Flow of cluster analysis.

<p>A total of 2,219 HIV-1C <i>env</i> sequences were included. After re-alignment of variable loops, the first step ML analysis was implemented by FastTree2, which selected 969 sequences based on the Shimodira-Hasegawa test for splits support ≥0.98. Two ML analyses implemented by PhyML and RAxML, and ME analysis using MCL model, were performed in the second step. Clusters were identified based on bootstrap support of ≥80% in at least 2 of 3 methods in the second step.</p

The HIV-1C <i>env</i> gp120 V1–C5 signature sequences across 22 clusters with 3+ members, VESPA analysis.

<p>The HIV-1C <i>env</i> gp120 V1–C5 signature sequences across 22 clusters with 3+ members, VESPA analysis.</p

Selected countries representation of HIV-1C <i>env</i> sequences included in analysis.

<p>Selected countries representation of HIV-1C <i>env</i> sequences included in analysis.</p

Phylogenetic relatedness of HIV-1C <i>env</i> sequences based on the ML_FastTree2 + ML_PhyML analysis.

<p>The identified clusters with bootstrap support of ≥80% are collapsed. The collapsed clusters shown in red represent Mochudi-unique clusters; in blue, Mochudi mixed clusters with other Botswana sequences; in orange, non-Mochudi clusters with Botswana sequences; and in green, non-Botswana clusters. Clusters with mother-infant pairs from two MTCT studies in Malawi are shown by asterisks <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080589#pone.0080589-Russell1" target="_blank">[78]</a> and green circle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080589#pone.0080589-Kumar1" target="_blank">[79]</a>.</p

Examples of individual clusters with 3+ members.

<p>The relevant patient-specific data (prevalent or incident HIV infection, gender, age, HIV-1 RNA, CD4 count and ART status) are connected by dotted lines with the tree branches corresponding to genotyped subjects. Note: Subjects reported receiving ART through the National ARV program during the household surveys. <b>A:</b> Mochudi-unique cluster #125 with 11 members. Subjects cohabited in the same household are indicated with asterisks. <b>B:</b> Mochudi-unique cluster #162 with 11 members. <b>C:</b> Mochudi-unique cluster #84 with 6 members. <b>D:</b> Mochudi-mixed cluster#151 with 6 members. Viral sequences from Mochudi are shown as yellow squares, while sequences from outside of Mochudi are denoted by orange circles. <b>E:</b> Mochudi-unique cluster #94 with 5 members. <b>F:</b> Mochudi unique cluster #120 with 5 members.</p

Phylogenetic relationships between Mochudi seroconverters.

<p>The tree was generated by ML_PhyML with 968 HIV-1C <i>env</i> sequences from Botswana. Clustered sequences with bootstrap support of splits ≥80% are shown in red. The incident HIV-1C cases from Mochudi are highlighted by circles: clustered seroconverters are indicated with filled red circles, while non-clustered seroconverters are shown with open circles.</p

CD4 levels in the prospective cohort of individuals with primary HIV-1C infection in Botswana and seroconveters in this study.

<p> post-seroconversion, p/s.</p

Comparison of HIV-1 RNA load within clustered and non-clustered sequences in ART-naïve individuals.

<p>The estimated p-values by Wilcoxon test are shown above each graph. <b>A:</b> The entire set of ART-naïve individuals. <b>B:</b> ART-naïve males. <b>C:</b> ART-naïve females.</p