HIV-1 subtype C accounts for nearly half (48%) of global HIV infections and is the predominant subtype in Sub-Saharan Africa and India.1 HIV-1 isolates can also be differentiated based on their ability to use different chemokine coreceptors to gain entry into host cells.2,3 Strains which use C-C chemokine receptor type 5 (CCR5) are termed as R5-tropic strains, and those that use C-X-C chemokine receptor type 4 (CXCR4) are classified as X4-tropic strains. Viruses that are able to utilize both receptors are termed as dual-tropic strains. X4-tropic viral strains have been extensively studied in subtype B and are thought to emerge in advanced stages of illness,4 however, they are considered to be rare in subtype C even in the later stage of disease.5–7 The factors limiting the emergence of X4 viruses in subtype C are not well known because of limited studies8 and low prevalence of X4-tropic strains in this subtype. Strain tropism is usually determined by phenotypic testing. However, genotypic tropism testing using population sequencing has advantages in clinical practice in resource-constrained settings because of its low cost, simpler technical demands, and faster turnaround time compared with phenotypic tropism testing.9,10 Furthermore, good concordance between population tropism genotyping and phenotypic tropism testing was observed in a number of studies involving subtype B and C viruses.10–12
In this study, we determined the genotypic coreceptor tropism in children with perinatally acquired HIV infection with known duration of infection of 2–17 years. In addition, we examined the clinical, demographic, host and viral genetic factors associated with coreceptor switch in HIV-1 subtype C infection among this population of infected children.
The study participants included perinatally infected children aged 2–17 years who had attended the Pediatric Infectious Disease Clinic, St John's Medical College and Hospital, Bangalore, India, between August 2007 and May 2011. The study participants are residents of Karnataka state and neighbouring states located in the southern part of India. Perinatal infection was confirmed by documentation of HIV infection in the mother. Sociodemographic and clinical data were recorded from the medical records. Cross-sectional samples were collected from 139 children who were antiretroviral therapy (ART) naive (n = 78) and ART experienced (n = 61). Viral load was measured by an in-house real-time polymerase chain reaction (PCR) with TaqMan assay method for 2007 and 2009 samples and Abbott m2000rt system (Abbott Molecular Diagnostics, Des Plaines, IL) for 2010 and 2011 samples. Plasma was stored at −80°C until used. Available samples whose viral load was >500 copies per milliliter (n = 76; ART naive, 68 and ART experienced, 8) were included in this study.
PCR and Sequencing
Viral RNA from plasma was extracted using a commercial kit (QIAamp Viral RNA extraction kit, Qiagen, Hilden, Germany). V3C3 regions of the envelope (env) gene was amplified using reverse transcriptase polymerase chain reactions followed by conventional nested PCR using the primers using the modified primer as described previously.13 First-round PCR was carried out using IN_ED5 5′-ATGGGATCAAAGCCTAAAGCCATGTG-3′ and IN_ED12 5′-AGTGCTTCCTGCTGCTCCCAAGAACCCAAG-3′. PCR conditions were as follows: 1 cycle at 94°C for 5 minutes; 39 cycles at 94°C for 20 seconds, 55°C for 30 seconds, and 72°C for 80 seconds; followed by 1 cycle at 72°C for 7 minutes. Nested PCR was carried out with IN_ES7 5′-CTGTTAAATGGCAGTCTAGC-3′ and IN_ES8-5′-CACTTCTCCAATTGTCCCTCA-3′ with following PCR conditions 94°C for 5 minutes; 39 cycles at 94°C for 20 seconds, 50°C for 30 seconds, and 72° for 60 seconds; followed by 1 cycle at 72°C for 7 minutes. The purified PCR products were subjected to bidirectional population sequencing in 3730xl DNA analyzer (Applied Biosystems, Carlsbad, CA). Among the 76 samples, the V3C3 region was amplified in 72 samples. For sequence interpretation, all major populations (>50% peaks) and minor populations with all possible combinations (>25% peaks) were included.
Subtyping and Sequence Quality Control
Subtyping was carried out using 2 different online tools REGA subtyping tools, and Recombination Identification Program version 3.0 followed by phylogenetic analysis in MEGA 5 software were used to ensure consistency of results. To check for PCR cross-contamination, phylogenetic analysis was carried out using Kimura-2 parameter in MEGA 5 software.14 All study sequences demonstrated well-separated branches indicating the absence of cross-contamination. All the reference sequences used in this study for phylogenetic analysis and sequences of Env V3 with known biological phenotype were downloaded from HIV Los Alamos Database (www.hiv.lanl.gov).
