1 Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, 08916 Badalona, Spain
2 Liver Unit, Department of Medicine, Institut d'Investigacions Biomèdiques August Pí i Sunyer (IDIBAPS), Hospital Clinic, Facultad de Medicina, Universidad de Barcelona, Spain
3 Unitat d'Epidemiologia i Bioestadística, Department of Medicine, Institut d'Investigacions Biomèdiques August Pí i Sunyer (IDIBAPS), Hospital Clinic, Facultad de Medicina, Universidad de Barcelona, Spain
4 Ifakara Health Research and Development Centre, Ifakara, Tanzania
Correspondence
Miguel Angel Martínez
mamartz{at}ns.hugtip.scs.es
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ABSTRACT |
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The nucleotide sequence data reported in this paper have been submitted to GenBank under accession numbers AF439652AF439710.
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INTRODUCTION |
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HIV-1 mother-to-child transmission rates vary from 15 to 45 % (De Cock et al., 2000). Many factors, such as maternal stage of the disease, maternal immunological status, virus load, mode of delivery, duration of breast-feeding and availability of antiviral therapies, can contribute to these differences (McGowan & Shah, 2000
). Nevertheless, the role of virus determinants in mother-to-child transmission has not been well established yet (Dickover et al., 2001
). Several studies have shown that maternal diversity is generally higher than that present in the infant (Scarlatti et al., 1993
), suggesting that maternal viruses are selected before transmission. However, it is still unknown which and how many genetic determinants drive this selection. Variation in the envelope (env) gene, particularly the V3 region, has been shown to correlate with coreceptor affinity and cell tropism, as well as immune evasion. Additionally, it has been suggested recently that the V3 region of env could be a key determinant for mother-to-child transmission (Renjifo et al., 1999
). Therefore, V3 seems to be an ideal coding region to identify biological differences in mother-to-child transmission between the different HIV-1 subtypes (Becker-Pergola et al., 2000
). Since differences in transcriptional regulation have been observed among different HIV-1 subtypes (Jeeninga et al., 2000
; Montano et al., 1997
; Rodenburg et al., 2001
), in addition to the V3 region, the long terminal repeat (LTR) region has been analysed also in previous studies of mother-to-child transmission of different HIV-1 subtypes (Blackard et al., 2001
).
To investigate whether the subtype of HIV-1 may affect HIV-1 mother-to-child transmission rates, we have carried out a sequence analysis of the env (C2V3C3) and LTR (U3) region of 31 samples from pregnant mothers from Ifakara, a semi-rural area of southeastern Tanzania. The genetic divergence between the different viruses studied was analysed also.
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METHODS |
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Fourteen children tested positive for HIV-1 antibodies by ELISA. However, children were only considered infected when they were positive for HIV-1 RNA or DNA, as assesses using LTR or env RT-PCR (see below). Eight children were found to be HIV-1 RNA- or DNA-positive and were consequently considered to be infected with HIV-1. Products from the children's HIV-1 PCRs were sequenced and the sequences matched with their corresponding mothers (data not shown).
Recovery, amplification and sequencing of viral RNA.
RNA was extracted from 140 µl of serum using the RNA Qiamp Blood kit (Qiagen) according to the manufacturer's protocol and stored at -70 °C. After viral RNA isolation, 1020 µl of resuspended RNA (corresponding to 28 µl of serum) were reverse-transcribed at 42 °C using the avian myeloblastosis virus RT (Promega). Two different HIV-1 genomic regions were targeted for PCR amplification: the U3 region from the LTR (223 bp) and the C2V3C3 env region (422 bp). Nested PCR was then performed with AmpliTaq Gold DNA polymerase and buffers (Perkin Elmer) and under the conditions recommended by the manufacturer. For first-round PCR, 5 µl of the RT product was amplified. LTR primers used for first-round amplification were NI 25 (HXB2 positions 5777) and NI 23 (HXB2 positions 389408) (Ibanez et al., 2001). The following amplification conditions were used: 10 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 55 °C and 40 s at 72 °C, and a final extension step at 72 °C for 10 min. LTR primers used for second-round amplification were NI 33 (HXB2 positions 350372) and NI 35 (HXB2 positions 81100), as described in Ibanez et al. (2001)
, following the same conditions to those in the first round. The same reagents and reaction conditions were followed for the amplification of the C2V3C3 region. The outer pair of primers for env amplification were env1 (HXB2 positions 68586878, 5'-CCAATTCCYATACATTATTGT-3') and env4 (HXB2 positions 75207539, 5'-ATGGGAGGGGCATACATTGCT-3') and the inner primers were env2 (HXB2 positions 68856904, 5'-GCTGGTTWTGCGATYCTAAA-3') and env3 (HXB2 positions 73657385, 5'-TGWATTRCARTAGAAAAATTC-3'). Both strands of the PCR fragments were sequenced directly using internal (nested) PCR primers and the ABI Prism Dye Terminator Cycle Sequencing Reaction kit (Perkin Elmer). The products of the reactions were then analysed on an ABI 310 sequencer. Sequence editing was performed using the program SEQUENCER, version 4.1 (GeneCodes).
Measurement of virus load.
Mother RNA levels are shown in Table 1. Serum HIV-1 RNA levels were measured by the Amplicor Monitor assay (Roche).
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Statistical analyses.
