Origin and evolution of HIV-1 in breast milk determined by single-genome amplification and sequencing

Abstract

HIV transmission via breastfeeding accounts for a considerable proportion of infant HIV acquisition. However, the origin and evolution of the virus population in breast milk, the likely reservoir of transmitted virus variants, are not well characterized. In this study, HIV envelope (env) genes were sequenced from virus variants amplified by single-genome amplification from plasmas and milk of 12 chronically HIV-infected, lactating Malawian women. Maximum likelihood trees and statistical tests of compartmentalization revealed interspersion of plasma and milk HIV env sequences in the majority of subjects, indicating limited or no compartmentalization of milk virus variants. However, phylogenetic tree analysis further revealed monotypic virus variants that were significantly more frequent in milk (median proportion of identical viruses, 29.5%; range, 0 to 61%) than in plasma (median proportion of identical viruses, 0%; range, 0 to 26%) (P = 0.002), suggesting local virus replication in the breast milk compartment. Moreover, clonally amplified virus env genes in milk produced functional virus Envs that were all CCR5 tropic. Milk and plasma virus Envs had similar predicted phenotypes and neutralization sensitivities to broadly neutralizing antibodies in both transmitting and nontransmitting mothers. Finally, phylogenetic comparison of longitudinal milk and plasma virus env sequences revealed synchronous virus evolution and new clonal amplification of evolved virus env genes in milk. The limited compartmentalization and the clonal amplification of evolving, functional viruses in milk indicate continual seeding of the mammary gland by blood virus variants, followed by transient local replication of these variants in the breast milk compartment.

FIG. 1.

HIV variants in milk do not appear to be compartmentalized from plasma virus variants in phylogenetic analyses yet display a higher frequency of genetically identical virus variants than that of plasma virus variants. Trees from maximum likelihood analyses are shown for full-length HIV env RNA nucleotide sequences amplified by single-genome amplification from milk (dark blue circles) and plasma (red circles) from 12 chronically HIV-infected Malawian women (A to L). HIV env sequences amplified from milk from the left breast are identified with filled circles, and those amplified from milk from the right breast are noted with stars. Open blue bars represent groups of identical viruses from the right breast, and filled blue bars represent groups of identical viruses from the left breast. Numerals at nodes indicate approximate likelihood ratio test values of ≥0.95. The scale bar represents 0.01 nuclear substitution per site. Groups of identical env sequences in plasma (red bars) or breast milk (blue bars) are indicated and numbered.

FIG. 1.

HIV variants in milk do not appear to be compartmentalized from plasma virus variants in phylogenetic analyses yet display a higher frequency of genetically identical virus variants than that of plasma virus variants. Trees from maximum likelihood analyses are shown for full-length HIV env RNA nucleotide sequences amplified by single-genome amplification from milk (dark blue circles) and plasma (red circles) from 12 chronically HIV-infected Malawian women (A to L). HIV env sequences amplified from milk from the left breast are identified with filled circles, and those amplified from milk from the right breast are noted with stars. Open blue bars represent groups of identical viruses from the right breast, and filled blue bars represent groups of identical viruses from the left breast. Numerals at nodes indicate approximate likelihood ratio test values of ≥0.95. The scale bar represents 0.01 nuclear substitution per site. Groups of identical env sequences in plasma (red bars) or breast milk (blue bars) are indicated and numbered.

FIG. 2.

Milk and plasma HIV variants evolve similarly over time, with clonal amplification of new virus variants in milk. Trees from maximum likelihood analyses are shown for full-length HIV env RNA nucleotide sequences amplified by single-genome amplification from milk (dark blue circles) and plasmas (red circles) obtained at 4 to 5 weeks and from milk (light blue squares) and plasmas (pink squares) obtained 3 months after delivery from two chronically HIV-infected, lactating women. Numerals at nodes indicate approximate likelihood ratio test values of ≥0.95. The scale bar represents 0.01 nuclear substitution per site. Groups of identical sequences in plasma at 4 to 5 weeks (red bars) and 3 months (pink bars) or in breast milk at 4 to 5 weeks (dark blue bars) and 3 months (light blue bars) are indicated.