|Year : 2008 | Volume
| Issue : 3 | Page : 353-359
|Divergent strains of human immunodeficiency virus type 1 circulating in India, subtyped by heteroduplex mobility assay
AK Sahni1, K Kapila2, RM Gupta2
1 Department of Pathology, Army Hospital (R and R), Delhi Cantt., New Delhi - 110 010, India
2 Department of Microbiology, AFMC, Pune - 411 040, India
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| Abstract|| |
Genomic variations in HIV-1 represent a major problem in understanding disease progression, studying drug resistance and developing effective vaccines. Heteroduplex Mobility Assay (HMA) was used for analyzing HIV-1 subtypes resulting from genetic similarity or divergence of C2 -V3 -V5 region of envelope gene between HIV-1 strains obtained from clinical samples in a tertiary care center at Pune. DNA from the PBMCs of infected individuals was amplified by nested PCR. Heteroduplexes were then formed by denaturing DNA from the unknowns with DNA from the reference strains. The results were analyzed by polyacrylamide gel electrophoresis. Out of 177 samples analyzed, 170 were of subtype C (96%). Four samples were found to be of subtype B (2.2%); in three samples, no definitive assignment of subtype was possible by HMA and these perhaps could be circulating recombinant forms (CRFs) of HIV-1. These findings may have significant implications toward development of a candidate vaccine for India.
Keywords: India, HIV-1 subtypes, heteroduplex mobility assay
|How to cite this article:|
Sahni A K, Kapila K, Gupta R M. Divergent strains of human immunodeficiency virus type 1 circulating in India, subtyped by heteroduplex mobility assay. Indian J Pathol Microbiol 2008;51:353-9
|How to cite this URL:|
Sahni A K, Kapila K, Gupta R M. Divergent strains of human immunodeficiency virus type 1 circulating in India, subtyped by heteroduplex mobility assay. Indian J Pathol Microbiol [serial online] 2008 [cited 2020 Jun 4];51:353-9. Available from: http://www.ijpmonline.org/text.asp?2008/51/3/353/42510
| Introduction|| |
The continuously evolving nature of human immunodeficiency virus in vivo gives rise to a pool of genetic variants. The geographical distribution of subtypes is a dynamic, unpredictable process and intermixing of human immunodeficiency virus type 1 (HIV-1) variants is inevitable. Extensive variability of HIV-1 results in high level of genomic diversity which has potential impact on disease epidemiology, diagnosis, therapy and prevention of infection. The marked diversity of HIV-1 is because of the introduction of genetically diverse simian immunodeficiency viruses (SIVs) into humans and error-prone reverse transcriptase (RT) which lacks proof reading function during viral replication. This results in 1-3 nucleotide change each time the provirus is synthesized.  There is a rapid turnover of virion in HIV-1 infected individuals (10 9 virions per day), which results in in vivo swarm of closely related viral variants called quasispecies . The mutant virus population increases exponentially, doubling every two days.  It has been seen that even a fairly modest mutation of 10 5 per base per generation would lead to approximately 10 8 -10 9 mutants per day.  Thus, there are endless possibilities for rapid adaptation of the virus to its environment, thereby resulting in immune escape mutants.
Recombinant viruses have already contributed substantially to the global pandemic. The likelihood of generating recombinant viruses will only continue to increase as the different HIV-1 subtypes spread to all continents, resulting in complex circulating recombinant forms (CRFs). However, the frequency of recombinant viruses is almost certain to increase; because recombination, once it has occurred, cannot be undone.
The study of genomic diversity of HIV-1 is significant due to several reasons. First, the sensitivity and specificity of the available diagnostic test kits are mainly based on HIV-1 subtype B, which is the predominant subtype in North America and Europe, whereas subtype C is the prevalent subtype in India. Second, there is independent spread of epidemics caused by different subtypes of HIV-1, which is evident by their diverse transmission patterns, infectivity and pathogenicity. Third, the response to vaccine and antiretroviral therapies will vary for different subtypes. The genetically distinct HIV-1 subtypes are also immunologically distinct though they share cross-reactivity due to similarity in their conserved regions. Last but not the least, there is biological differences among groups and subtypes of HIV-1, which is determined by the virus and the various immune responses mounted by the host. Systematic collection and analysis of HIV-1 genetic variants from different epicenters of the HIV-1/AIDS pandemic is a critical step in the development of a globally effective vaccine. ,,
The third hyper variable region [V3 loop] in the envelope gene of HIV-1 has been shown to be a principal neutralizing domain and a major determinant influencing a number of biological characteristics of the virus. Based on phylogenetic analysis of envelop (env) and group-specific antigen (gag) gene sequences, HIV-1 is divided into three groups, viz., M [main] group, O [outlier] group and N [non M, non O group]. The M [main] group is further subdivided into various subtypes as clades designated A to H, J and K. ,, Subtype "A" is found primarily in central Africa, Subtype "B" in North America, Western Europe, Australia and Japan, Subtype "C" in South Africa and India, Subtype "D" in Central Africa, Subtype "E" in Thailand, Subtype F includes isolates from Brazil and Romania. Other sequence subtypes ("G, H, I") include viruses from Africa, Russia and Taiwan. Subtype "K", whose env C2-V5 sequence branched within group "M" but remained from all known HIV-1 subtypes, was reported from Cameroon. 