To test for differences in the mother-to-child transmissibility of HIV-1 subtypes A, C and D and recombinant forms, we used Fisher's exact test analysis. Since intrasubtype genetic distances variation was not normally distributed and the studied cohort was relatively small, non-parametric analyses were performed to test for differences between intrasubtype genetic distances. Consequently, the MannWhitney test was used to check whether the genetic means were significantly different. The MannWhitney test was also used to search for significant differences between the virus loads of transmitters and non-transmitters. P values of less than 0·05 were considered statistically significant. Analyses were performed using the SPSS software package, version 10.0.
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RESULTS |
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LTR and envelope subtyping
HIV-1 genetic subtyping of the 31 Tanzanian isolates was performed by direct sequencing of the U3 LTR and C2V3C3 env regions (Table 1). To determine the LTR and envelope subtype of these 31 isolates, phylogenetic trees with the study samples and the Los Alamos subtype reference sequences were constructed (Figs 1
and 2
). Thirteen samples (3, 7, 8, 10, 12, 13, 15, 16, 18, 25, 26, 28 and 30) were classified as subtype A, six (1, 5, 6, 14, 23 and 29) as subtype C and two as subtype D (9 and 19) (Table 1
). Although no amplification of the env region was achieved in three samples (21, 22 and 24), they were subtyped in the LTR region as C (21 and 22) and A (24). Finally, seven samples presented a different subtype in LTR and envelope (2 A/C, 4 C/D, 11 C/A, 17 A/C, 20 D/A, 27 A/C and 31 C/A) (Table 1
). In short, among the 28 samples amplified in both regions, we have found that 13 (46 %) of the samples were subtype A, six (21 %) belong to subtype C, two (7 %) were subtype D and seven (25 %) were recombinants (Tables 1 and 2
).
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Genetic divergence between subtypes
To assess the intrasubtype genetic diversity, genetic distances between the sequences of each clade were calculated. The mean intrasubtype LTR genetic distances observed among A, C and D sequences were 9 % (range 417 %), 14 % (range 525 %) and 14 % (range 1315 %), respectively (Fig. 3). The observed C2V3C3 env mean intrasubtype genetic distances were 13 % (range 222 %), 13 % (range 622 %) and 21 % (range 1727 %) for subtype A, C and D, respectively. Therefore, subtype D was the one with more divergent C2V3C3 env sequences, followed by subtypes C and A. (Fig. 3
). The mean LTR intrasubtype genetic variation was significantly different between each subtype (P>0·0001, MannWhitney test), except between subtypes C and D. Likewise, we also calculated the C2V3C3 intrasubtype genetic variation, which was statistically significant between subtypes A and D (P>0·0001) but not between subtypes C and D or A and C. Therefore, the intrasubtype genetic distances observed in the Ifakara (Tanzania) cohort were relatively high.
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DISCUSSION |
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The present study was designed to assess whether LTR and envelope HIV-1 subtypes could affect the mother-to-child transmission rate. This transmission rate is similar to that reported in recent clinical trials in Africa (Eshleman et al., 2001). Regarding the influence of the HIV-1 subtype in the rate of mother-to-child transmission, our results suggest that there is no difference in the relative rate of transmission between subtypes A, C or D. However, the low number of mothers infected with subtype D limits the power of the study. Similar results have been documented from other subSaharan countries, where the major strains seem to be A, C and D (Becker-Pergola et al., 2000
; Murray et al., 2000
). In contrast, a study from Tanzania suggested that subtype D is less likely to be transmitted mother-to-child than subtypes A, C or intersubtype recombinants (Renjifo et al., 1999
, 2001
). The former study, based on the analysis of the env V3 region, also suggested that the fitness of subtype D V3 might be reduced in mother-to-child HIV-1 transmission. Since subtype-specific differences among LTRs have been observed (Jeeninga et al., 2000
; Naghavi et al., 1999
) and the LTR subtype has been also associated with different rates of mother-to-child transmission (Blackard et al., 2001
), the LTR region of the 31 HIV-1-infected mothers included in the present study was also analysed. However, the LTR subtype did not seem to influence the rate of mother-to-child transmission in this study. It has to be mentioned that other variables, such as maternal virus load or maternal immunological status, may affect the rate of mother-to-child transmission. Nevertheless, no significant differences were observed in the present study between the virus loads of transmitter and non-transmitter mothers (Table 1
).
An interesting finding is the high intrasubtype genetic diversity observed among viruses cocirculating in a relative small village, Ifakara, located in southeastern Tanzania. In particular, the high genetic diversity found within subtypes C and D is remarkable. The degree of intrasubtype diversity is equivalent to that reported when comparing viruses isolated from different subSaharan countries (Vidal et al., 2000). This result may be informative of the rapid spread of different genotypes through southeastern Africa, as it seems to be the case for subtype C which is increasing in prevalence in this geographical area (Renjifo et al., 1999
). However, it has also been suggested for other subSaharan countries that this high intrasubtype genetic diversity can be also due to an old epidemic of these subtypes in this particular geographical area (Vidal et al., 2000
). In any case, the high intrasubtype genetic diversity observed here in a very localized geographical area, together with the increasing prevalence of intersubtype recombinants, denotes the difficulty for future vaccine development as well as for an efficient antiretroviral treatment.
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ACKNOWLEDGEMENTS |
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Received 6 August 2002;
accepted 20 November 2002.
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