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## Results: 6

1.

**Neighbor-joining tree representing full-length SGA**. Blue and red sequences represent the env SGA sequences from acutely infected ZM247F (blue) and superinfected (red) viral sequences. Black sequences are derived from ZM247M, the epidemiologically unlinked chronically infected partner. Bootstrap values > 80 are considered statistically significant. Sequences denoted by circles indicate the parental sequences (blue, red circle), and stars denote potential recombinant daughter sequences (purple stars) that were used for recombination analyses (below).

*env*sequences for ZM247F2.

**Neighbor-joining trees of SGA**. (

*env*and population gp41 sequences for the cohort**A**) Neighbor-joining phylogenetic tree of single genome amplified

*env*gene sequences from each superinfected individual in the context of Zambian sub-type C

*env*sequences. The Zambian subtype C sequences include twelve of the non-superinfected, newly infected individuals from this study. Superinfected individuals are assigned discrete colors and the superinfecting quasispecies is denoted by SI. (

**B**) Neighbor-joining tree of gp41 sequences for all 22 couples. Superinfected individuals are assigned discrete colors.

3.

**Highlighter recombination plots of full-length**. Highlighter recombination analysis of sequences from ZM282M (

*env*for each individual who was superinfected**A**), ZM247F (

**B**), and ZM211M (

**C**) and ZM211F (

**D**). The parental virus sequence from the acutely infected or a representative parental sequence from the chronically infected partner (ZM211M) are shown in blue in the first bar of each panel. The superinfecting parental sequence shown in red is the second bar. The presumptive recombinant daughter sequences from three representative time points are shown below the parents for each case of superinfection.

4.

**Neighbor-joining tree of full length SGA**. (

*env*sequences for ZM211M and ZM211F**A**) The chronically infected ZM211M sequences are depicted in blue, and the superinfecting ZM211F sequences are depicted in red, while those in purple represent potential recombinant sequences between the blue and red sequences. (

**B**) An expanded phylogenetic tree showing time points 0-36 months for the acutely infected ZM211F initial infecting virus (blue) is distinct from ZM211M (black). Sequences denoted by circles indicate the parental sequences (blue, red circle) and stars denote potential recombinant daughter sequences (purple stars) that were used for recombination analyses described below.

5.

**Neighbor-joining tree of full-length SGA**. Blue and red sequences represent the initial infection and superinfection sequences, respectively, from the acutely infected subject ZM282M; black sequences are derived from the epidemiologically unlinked cohabiting partner ZM282F, who is chronically infected. The time points are indicated along with M or F for each sequence (i.e. M_8 is the acutely infected ZM282M at 8 months post seroconversion). The ZM282F "0" time point corresponds to seroconversion of ZM282M. The duration of infection for ZM282F is not known. Bootstrap values > 80 are considered statistically significant. Sequences denoted by circles indicate the parental sequences (blue, red circle) and those denoted by stars identify potential recombinant daughter sequences that were used for recombination analyses (below).

*env*sequences for ZM282M6.

**Sequence and Highlighter Analysis of Longitudinal Samples Provides Evidence for Superinfection**. Comparison of maximum sequence divergence in gp41 versus the maximum number of degenerate bases at any time point within acutely infected individuals (

**A**) and chronically infected individuals (

**B**). The maximum percentage of degenerate bases is plotted on the y-axis; the maximum percentage of genetic distance is plotted along the x-axis. Black arrows indicate superinfected subjects; arrowhead indicates a subject co-infected with two variants from a single donor. Highlighter plots for gp41 sequences of ZM289M (

**C**), ZM282M (

**D**), ZM247F (

**E**), and ZM211 F (

**F**) sampled at 0 to 36 months post-seroconversion (shown on right of plot). The sequence at each time point is compared to the initial infecting HIV-1 gp41 sequence. Tick marks denote nucleotide changes from the seroconversion sequence (T = red, A = green, C = blue, G = yellow), with dark blue indicating degenerate bases (See Methods).