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

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

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-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

Estimated tMRCA, days.

1<p>Days post-seroconversion.</p>2<p>HPD is the highest posterior density interval, which represents the most compact interval on the selected parameter that contains 95% of the posterior probability. It is a Bayesian analog to a confidence interval.</p>3<p>ESS: Effective Sample Size – should be higher than 100, and characterizes the posterior distribution.</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

Patient characteristics, time of sampling, and number of analyzed <i>gag</i> and <i>env</i> sequences

1<p>Years at the time of sampling.</p>2<p>log₁₀ copies/ml.</p>3<p>log₁₀ copies/10⁶ PBMC.</p>4<p>Date of sampling (first sampling for patients with dual dates of sampling).</p>5<p>Seroconversion.</p>6<p>Fiebig EW, Wright DJ, Rawal BD, Garrett PE, Schumacher RT, et al. (2003) Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection. AIDS 17: 1871-1879.</p>7<p>Subjects A and B had viral sequences available at two time points within 50 days p/s. Both sets were included in analysis.</p

Decision thresholds for transmission of single and multiple HIV-1 variants

<p>Note: The following decision strategy was applied for each analysis of subject's maximum and mean distances within <i>gag</i> and <i>env</i>: A sample with value exceeding the “Multi” threshold was associated with transmission of <i>multiple</i> HIV-1 variants. A sample with value smaller than the “Single” threshold was associated with transmission of a <i>single</i> HIV-1 variant. A sample with value exceeding the “Single” threshold but less than the “Multi” threshold was considered <i>undetermined</i> in relation to multiplicity of HIV-1 transmission.</p

HIV-1 subtype C diversity within 50 days p/s among acutely and recently infected individuals from the Tshedimoso cohort in Botswana: <i>env</i> sequences, NJ tree.

<p>Acutely infected individuals are denoted by a single letter A through H. The patient ID of recently infected individuals has two letters, OC to QT. A subscript next to the patient ID denotes time of sampling in days p/s. Asterisks denote bootstrap values ≥80. The horizontal bar represents genetic distance.</p

HIV-1 subtype C diversity within 50 days p/s among acutely and recently infected individuals from the Tshedimoso cohort in Botswana: <i>gag</i> sequences, NJ tree.

<p>Acutely infected individuals are denoted by a single letter A through H. The patient ID of recently infected individuals has two letters, OC to QT. A subscript next to the patient ID denotes time of sampling in days p/s. Asterisks denote bootstrap values ≥80. The horizontal bar represents genetic distance.</p