Recognition of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine study by enabling the direct analysis of those viruses actually responsible for productive clinical illness. empirical dataset of 3,449 SGA-derived full sequences (11). The results supported the model and its assumptions. Importantly, the model and the empirical findings allowed us to infer that in 70C80% of the instances of sexual tranny of HIV-1, a single disease (or infected cell) is responsible for establishing productive medical infection, a summary now supported by studies in seven additional patient cohorts infected by HIV-1 subtypes A, B, C, or D (unpublished data) (8C10, 12). In the present study, we asked if the experimental strategy for identifying transmitted/founder sequences can be applied successfully to full-length HIV-1 vRNA genomes, which are nearly four instances longer than genes (9 vs. 2.6 kb), and whether recognition of such genomes can provide new insight into the biology of HIV-1 tranny, and the kinetics and pathways of disease diversification and adaptation leading to viral persistence. RESULTS Study subjects Plasma specimens from 12 adult subjects (10 male and 2 woman) with acute HIV-1 infection were analyzed with this study (Table I). Nine subjects were infected by HIV-1 subtype B and three were infected by subtype C. At the initial sampling time point, 10 subjects were plasma vRNA+/Ab? (Fiebig stage II; the HIV-1 clinical staging system is discussed in recommendations 11, 17), and two subjects were vRNA+/ELISA+/WB indeterminant (Fiebig stage IV). Three subjects were TEL1 sampled longitudinally through as many as 85 wk of follow-up. Maximum plasma viral lots ranged from 394,649 to 26,700,000 vRNA copies per ml. Four subjects admitted to heterosexual publicity as their only HIV-1 risk element, and eight were men who experienced sex with males. No subject admitted to injecting drug use. Table I. Subject demographics, risk group, and baseline laboratory data SGA and sequencing Between 5 and 18 full viral genomes (median = 9) were derived by amplification of individual plasma vRNA/cDNA molecules from each subject (108 amplicons in total; Table II). Each of the 108 amplicons was sequenced directly without interim cloning. Sequence chromatograms of 62 amplicons were unambiguous at every position. Sequence chromatograms of 46 amplicons experienced combined bases at one to five positions per sequence. Because the proportion 210755-45-6 IC50 of PCR-positive wells at endpoint cDNA dilution was <20%, and because combined bases generally displayed only a subset of polymorphisms in any one sequence, we could infer that most combined bases on chromatograms resulted from polymerase errors in the initial PCR cycles and not from amplification from more than one unique vRNA/cDNA template; in such cases, a correct task of the ambiguous foundation could be made. 210755-45-6 IC50 In five instances where one or more mixed bases displayed the only polymorphisms inside a sequence, this was not possible. Therefore, we could make an unambiguous task of nucleotides at each position in the nucleotide sequences of 103 HIV-1 genomes and at all but nine positions in five others. From three subjects 210755-45-6 IC50 (CH40, CH58, and CH77), an additional 209 overlapping half genomes and 177 shorter sequence fragments were identified from time points beginning before 1st antibody detection (Fiebig stage II) and extending to 350C592 d later on (Fiebig stage VI). Table II. Diversity analysis of full-length HIV-1 genomes derived from individuals with primary illness HIV-1 diversity Inside a maximum probability phylogenetic tree, viral sequences from your nine US subjects clustered significantly with prototype B clade viruses, whereas sequences from your 210755-45-6 IC50 three Zambian subjects clustered with prototype C clade viruses (Fig. 1). Maximum interstrain diversity among all 108 full-length genomes was >25%, reflecting variations typically observed between different clade B and C viruses. Within individual subjects, maximum disease diversity was far less, ranging from 0.04% in subject SUMA0874 to 2.46% in subject ZM247F (Table II). There was no interspersion of sequences among study subjects. Maximum within-patient viral diversity was distinctly reduced 11 subjects (<0.14% in each) compared with the 12th subject, ZM247F (2.46%). We postulated the observed variations in maximum viral diversity observed within individuals might reflect the numbers of viruses responsible for establishing productive illness in these subjects, as demonstrated previously for diversity (11). We formally tested this hypothesis by comparing observed viral genome diversities in each subject with estimates, based on model predictions, of the maximum diversity one could expect within 100 d after tranny of a single disease (0.60%; 0.54C0.68% confidence interval [C.I.]) (11). 11 out of the 12 subjects experienced sequences that fell well below the.