Three observations are noteworthy from our study. First, we found only limited compartmentalization of HIV-1 in breast milk specimens, suggesting substantial interchange of viruses between the milk and blood specimens obtained from the participants we studied. Second, monotypic sequences were overrepresented in the HIV-1 populations from milk specimens compared to those from blood specimens, suggesting local HIV-1 production within the breast, possibly from the proliferation of infected cells. Third, the mean genetic distance between milk and blood viruses correlated positively with breast milk Na+ concentrations, suggesting full cycles of viral replication within the breast and that the increased breast milk HIV-1 concentrations observed with mastitis are not simply due to the passage of virus from blood into breast milk.
Viral compartmentalization was detected in a minority of breast milk specimens using a panel of statistical methods commonly used to evaluate population structure. However, among the participants evaluated, no tissue-specific clades were observed in phylogenetic trees. Rather, viral sequences from milk and blood specimens were intermingled, suggesting that viruses mix readily between these fluids. Similarly, studies of HIV-1 populations in the genital tract and lung have found compartmentalization by statistical testing when segregation by fluid/tissue is not apparent in phylogenies (7
). The importance of such compartmentalization is unclear but is consistent with replication or proliferation within a small virus population (i.e., limited effective population size) (7
Detection and quantification of minority sequence variants are dependent on the method of sampling and the number of sequences obtained. We sequenced virus derived from single PCR amplicons, which prevents bias due to resampling of individual variants (9
). Resampling of viral sequences may explain the apparent compartmentalization of HIV-1 in breast milk specimens previously reported for some women (3
). In support of our findings, a study using a heteroduplex tracking assay was unable to distinguish differences between HIV-1 populations from breast milk and blood specimens obtained from 8 women (22
), as did a recent study that cloned viruses but employed methods to avoid resampling of viral templates (20
). The time and expense required for the generation of single-amplicon sequences limits the sample size feasible for phylogenetic analyses and therefore affects the precision of estimates of population differences. We analyzed a large number of sequences, but these sequences were derived from a small number of nonrandomly selected participants, potentially limiting the generalizability of our findings. In addition, it was not possible to perform analyses on cell-free virus populations separately, as few sequences were generated from breast milk specimens with low concentrations of HIV-1 RNA. Finally, because the sequences were derived at a single time point early in lactation, we cannot rule out the possibility that HIV-1 compartmentalization in an individual's breast milk changes significantly over time. Relevant to this last point, a comparison of the compartmentalization of blood and milk when these specimens were collected on the same or different dates gave discrepant results (20
). Viruses in specimens collected on the same dates were rarely compartmentalized, whereas viruses in those collected more than 100 days apart were consistently compartmentalized across statistical analyses, suggesting that significant evolution occurred in this time frame.
Even using methods that preclude resampling, identical sequences were found in the majority of specimens. Indeed, we may have slightly underestimated the number of identical sequences, as approximately half of the sequences were generated by cloning, which we estimate could introduce an average of <1- to 3-base-pair changes per sequence (14
). These monotypic sequences likely reflect very recent viral replication or, in the case of DNA sequences, the clonal expansion of HIV-1-infected cells (2
). Thus, the disproportionate prevalence of monotypic sequences in breast milk specimens compared to those in blood specimens may represent a relatively greater production of HIV-1 in milk. Alternatively, the likelihood of sampling identical sequences from blood specimens compared to that from breast milk specimens may differ due to the large total circulating volume and higher HIV-1 concentrations in blood than in milk. It is also possible that the fact that slightly more blood sequences than milk sequences were obtained by cloning and may include additional errors introduced during PCR resulted in an underestimate of monotypic viruses in blood specimens. In either case, the presence of identical sequences can bias statistical estimates of population structure (2
). Of note, monotypic sequences accounted for half of the instances of compartmentalization detected. Our findings are remarkably similar to those from studies of rhesus macaques (42
) that had large numbers of monotypic SIV env
sequences detected in the breast milk specimens but not in blood specimens. Furthermore, although breast milk and blood simian immunodeficiency virus (SIV) sequences were interspersed in phylogenetic trees, SM testing detected compartmentalization that was again diminished by exclusion of monotypic sequences. We contend that compartmentalization based on monotypic or nearly identical sequences may not reflect a separate viral population but rather might be a snapshot of recent events, namely, a burst of viral replication and/or proliferation of HIV-1-infected cells.
Mastitis is associated with increased breast milk HIV-1 loads, presumably by either influx of virions from the blood and/or local viral production within the breast (15
). We observed a statistically significant positive correlation between breast milk Na+
concentrations and the genetic distance between milk and blood HIV-1 subpopulations. This is consistent with increased replication of HIV-1 within the breast due to mastitis, resulting in divergence of breast milk HIV-1 subpopulations from those in blood. The plausibility of mastitis stimulating local HIV-1 replication in the breast is supported by observations of the effects of infection and inflammation in other settings. For example, increased production of HIV-1 in lungs (29
) and lymph nodes (39
) has been associated with opportunistic infections, and viral replication can be stimulated in T cells by various inflammatory cytokines (27
When inflammation renders mammary epithelial tight junctions permeable, HIV-1 from the blood may more easily enter breast milk, as occurs with other blood components (15
). Although the passage of HIV-1 from blood into breast milk may contribute to the increase in milk HIV-1 concentrations due to mastitis, our observations suggest that this is not the sole mechanism, because an influx of blood viruses would decrease the genetic differences between these subpopulations. While the absolute divergence of breast milk HIV-1 we detected in mastitic milk was small, it is important to note that the duration of increased viral replication would at most be 6 to 16 weeks in duration due to the timing of sample collection after the onset of lactation. In addition, the magnitude of the genetic differences between milk and blood HIV-1 may have been diminished by the countervailing contribution of blood virus leaking into milk. Of note, although there was a significant association between milk Na+
concentrations and the genetic distance between HIV populations in blood and milk specimens, we did not detect an association between mastitis and the prevalence of monotypic sequences in milk specimens. This discrepancy suggests that mastitis may stimulate viral replication across a larger population of virus in the infected breast compared to that in the healthy breast, perhaps due to an influx of infected inflammatory cells. Furthermore, compartmentalization of virus in breast milk specimens was not associated with mastitis, suggesting either that a relatively diverse group of viruses replicated in milk specimens or that the net effect of inflammation on replication within the breast was not sufficient to affect HIV-1 population structure by these measures.
In summary, phylogenetic analyses of HIV-1 genomes from breast milk and blood specimens obtained from lactating women found limited viral compartmentalization, indicating relatively free mixing of viruses between milk and blood specimens. The greater prevalence of monotypic HIV-1 in breast milk specimens suggests viral replication and/or proliferation of cells containing proviruses within the breast. Statistical modeling of the effect of inflammation on genetic distance between HIV-1 subpopulations in milk and blood specimens found evidence for increased HIV-1 replication in breasts with mastitis.