In India, several subtypes of HIV-1 have been detected, A, B, C and D including recombinant strains. , Subtype B, the Thai variety, is mostly seen in the North-east where the borders are common with Myanmar. In India, subtype C is predominant. ,, Globally, subtype-C strains cause half the total infections.
All the subtypes show an unprecedented degree of antigenic variation. This implies that an effective HIV-1 vaccine candidate should ideally be drawn from the predominant viral genotype circulating in the population.  In addition, recent findings highlight that current HIV antibody screening assays, which are based on subtype B amino acid sequence data, cannot reliably detect more divergent non-subtype B HIV-1 strains especially during the early part of seroconversion. ,
In India, the epidemic was first documented in 1986 when six female sex workers were found positive for anti-HIV antibodies in Tamil Nadu, in South India.  Since then, the epidemic has spread rapidly. There are more than 2.5 million individuals infected with HIV in this country of over 1 billion people. Andhra Pradesh, Goa, Karnataka, Maharashtra, Manipur, Mizoram and Nagaland are the seven states in India, representing 22% of the population, which have generalized epidemics, as indicated by a 1% or higher prevalence rate among pregnant women in prenatal clinics.  India has seen a rapid spread of HIV-1 primarily via heterosexual transmission and certain HIV-1 subtypes may be adapted to more efficient heterosexual transmission. 
In this study, we collected clinical samples from individuals from different geographical regions of India (particularly western India) reporting for HIV testing in the virology section of a tertiary care center and analyzed subtypes of HIV-1 on the basis of the results obtained by HMA of C2- V3- V5 regions of the env gene.
| Materials and Methods|| |
Study group: The study comprised 177 randomly selected HIV-1 seropositive individuals referred to HIV Reference Center at the Department of Microbiology, Tertiary Care Center at Pune from March 2002 to August 2006. Informed consent was obtained from each patient included in the study. Ethical clearance was obtained from the committee constituted by the institute where this present study was conducted. In case of children, the approval for collection of blood and subtyping of HIV-1 strain was obtained from their parents. The majority of patients were symptomatic at the time of collection of samples.
Specimen collection: A sample of 10 ml of blood was collected in EDTA from each patient. The PBMCs were separated by Ficol-Hypaque [Sp Gr 1.076, Sigma] density gradient centrifugation. DNA was extracted from PBMCs by a method previously described. 
DNA-PCR: Nested PCR protocol was followed. 1 µg of DNA was amplified in a 100-µl reaction containing 1.25 mM MgCl 2 ; 200 µM each of dATP, dCTP, dGTP and dTTP; 20 pM of each primer; and 2.5 U of Taq DNA polymerase  (Promega Corp. USA). The sequences and positions of the primers with their expected amplicon size are given in [Table 1]. The samples were denatured at 95°C for 2 min. The amplification was done for 30 cycles with each cycle consisting of denaturation (95°C for 15 seconds), annealing (55°C for 45 seconds) and extension (72°C for 60 seconds). Final extension was carried out for 10 min at 72°C. In the first-round amplification, primers ED3 and ED14 were used to generate a 2 Kb fragment spanning from the first exon of rev to the transmembrane protein gp 41 region of env . In the second-round amplification, 2 µl of first round amplified product was re-amplified by using primers ES7 and ES8 under similar reaction conditions to generate a final amplified product of 0.7 Kb in size spanning the V3-V5 coding region of glycoprotein (gp) 120.
In 15 samples, sufficient amplified product could not be obtained even after two rounds of amplification. In these samples, a third round of amplification was carried out. After the first round of amplification with ED3 and ED14 primers, a second-round amplification was done with another set of primers ED5 and ED12 (to obtain a 1.2-Kb fragment spanning the V1-V5 coding region of gp 120). This was followed by a third round of amplification using ES7 and ES8 primers as described above. The final 0.7-Kb amplified product was purified by using the wizard PCR Preps DNA purification system (Promega Corp) and used for analysis by HMA [Table 1].
Heteroduplex mobility assay (HMA): HMA was performed as described by Delwart et al.  using HMA HIV-1 env subtyping kit version 3 (NIH AIDS Research and Reference Reagent program, Bethesda, USA). The kit contained reference plasmids representing subtype A (Rwanda), B (Brazil), C1 (Malawi), C2 (Zambia), C3 (India), C4 (Brazil), D (Uganda) and E2 (Thailand). These plasmids were also subjected to PCR amplification by using primers ES7 and ES8, as described above, to yield a 0.7-Kb amplified product.
