Dual Transmission of Subtype A and D HIV Type 1 Viruses from a Ugandan Woman to Her Infant

217 GROUP M HIV-1 VIRUSES have been categorized into subtypes A±J based on phylogenetic reconstruction using DNA sequences. Sequence variation between these subtypes generally ranges from 25 to 35% within the env gene. In most cases of HIV-1 infection, viruses of a single subtype are detected. While the identification of recombinant viruses derived from different subtypes suggests the occurrence of dual infection, there are relatively few reports in which HIV-1 viruses of two different M group subtypes were detected in a single individual. In most cases, it is not possible to determine the timing of infection with viruses of different subtypes or whether both subtypes originated from the same source. The setting of vertical (mother-to-infant) HIV-1 transmission provides a unique opportunity to study dual transmission. In this setting, the time available for transmission is relatively limited and the mother is usually the only potential source of infection. There is little known about dual HIV-1 infection in the setting of vertical transmission. In one study, subtype A and C viruses were detected in a Rwandan woman; however, only subtype A was detected in her infant. In a second study, subtype B and C viruses were detected in a Brazilian woman and in her infant. To examine the potential transmission of HIV-1 viruses of different subtypes, we analyzed V3 cDNAs from Ugandan women and infants. The presence of diverse HIV-1 subtypes in Uganda increases the potential for dual infection. HIV-1 subtypes found in Uganda include A, B, C, D, and G; subtypes A and D account for the majority of infections. Plasma samples were collected from a Ugandan woman and her infant at the time of delivery. An additional sample was collected from the infant at 6 weeks of age. These samples were collected in 1993 as part of a natural history study of vertical HIV-1 transmission in Kampala, Uganda. Plasma was shipped to the United States for analysis. HIV-1 RNA was extracted from 200 m l of plasma using the Amplicor HIV-1 Monitor test kit (Roche Diagnostic Systems, Branchburg, NJ) according to manufacturer instructions. RNA extracts were resuspended in a final volume of 400 m l. Twenty-five microliters of each RNA extract was used for reverse transcription (RT) with Moloney murine leukemia virus reverse transcriptase. Each 25-m l aliquot contained at least 50 copies of HIV-1 RNA. To minimize the potential for bias due to primer binding, random hexamer oligonucleotide primers [pd(N)6; Pharmacia, Piscataway, NJ] were used in the RT reactions. The V3 region of the env gene was amplified in a nested polymerase chain reaction (PCR) using primers that correspond to highly conserved regions of the env gene.23 To reduce the potential for sample contamination, each step of the RT-PCR amplification was assembled in a separate, enclosed workstation using aerosol-resistant pipette tips and dedicated equipment. Equipment in each workstation was ultraviolet (UV) irradiated after each experiment. Negative controls without template were included for each RT and PCR reaction. Each step of the analysis (including RT, PCR, cloning, and plasmid analysis) was performed on a separate day for each sample. Comparison of V3 cDNA sequences obtained from these plasma samples and from unrelated samples analyzed in our laboratory showed no evidence of contamination. PCR products were purified using a spin column technique and cloned into the pCR2.1 vector, using the TA cloning kit (Invitrogen, Carlsbad, CA). The resulting plasmids were isolated and sequenced using the 2 21 M13 forward primer and the BigDye terminator cycle sequencing ready reaction kit (Perkin-Elmer Applied Biosystems, Foster City, CA). At least 10 cDNAs from each sample were analyzed by DNA sequencing and the predicted amino acid sequences were determined (Fig. 1). V3 sequences were used for subtype analysis. Sequences were aligned manually in the program VisEd (provided by Dr. Ken Peters). For regions of high variability, the alignment was based primarily on the most conservative amino acid substitutions. The full sequence alignment contained 18 unique Ugan-