Since the first stages of HIV-1 infection might be crucial for the characterization of viral diversity and the antibody response against viral epitopes might be the leading force driving HIV diversification in the initial phase of infection 
. Thus, the observation that monoclonal antibodies preferentially neutralize X4 variants 
might explain the initial advantage of R5 viruses. However, it has been shown that recent emerged X4 variants are more sensitive to antibody neutralization than late emerged X4 variants 
. Consequently, X4 variants might effectively adapt to escape from antibody neutralization and establish as new lineages in the late stages of HIV infection. In addition, the HLA profile would be insightful to the further understand the pattern of selective pressure in the quasispecies within each patient, unfortunately this information not available in our study. Of note, X4 viruses observed within the recipient RB presented reduced number of glycosylation sites compared to other variants, thus suggesting that to infect host cell newly emerged X4 variants lessen the protection of the “glycan shield”. X4 viruses isolated in late stages of RB infection maintained low levels of glycosylation, making them more vulnerable to antibody neutralization. Furthermore, it has been suggested that structural features of V3 loop might also constraint the R5-to-X4 shift within-hosts 
. Thus, the background of the virus that initiates the infection might also contribute to the appearance and establishing of X4 variants.
On the other hand, considering that HIV has extremely high rates of adaptive mutations of HIV 
and the emergence of X4 variants is associated with the acquisition of a reduced positively charged amino acids in V3 loop 
. Then, the selective pressure imposed solely by antibody neutralization might partially explain the late emergence of X4 viruses, but not the replacement of R5 variants or the decline of CD4 levels associated with of X4 viruses. Our results showed relaxation of positive selection, in the patient RB, occurred equally in X4 and R5 viruses. Consequently, there is no evidence of distinct adaptive evolution between these variants that would favor X4 variants to predominate in the infection.
Our results also showed that appearance of distinct HIV viruses occurs even when the overall diversity is low (i.e, GSGR variants). Thus, the genetic divergence (measured by the pairwise diversity) in the epidemic cluster has no relation to the dynamic process of lineages replacement. The intra-host HIV population of the donor DO was characterized by the emergence of GSGR variant two years after initial infection. This less divergent strain was the main variant between 1989 and 1991, while the GPGR strain (that initiated the infection and was highly divergent) reappeared and was the only variant detected in 1993. Although selective pressure has been pervasive, including in V3 loop sites associated with R5-to-X4 shift (), the pattern of X4 emergence was quite distinct among the individuals of the epidemic cluster. Interestingly, X4-strains were not detected in the viral population of the recipient RA and in the sexual partner SC during the study. These results suggest that although the genetic background of the virus is important to determine the emergence of X4 strains, the cellular environment of hosts might be the key factor to control the timing of R5-to-X4 shift. Interestingly, the emergence of X4 strains in the donor (DO) and recipient B (RB) coincided with the high peak of overall pairwise diversity.
Although the patient RA was treated with zidovudine since 1988 and the patient RB started with zidovudine monotherapy in 1989, the pairwise diversity of HIV was not affected. shows that viruses within the patient RA presented low diversity prior to the therapy, whereas viral diversity in the patient RB continuously increased since 1987.
Detailed analysis, performed in an known HIV transmission chain showed that trees inferred using the pol
gene were unable to rescue the real evolutionary history of the virus because the appearance of convergent drug-associated mutations. On the other hand, trees constructed using the env
gene were not affected by reverse-transcriptase and protease inhibitors 
. The effect of antiretroviral therapy on the HIV diversity is highly specific: antiviral drug will increase the adaptive value of mutations that confer resistance to the virus. In fact, codons associated with drug resistance in the RT gene of HIV evolve under purifying selection in absence of therapy and shift to positive selection when therapy initiates, while the overall intensity of selective pressure remained unchanged 
It has been observed that selective pressure declines over time in the intra-host HIV-1 population 
. Previously, we found that intensity of positive selection in HIV-1 is directly related with the number of CD4+ cell 
. Lemey et al. 2007, noticed an increase in the rate of neutral mutations associated with disease progression and suggested it was affected by the rate of HIV-1 replication (viral generation time). It is noteworthy that the increasing diversity detected in 1989 in the recipient RB coincides with the severe decline of CD4+ T cell counts. This reduction of CD4 cells might indicate loss of integrity of the immune system to mount response directed to new viral epitopes. In this regard is tempting to suppose that relaxation of immune pressure creates an ideal cellular environment that increases the changes of newly emerged variants not to be eliminated from intra-host viral population. The replication rate observed in the patient RB coincide with the possibility that elevated rate of naïve cells divisions facilitate X4 overgrowth 
and provide further support to the importance of host cellular environment to the dynamics of R5-to-X4 shift. Indeed, random processes are likely to define HIV variants that will prevail in the viral population of recently infected individuals 
Taken together all these features, it is quite clear that HIV-1 evolution within host is a complex process that might involves subtle equilibrium between adaptation to the cellular environment, escape of the immune response and perhaps random fixation of variants. By using pairwise distances and branch-site models we showed that HIV-1 diversity in the late stages of infection increases and positive selection is relaxed. It is likely that the observed increase in diversity occurred due to the accumulation of neutral mutations. Although the decline of positive selection and the increase in of genetic diversity observed in the late stages of HIV-1 infection have been associated with humoral immune response 
. Here we show varying levels of humoral immune response over infection time had little impact to HIV diversity, however reduction of CD4+ T cells counts is the main cause of the low intensity of positive selection.