Recombination plays a major role in HIV-1 evolution and represents a powerful mechanism to produce genetic diversity during the viral lifecycle. Although recombination in HIV-1 viruses has been well documented, especially between different subtypes within an individual or in the context of circulating recombinant forms in a population of infected subjects, it has been less studied in the context of its occurrence within individuals. The present study represents the most extensive investigation of HIV-1 intra-host recombination within different tissues performed to date. Seven hundred eighty gp120 sequences amplified from 53 tissues with either normal or abnormal histopathology were collected from seven patients who died of different AIDS-associated illnesses.
One characteristic of end-stage AIDS is the presence of HIV p24 expressing macrophages along with T-cell depletion. At end-stage disease, Mack et. al. 
, found that in a large variety of autopsy tissues classified as diseased tissues, including lymph nodes, spleen and brain, with amplifiable amounts of DNA, p24 antigen staining was predominantly localized to macrophages interspersed in a background of p24-negative lymphocytes. This type of staining was not seen in non-diseased tissues in HIV positive patients 
. We found a similar staining pattern in the tissues used for this study. The higher frequency of recombinant sequences consistently found in tissues with abnormal histopathology is likely explained by the fact that such tissues display increased macrophage proliferation due to an inflammatory response. HAART therapy, which was given to four of the seven patients prior to death, appeared to have little effect on overall recombination rates. This is not unexpected, since macrophages are one of the most important cellular reservoirs sustaining virus replication during HAART 
. In fact, several studies have implicated HAART therapy in the development of metabolic lipid dysfunction and other disorders that can lead to tissue abnormalities 
. The results strongly implicate macrophages as the primary producers of recombinants in damaged tissues. Tissue samples taken over time from an animal model would confirm if a similar association between recombination and abnormal tissues are typical in early and mid-stage, rather than only at end-stage disease.
HIV integration within host cell genomic DNA is a required step of the viral infection cycle. HIV integration site mapping and laser capture micro dissection analysis of infected macrophages have shown that viral integration usually occurs within introns of genes related to or near cellular activation loci 
. It is interesting to note that the brain specimens used to map insertion sites in Mack et al. 
were also analyzed in the current study (patient CX), demonstrating that high recombination rates occur within brain macrophages that contain inserted forms of HIV. Sequences from patient CX were also analyzed using a phylodynamic approach 
. This study showed that activated macrophages migrate between HIV infected brain tissues and especially to areas of the brain where there is an abundance of tissue damage. Persistent macrophage activation is associated with an inhibition of apoptotic signals, giving end-stage HIV-infected macrophages a survival advantage, the ability to act as a continuous source of HIV and to serve as a long-term reservoirs/sites of viral recombination 
. Furthermore, tissue macrophages co-infected with opportunistic pathogens such as Mycobacterium Avian Complex
(MAC) or Pneumocystis carinii
dramatically increase viral production and the likelihood of macrophage-mediated tissue destruction 
The occurrence of HIV-1 recombination in vivo
can be explained by the ability of the nascent viral strand to switch RNA templates during reverse transcription 
, and it necessitates super-infection of the target cell with two or more viruses, each carrying a different HIV-1 genome. Certain cell-types may be more prone to multiple infections than others. For example, cells that live longer or tissue sites of continual feeding of new viral populations would be more likely to be super-infected. The continuous recruitment of macrophages at infection sites and their long lifespan makes them the perfect target for super-infection. While the turnover of HIV-infected activated T-cells is 2–3 days, infected macrophages survive considerably longer, as discussed above, which increases both the probability of super-infection and the probability of recombination once super-infection has occurred. Our finding of a large number of recombination breakpoints distributed across the gp120 envelope protein in abnormal tissues with high levels of replication-competent macrophages is consistent with in vitro
studies, which showed that while a single round of viral replication in T-lymphocytes in culture generated an average of nine recombination events, the infection of macrophages led to approximately 30 crossover events per cycle 
Other studies have identified macrophages as a source of the production of recombinant viruses 
, but their role as a major contributor to this process remains a subject of debate. The combined results from our study demonstrate an increase in the potential for macrophage-mediated immune evasion during HIV disease because: 1) abnormal or damaged tissues commonly occur during prolonged HIV disease, 2) damaged tissues recruit macrophages that are clearly a replication site for HIV, whereas normal tissues do not, 3) as activated macrophages accumulate within abnormal tissues, they may become super-infected, thus increasing in the potential for the generation of recombinants, 4) any HIV-associated disease process or HAART-associated side effect that generates tissue damage has the potential to increase the production of recombinants, 5) the degree of recombinants produced within an individual may increase during HIV- or HAART-associated tissue damage within an individual. Importantly, if macrophages are a continued reservoir for the generation of HIV-1 intra-patient recombinant sequences in vivo
, then they are also source of continued viral evolution and diversification over time. The current study provides additional evidence that successful eradication of HIV-1 is unlikely to be achieved unless new therapeutic approaches specifically targeting tissue macrophages are developed.