Primary isolates of human immunodeficiency virus type 1 are usually dominated by the major variants found in blood.
The isolation of primary strains of human immunodeficiency virus (HIV) is an invaluable tool for assessing properties of viruses replicating in HIV-infected subjects. A common method for obtaining a primary isolate is coculture of peripheral blood mononuclear cells (PBMCs) from HIV-infected subjects with PBMCs from uninfected donors. However, such in vitro expansion may disturb the composition (identities and relative proportions of constituting viral species) of the original viral population. We developed a GeneScan assay to monitor HIV populations by detecting variants that differ in the length of the V1/V2 coding region of the envelope gene. This assay was used to compare proviral DNAs from the PBMCs of eight subjects to the corresponding primary isolates. Major variants found in uncultured PBMCs usually persisted during culturing, while the minor variants frequently disappeared, resulting in a reduction in viral diversity. The outgrowth of the initial (2 to 4 days) viral population appeared to be determined by random events. However, subsequent changes in the population were deterministic, and as a result, the compositions of primary isolates from parallel cultures were often very similar. For two of three subjects studied, the source of HIV-negative PBMCs had little effect on the composition of primary isolates, while for the third subject donor-dependent effects were observed. Overall, our results show that most primary isolates accurately represent the major viruses found in a subject's blood and that rapid population-based genotyping methods are useful for detecting isolates with perturbed viral populations.
Voronin
Yegor
Y
Division of Human Biology, Mail Stop C2-023, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N, Seattle, WA 98109-1024, USA.
Chohan
Bhavna
B
Emerman
Michael
M
Overbaugh
Julie
J
D001769:Blood / Q000821:virology; D018920:Coculture Techniques; D005260:Female; D014644:Genetic Variation; D015699:HIV Envelope Protein gp120 / Q000235:genetics; D015497:HIV-1 / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D015699:HIV Envelope Protein gp120