Genotypic Tropism Testing
We used an advanced interpretation system, geno2pheno(coreceptor) (G2P), which uses support vector machine technology trained with a set of env V3 nucleotide sequences with corresponding R5, X4, dual, or mixed tropism phenotypes. The result is interpreted as a quantitative value. Also considered is the false-positive rate (FPR), which defines the probability of falsely classifying an R5 virus as X4. There is currently no consensus on the ideal FPR threshold to be used in clinical practice. We followed the clinical interpretation scheme based on the European guidelines for HIV-1 tropism testing which uses 10% FPR as the threshold for X4 predictions.10 Minor populations with all probable combinations of V3 sequences were also included. A total of 473 sequences were obtained from 72 amplified samples. The tropism interpretation was as follows: the sample was considered “X4 tropic” if all sequences from that patient showed FPR ≤10%; or “R5 tropic” if all the sequences from the patient showed FPR >10%. Samples which contained sequences with differing FPRs that were both ≤10% and >10% were classified as “R5/X4-tropic” samples.
An India-specific consensus sequence of V3C3 region was created as described by us previously from the downloaded reference sequences reported from India on the Los Alamos Database.15 Intrapopulation divergence, defined as the genetic distance to the Indian consensus C sequence, was calculated in MEGA 5.
To examine the association of the CCR5Δ32 deletion with HIV-1 coreceptor tropism, CCR5Δ32 genotyping was performed by PCR with subsequent gel electrophoresis as described previously.16 PCR amplicons of either 262 bp (CCR5Δ32 wild type) or 230 bp length (CCR5Δ32 deletion) were visualized in a 5% ethidium bromide-stained agarose gel.
Descriptive statistics and frequencies were used to describe the characteristics of the study participants. The demographic, clinical, and viral genetic differences between the 2 groups (R5 tropic and X4 tropic) were evaluated by Student t test and χ2 analysis. Multivariate logistic regression was used to find the significant predictors of X4-R5/X4 tropism.
Study has been approved by institutional ethical review board, St John's Medical College Hospital, Bangalore, India. Written inform consent has been obtained from all participants and their care givers.
Among the 72 children included in the final analysis, median age was 7 years (range 2–17 years), and 55.6 % (40 of 72) were male. The median CD4 count 554 cells per cubic millimete (range: 52–2163 cells/mm3) and mean log10 viral load was 5.28 copies per milliliter (±0.88). There were 9.7% (7 of 72) children who were on ART for a median duration of 22.6 months (range: 2–78 months).
Subtyping, Genotypic Tropism Testing, and Characteristics of Indian V3 Sequences
Among the samples, 98.6% (71 of 72) of the strains were subtype C, and 1 was identified as subtype A1. A total 473 sequences from 72 patients were used to determine the tropism analysis in G2P system with FPR 10%. Among the patients with subtype C strains (n = 71), 81.7% (58 of 71) were identified as R5-tropic strains with median FPR of 68.1% (range: 10.5–99.2) and 9.9% (7 of 71) were identified as X4 tropic with median FPR of 3.15% (range: 0.10–9.70). The presence of both R5-tropic and X4-tropic sequences were found in 8.5% (6 of 71) (Table 1), and these were termed as R5/X4-tropic strains. The subtype A1 strain was classified as an R5/X4 strain. To compare how the Indian genotypically predicted X4-tropic sequences differed from other globally-derived X4-tropic strains, we analyzed the degree of conservation of amino acid residues and frequency of polymorphisms within biologically determined X4-tropic strains obtained from different subtypes from an international panel17,18 (Table 2). The first 3 amino acid residues in 15GPGQ18 motif, an important determining factor for viral tropism, were significantly conserved. The 18Q position was mutated to R in 23% (n = 10) of sequences, which was observed in previously biologically determined X4-tropic Indian C sequence and other non-C sequences including subtype A1, B, and 01_AE.17,18
The analysis of CCR5 genotype identified all the PCR amplicons at 262 bp position indicating that all the children included in the study bore the wild-type CCR5[INCREMENT]32 genotype, indicating the absence of heterozygosity or homozygosity for the [INCREMENT]32 deletion in this population.
