results after assessment of each individual data and elimination of VL outliers after seroconversion and before antiretroviral therapy [3]. PBMC-associated DNA was obtained from 107 cells by standard methods. HIV DNA was amplified in the C2-V5 region of the env gene as described [11,12] and sequenced in both directions. Quantification of DNA, estimated by limiting dilution PCRs, was expressed as number of copies per million peripheral mononuclear cells (PBMC). Phylogenetic analysis of the env gene For viral phylogenetic analysis and X4/R5 genotype, a 210 bp fragment in env gene from the distal position of C2 to the middle of C3 was obtained for each participant. Bulk nucleotide sequences were edited using the SeqMan version 3.61 (Inc. Dnastar, Madison, Wis). An estimation of the ‘‘viral dating’’ of nucleotide sequences was established, as previously reported by Bello et al [11,12], according to the genetic distance to the reconstructed origin of the HIV-1 subtype B Spanish epidemic and assuming a relaxed molecular clock. The estimated dating time of the ancestral samples studied differs from the sampling time up to 15 years. A similar dating time was obtained when considering as most recent common ancestor (MRCA), the ancestral virus of the subtype B epidemic in the Los Alamos Database (USA). The CXCR4 or the CCR5 phenotype was inferred from the V3 amino acid bulk sequences by the PSSM algorithm in Jensen et al [17] in (http://indra.mullins.microbiol.washington.edu/pssm/) site. Results The study individuals represent the five clinical definitions summarized in Table 1: LTNP-EC (n = 9), LTNP-VC (n = 7), LTNP-NC (n = 14), P (n = 10) and RP (n = 24). The characteristics of the study participants are presented in Table S1. From LTNP-EC to RP, clinical definitions were associated with changes in the frequency (depletion) of protective host factors, in particular the CCR5 protective haplotypes (CCR5_H+/H+, proportion decreasing from f = 0.78 in EC to 0.33 in RP), the HLA-B protective alleles (B*2705, *5701, *5101, *1302, codified as HLA-B+, decreasing from f = 0.39 to 0.04), and the HLA-C-35 rs9264942 variant (decreasing from f = 0.83 to 0.13). The inverse situation occurred with host markers related with rapid progres- sion, in particular CCR5_P1 homozygosity (proportion increasing from 0 in EC to 0.13 in RP), and HLA-B risk alleles (B*1801, HLA- B*35Px alleles and B22 serogroup, increasing from f = 0.11 to 0.25), Figure 1A. In regression analysis, several of the markers displayed significant statistical association in their frequency distribution across clinical definitions: protective HLA-B+ (r2 = 0.93, p = 0.01) and CCR5_H+/H+ (r2 = 0.79, p = 0.02), and trend association for HLA-C-35 (r2 = 0.75, p = 0.06) and CCR5 D32 (r2 = 0.72, p = 0.07); Figure 2. Consistent with recent data [18], copy number of CCL3L1 did not discriminate among clinical groups (average copies per diploid genome; LTNP-EC, 2.1; LTNP-VC, 2.6; LTNP-NC, 2.1; P, 1.6; RP, 2.2). Selected viral factors previously associated with HIV clinical progression were included in our analysis: viral load, viral DNA load, viral dating, and X4/R5 genotype (Table S1). As expected, there was a progressive increase in the viral load from LTNP-EC to the RP. The increase in viral load was significantly associated (p value,0.0001) with a simple additive score that included the most valuable genetic markers (HLA-C -35, CCR5 D32, CCR2 V64I, CCR5_P1/P1, and HLA-B+ and B- alleles), Figure 1B. The average additive genetic score was 3.0 for EC, 1.5 for LTNP viremic controllers, 1.3 for LTNP-NC, 0.9 for P, and 20.3 for RP (p,0.0001). An increase in proviral DNA values was also HIV-1 Progression Definitions associated with the various clinical definitions. Proviral load was extremely low in LTNP-EC and LTNP-VC (in general, ,5 copies/106 PBMCs). LTNP-NC were, in general, above 30 copies/106, and RP showed values 100 times higher values than the LTNP-EC and LTNP-VC (Table S1). Thus, although a precise discrimination across groups is limited by evidence of overlap of close disease strata, the emphasis is placed on the dosing of multiple protective and risk alleles that define progression. An important difference between groups was the presence or absence of evolution in the viral quasispecies. Chronic and rapid progressors, presenting a continuous high viral replication carried, as expected, modern viruses. In contrast, among LTNP-EC and LTNP-VC, individuals maintained ancestral virus genotypes, close to the transmitted virus [11,19,20] although residual viral replication may occur [21,22,23]. The switch from the ancestral to the modern genotype occurred within the group of LTNP-VC (Table S1); differences in allelic frequency of various host factors distinguished these two groups. The additive genetic score was 2.6 among individuals carrying ancestral viruses, vs 1.5 for LTNP carrying modern viruses, p = 0.08 (Figure 1B). The possible contribution of the X4/R5 genotype to explain the differences observed between groups was also assessed. Receptor use, deduced from the V3 amino acid sequence, was statistically consistent with an R5 phenotype in viruses from all the studied patients (Table S1). Discussion There are different classifications of HIV-1 patients because of the distinct criteria used [13]. Classifications based in clinical data differentiate LTNPs, P and RP [1]. Consideration and inclusion of viral load measurements allows the definition of additional sub- categories: LTNP elite controllers, LTNP viremic controllers and LTNP viremic non-controllers [2]. This study shows that these broadly used clinical definitions of HIV-1 disease progression are generally supported by the pattern of distribution and enrichment of viral and host genetic factors. Among them, the frequencies of protective HLA-B alleles, HLA-C- 35 rs9264942, and protective CCR5/CCR2 alleles as well as the ancestral/modern genotype were the factors that best discrimi- nated among groups. The least distinct groups are the LTNP with (LTNP-VC) or without (LTNP-NC) effective control of viremia that cannot be readily separated on the basis of host genetic markers. Analysis of the characteristics of viral sequences permitted, however, to distinguish within LTNP-VC a subset of individuals with viruses with ancestral dating – a characteristic of the LTNP-EC. The lack of viral evolution, which is the basis of the ancestral characteristic of the sequences, reflects the strict control of viral replication. The ancestral genotype in LTNP-VC could have prognostic value; but the limited number of individuals in this group, that differ solely on the basis of viral dating, does not allow for greater precision on whether the split within this category will be of clinical consequence. The study emphasizes the characteristics of the group described as RP: very high viral load, few protective host factors and an increased presence of host genetic progression factors. Depending on the definition used, approximately 10% of HIV-infected individuals progress to AIDS within the first two or three years of HIV-1 infection. Recently, Dalmau et al described detailed genetic, virologic and clinical analyses of two who progressed in less than one year [24]. A combination of immunological, genetic, and viral factors were found contributing to the extremely pathogenic infection, including the detection of highly replicative dual tropic X4/R5 viruses [24]. None of the rapid progressors in PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11079