Genetic analysis of simian immunodeficiency virus expressed in milk and selectively transmitted through breastfeeding

Abstract

To develop effective intervention strategies that prevent breast milk transmission of human immunodeficiency virus (HIV), we must understand the specific viral properties and mechanisms responsible for infant infection. We have used lactating rhesus macaques infected with a pathogenic simian immunodeficiency virus (SIV) stock to analyze the viral genotypes expressed in plasma and milk throughout the disease course and to identify those variants ultimately transmitted to infants through breastfeeding. In these studies we observed mother-to-infant transmission of SIV/Delta(B670) by eight females during the chronic phase of disease, and we analyzed by heteroduplex tracking assays and sequence analysis the distribution and fluctuations in viral genotypes expressed. Each female expressed multiple V1 envelope genotypes in milk near the time of transmission, while a single genotype was found in each of the infants. Variants transmitted to infants were not expressed throughout the maternal disease course but were only detected near the time of transmission. The emergence of the transmitted genotype in the dam typically occurred in plasma before milk and was coincident with increased milk viral loads. Transmitted genotypes tended to be longer and more glycosylated and had a less negative charge over the V1 region compared to viral genotypes expressed in milk but not transmitted. These observations demonstrate that specific viral genotypes are selectively transmitted to infants through breastfeeding and support the hypothesis that transmission occurs as genotypes adapt for efficient expression in milk.

FIG. 1.

Deduced amino acid sequences from the envelope V1 region of virus expressed in milk of dams inoculated with SIV/Delta_B670. Viral sequences were amplified from milk supernatant by RT-PCR at the indicated time point postinoculation, cloned, and sequenced. The numbers of clones with identical amino acid sequences are indicated in parentheses. Infant sequences were amplified from PBMC DNA at the first time point of SIV detection, shown as days p.i. of the dam. (A) Alignments of milk virus sequences found in eight females that transmitted virus to their infants during the chronic phase of disease. Sequences are aligned with the viral genotype found in the infant. (B) Milk sequences found in two rapid transmitting-rapid progressor dams at 14 days p.i. aligned with genotypes found in the infant. (C) Sequences found in milk of three nontransmitting females at time points later in the disease course aligned with sequences from the SIV/Delta_B670 stock.

FIG. 2.

Comparison of V1 sequence characteristics of transmitted genotypes (black bars) and nontransmitted genotypes (white bars) expressed in milk of eight transmitting dams near the time of infant infection (sequences shown in Fig. 1A). V1 sequences transmitted to the infant were compared to genotypes expressed in milk that were not transmitted to evaluate (A) the number of amino acids in the V1 loop, (B) the number of potential N-linked glycosylation sites, and (C) the net charge of the V1 region.

FIG. 3.

Heteroduplex tracking assay gels of envelope V1 genotypes expressed in milk samples throughout the disease course of four transmitting females. Single-stranded ³²P-labeled probes (SS probe) were made from genotypes identified in infants. Homoduplex bands, indicating sequence identity to the probe, are shown in lane 1 of each gel. The time when transmission to the infant occurred is indicated (↓).

FIG. 4.

Analysis of viral expression in transmitting female P168. (A) Heteroduplex tracking assays evaluating V1 genotypes expressed in plasma at indicated times p.i. A single-stranded ³²P-labeled probe (SS probe) was made from the infant genotype. The time point when transmission to the infant occurred as indicated (↓). (B) SIV RNA copies per milliliter of milk or plasma as determined by real-time RT-PCR amplification at the indicated time points p.i. (C) Deduced amino acid sequences of envelope VI genotypes expressed in milk or plasma and proviral sequences from PBMC. Sequences were obtained by PCR amplification, followed by cloning and sequencing. The numbers of clones containing identical sequences are indicated in parentheses. Sequences identified in infants are also shown. Material and infant sequences are aligned with the most similar B670 stock genotype as determined by Clustal W alignments.

FIG. 5.

Analysis of viral expression in transmitting female P403. See the legend to Fig. 4.