplicity of HIV-1 infection determined by analysis of the <i>env</i>/<i>gag</i> genes.

<p>A maximum likelihood phylogenetic tree was reconstructed using Fastree2 (Price <i>et al</i>., 2010) using the GTR+G model for nucleotide substitution and visualized in Figtree v.1.1.3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035491#pone.0035491-Rambaut1" target="_blank">[54]</a>. Alternative likelihood ratio tests <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035491#pone.0035491-Anisimova1" target="_blank">[55]</a> >0.95 are shown by an asterisk. Subjects infected with multiple viral variants are colored red. Patient B and D subtrees (individual trees on grey background) show branching topology of earliest sampling (0–90 days p/s) and represent examples of single (subject B) and multiple (subject D) HIV-1 transmission.</p

Transmission of Single and Multiple Viral Variants in Primary HIV-1 Subtype C Infection

To address whether sequences of viral gag and env quasispecies collected during the early post-acute period can be utilized to determine multiplicity of transmitted HIV’s, recently developed approaches for analysis of viral evolution in acute HIV-1 infection [1,2] were applied. Specifically, phylogenetic reconstruction, inter- and intra-patient distribution of maximum and mean genetic distances, analysis of Poisson fitness, shape of highlighter plots, recombination analysis, and estimation of time to the most recent common ancestor (tMRCA) were utilized for resolving multiplicity of HIV-1 transmission in a set of viral quasispecies collected within 50 days post-seroconversion (p/s) in 25 HIV-infected individuals with estimated time of seroconversion. The decision on multiplicity of HIV infection was made based on the model’s fit with, or failure to explain, the observed extent of viral sequence heterogeneity. The initial analysis was based on phylogeny, inter-patient distribution of maximum and mean distances, and Poisson fitness, and was able to resolve multiplicity of HIV transmission in 20 of 25 (80%) cases. Additional analysis involved distribution of individual viral distances, highlighter plots, recombination analysis, and estimation of tMRCA, and resolved 4 of the 5 remaining cases. Overall, transmission of a single viral variant was identified in 16 of 25 (64%) cases, and transmission of multiple variants was evident in 8 of 25 (32%) cases. In one case multiplicity of HIV-1 transmission could not be determined. In primary HIV-1 subtype C infection, samples collected within 50 days p/s and analyzed by a single-genome amplification/sequencing technique can provide reliable identification o

Individual distribution of pairwise distances for each of the non-stuctural genes,<i>vif</i> (HXB2 start 5041 to 5619), <i>vpr</i> (HXB2 start 5559 to 5850), <i>vpu</i> (HXB2 start 6062 to 6310), <i>tat</i> exon 1 (HXB2 start 5831 to 6045), and <i>rev</i> exon 1(HXB2 start 5970 to 6045).

<p>Individual distribution of pairwise distances for each of the non-stuctural genes,<i>vif</i> (HXB2 start 5041 to 5619), <i>vpr</i> (HXB2 start 5559 to 5850), <i>vpu</i> (HXB2 start 6062 to 6310), <i>tat</i> exon 1 (HXB2 start 5831 to 6045), and <i>rev</i> exon 1(HXB2 start 5970 to 6045).</p

HIV-1 subtyping by analysis of phylogenetic relationships of HIV-1 non-structural genes.

<p>The analyzed region of HIV-1 genome corresponded to nucleotide positions 5,041 to 6,310 in HXB2. Three sequences were randomly selected for each study subject (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035491#s2" target="_blank">Methods</a>). A phylogenetic tree was inferred by Mr. Bayes using GTR model. The convergence was reached after 10 M MCMC run. The consensus tree was visualized in Figtree v.1.3.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035491#pone.0035491-Rambaut1" target="_blank">[54]</a>. Clade credibility values of >0.95 shown by asterisk, Subtype D cluster showed the support of 0.93 indicated by + symbol. HIV-1 subtype C reference sequences are shown as blue circles. All non-subtype C group M reference sequences are shown at the bottom of the phylogenetic tree. SIV sequence (CPZ.CM98.CAM3.AF115393) was used as an outgroup.</p

HIV-1C diversity, mean and 95% confidence intervals for non-structural genes <i>vif</i>, <i>vpr</i>, <i>vpu</i>, <i>tat</i> exon 1 and <i>rev</i> exon 1 over the first 500 days p/s.

<p>Viral diversity for each subject was calculated using maximum composite likelihood model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035491#pone.0035491-Tamura2" target="_blank">[56]</a>.</p

Summary table of second-step analysis for transmission of multiple HIV-1 variants in five subjects.

<p>The table includes the following sections: study subjects with corresponding Fiebig stage and time of sampling in days p/s; cumulative results of distribution of pairwise distances based on six analyses per gene (ML-corrected distances, K2P-corrected distances, Hamming distances, ML-corrected distances to MRCA, K2P-corrected distances to consensus sequence, and Hamming distances to consensus sequence); analysis of highlighter plots; cumulative results of recombination analysis; tMRCA; number of methods suggesting transmission of single and multiple viral variants; and conclusion regarding multiplicity of HIV-1 transmission. Numeric coding: 0 – transmission of single viral variant; 1 – undetermined; 2 – transmission of multiple viral variants. Numeric coding of “1” and “2” are further enhanced by blue and light red colors.</p