Abstract
Strategies to limit perinatal transmission of human immunodeficiency virus (HIV) include antepartum/intrapartum antiretroviral therapy and pre-labor cesarean section with resulting reduction in the vertical transmission rate from 25% to 2%. In the current study we examined fetal cord blood cells for the presence of integrated provirus which is generally not detectable by current methods used to screen neonatal blood. Placental tissue and cord blood were obtained from 21 HIV infected women who received prenatal care and delivered at Grady Memorial Hospital in Atlanta, Georgia. Prenatal care was provided in accordance with current guidelines for care of HIV+ parturients. All specimens were obtained under Emory University IRB approved protocols. RNA and DNA were prepared from isolated placental or cord blood mononuclear cells (CBMCs) by standard methods. Integrated provirus or HIV cDNA standard concentrations were then determined by a two step modified absolute concentration Nested Real-Time qPCR assay. Realtime PCR measurements of multiply spliced (MS) or unspliced (US) HIV mRNA transcripts were conducted to determine active transcription. Quantitative measurements of Y chromosomal DNA by real-time PCR were performed for select maternal/fetal blood pairs using DYS14 primer pairs to confirm the absence of maternal contamination of fetal cord blood. Placental cells isolated from these tissues all contained detectable levels of integrated HIV proviral DNA. Five (24%) of the corresponding samples of fetal CBMCs also had detectable levels of HIV proviral DNA, possibly suggesting a higher rate of intrauterine transmission than has been reported. Furthermore, all HIV+ CBMCs had detectable levels of MS and US proviral mRNA transcripts indicating that integrated HIV proviral DNA in all infected CBMCs was transcriptionally active. Findings suggest that even with use of antiretroviral therapy in the ante-and intrapartum periods some fetuses harbor integrated HIV proviral DNA that may retain the potential to become transcriptionally active.
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