Clinical, Demographic, and Genetic Characteristics of R5-Tropic and X4 Dual/Mix Tropic–Strains
Clinical and demographic characteristics of R5-tropic and X4-tropic strains are shown in Table 3. Children with X4 or R5/X4 strains were more likely to be older than those with R5-tropic strains (10 years vs. 7 years, P < 0.05), have lower CD4 counts (371 vs. 562, P < 0.05), and have viral populations with greater intrapopulation viral divergence (0.10 vs. 0.08; P < 0.01). We further divided the children into 3 age groups (≤5 years, 6–10 years, and ≥11 years) and observed that in the middle age group, the prevalence of the R5/X4-tropic or dual strains were higher (17.9%) than among the younger children, whereas in the older age group, the prevalence of pure X4-tropic strains was high (21.1%) (Fig. 1). The presence of tuberculosis infection was documented in 12 children (tuberculosis coinfection rate 17%); however, no significant association between tuberculosis and X4 tropism was seen. Using multivariate logistic regression and adjusting for other parameters such as gender, ART-naive status, CD4 count, log viral load, and viral divergence, the single independent predictor for X4 and R5/X4 tropism was older age (P < 0.05). Children who were ART naive had a 3.3-fold higher risk of having X4-tropic or R5/X4-tropic virus than ART-experienced patients although statistical significance was not obtained within this small sample.
This study is one of the largest described analyses of coreceptor tropism determination among perinatally infected children aged 2–17 years and highlights for the first time the prominent presence of X4-tropic viral strains in HIV-1 subtype C. The findings also indicate the likelihood of coreceptor transition from R5 to X4 tropism occurring in older age groups among children with increasing duration of HIV infection.
Previous studies on HIV-1 subtype C viruses have revealed the predominance of R5-tropic viruses at various stages of disease, including advanced HIV disease.19–22 Studies on subtype C isolates from Ethiopia and Malawi show the absence of altered coreceptor usage.19,21 The in vitro studies suggested restricted coreceptor switch from R5 to X4 in Indian subtype C strains even in later stages of disease.20 Most of these studies were conducted on adults with sexual transmission or those with known intravenous drug use where the actual duration of HIV infection was uncertain.23,24 Our study was unique in its inclusion of proven perinatally infected children of different ages where the timing of infection was known. The higher prevalence of X4-tropic or dual/mixed-tropic strains in older children suggest that, like subtype B, coreceptor transition can occur with greater duration of infection and higher disease progression in subtype C, although an exact comparison of the duration required for transition between the subtypes could not be concluded from this cross-sectional study.
A longitudinal analysis of samples obtained at different time points from a single patient showed that extreme genetic divergence and more number of mutations are required for coreceptor transition from R5 to X4 in HIV-1 subtype C strains compared with other subtypes, which may partially explain why only a small number of X4-tropic subtype C strains have been described worldwide.25 X4-tropic strains from our study were associated with greater intrapopulation divergence (from Indian consensus sequences), supporting the hypothesis that greater viral replication and divergence is necessary for tropism transition.
A recent study from western India reported a high prevalence of CCR5Δ32 genotype in a well-defined ethnic population.26 CCR5Δ32 genotyping performed on our study population revealed no patient with homozygous or heterozygous CCR5Δ32deletion, which was consistent with previous findings from India27,28 and eliminated the possibility of correlation or excessive selectivity of X4 tropism because of host genetic factors such as the presence of CCR5Δ32 deletion in these children.
Our study was limited by the cross-sectional nature of analysis, which included different children at different ages studied at a single time point. The small numbers of ART-experienced patients included in the analysis limited our interpretation of the effect of ART on coreceptor tropism transition in patients with HIV-1 subtype C. Though the current scientific literature supports good correlation between population sequencing and interpretation of viral tropism using the Geno2Pheno system with phenotypic tests, the main disadvantage with this approach is the limitation in determining the coreceptor tropism in minor viral populations. The approach used in this study attempted to include minor populations, although a more inclusive method would be the use of deep sequencing technology.29
In conclusion, our study provides insight into the prevalence of viral X4 tropism among children with perinatally acquired infection with HIV-1 subtype C in India. The higher prevalence of X4-tropic strains in the older children suggest that coreceptor transition can occur with longer duration of infection and greater disease progression and can have therapeutic implications for the future. Further longitudinal cohort analysis of viral characteristics can validate these findings in future.
We thank the staff of the Infectious Disease Clinic, St John's Hospital, Bangalore, for assistance with patient recruitment. We acknowledge Sven Gard's Fund for Virology Research and Erasmus Mundus External Co-operation Window 13 (India) Scholarship. Authors would like to thank Alexander Thielen, Max-Planck-Institutfür Informatik, Germany, and Prof. Rolf Kaiser, University of Cologne, Cologne, Germany, for a fruitful discussion and technical assistance for genotypic tropism testing.
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Keywords:© 2012 Lippincott Williams & Wilkins, Inc.
coreceptor tropism; HIV-1 subtype C; vertical transmission; viral divergence