For HMA, 5µl of amplified product of the sample was mixed with 5µl of amplified product of individual reference plasmid clones and 1.1µl of HMA annealing buffer (100 mM NaCl, 10mM Tris pH7.2, 2 mM EDTA). The mixture was denatured at 95°C for 2 min and reannealed by snap chilling on ice. The resultant homoduplexes (sample homoduplex and reference homoduplex) and heteroduplexes formed between sample and reference plasmid clones were then subjected to electrophoresis on a 5% non-denaturing polyacrylamide gel (PAGE) at a constant voltage (250 volts) for 3 hr. The gels were stained with ethidium bromide for 30 min and then destained with two changes in de-ionized water. The gel was then examined under UV transillumination and the video captured images of the mobility patterns of the homoduplexes and the heteroduplexes band position was recorded. The heteroduplex mobility ratio was then calculated by taking the ratio of the distance of the heteroduplexes band from the well bottom and distance of the homoduplexes band from the well bottom. The mobility migration ratio patterns of the sample with the different reference plasmid subtypes was calculated and the one showing the highest ratio with any one particular reference plasmid clone, indicated the HIV-1 subtype of the unknown sample.
Homoduplexes, representing 100% homology, showed the fastest mobility on PAGE. Retarded mobility of heteroduplexes reflected proportionate nucleotide mismatches between the sample and the reference strain. The sample/reference heteroduplex, which migrated closest to the corresponding homoduplex, indicating the highest mobility ratio, determined the subtype designation
[Figure 1a and b]. In the first well of the gel, the sample was mixed with equal volume of de-ionized water instead of reference plasmid clone to look for in vivo quasispecies and heterogeneity of HIV-1.
In 16 samples, sequence analysis of the HIV-1 samples already subtyped by HMA was carried out at the National AIDS Research Institute and there was concordance of the results obtained earlier by HMA in these samples.
| Results|| |
A total of 177 HIV-1 seropositive samples were included in the study. Of these, 153 samples were from males and 24 were from females. The maximum number of samples was constituted by the individuals belonging to the age group 31 to 40 years. The majority of patients had heterosexually acquired infection (91.5%). Recipients of blood and blood products comprised only 0.5% of the study group. Intravenous drug users (IVDUs) (0.5%) were from the northeastern region of the country, five children acquired infection from their mothers and in eight individuals no proper history of transmission could be elucidated. These patients denied any history of heterosexual promiscuity or homosexual behavior or intravenous drug use or having received any blood or blood product transfusion [Table 2].
Out of the 177 samples analyzed, 170 were of subtype C (96%). Further analysis of HMA data suggests that the majority sample/reference heteroduplexes migrated closest to the corresponding subtype C3 homoduplexes, thereby suggesting that subtype C3 is the predominant strain of HIV-1 circulating in India (89.2%). Heteroduplexes of fewer samples with reference strains migrated closest to subtype C2 (6.2%) or subtype C4 (0.5%). Four samples were found to be of subtype B (Thai B2) (2.2%). Two of these patients were asymptomatic antenatal cases, both in the first trimester of their pregnancy from Maharashtra (western India), who had got their HIV status checked as part of a routine antenatal check-up. The spouses of both of these patients could not be included in the study as their samples could not be made available. The other two cases of subtype B were from Manipur. One of them was an intravenous drug user and the other presented initially as a case of pulmonary tuberculosis [Table 3].
In three samples, HMA could not clearly identify the subtype of the envelope sequence as two heteroduplexes co-migrated in the PAGE. It is possible that these samples could be containing recombinant HIV (homotypic or intra-clade' recombinants and heterotypic or inter-clade' recombinants). These were considered to be untypable samples by HMA. In two samples, the observed pattern obtained was of homotypic recombinant of C2 and C4 subtypes and in one sample, it was homotypic recombinant of C1 and C2 subtypes [Table 2] and [Table 3].
The study also included five husbands and wives' epidemiologically linked samples and their HIV-1 subtypes were C3, suggesting the transmission of HIV to spouses from infected partners.
| Discussion|| |
Heteroduplex mobility assay (HMA) was used in this study for detecting and estimating the degree of genetic similarity or divergence between HIV strains.  The sample showing the fastest mobility with the appropriate reference strain was the subtype of the unknown strain [Figure 2]. Using the regions of significant base mismatch and length variation, this relationship could be used to rapidly classify HIV-1 strains into subtypes.
HMA enables us to determine uncharacterized HIV-1 env gene subtypes. Envelope gene has V1 to V5 loops with constant and variable regions. The inter-subtype variation between these loops is 25-30% and the intra-subtype variation is 20%.  Since the env gene shows more sequence heterogeneity and variation as compared to pol and gag genes, it is more suitable for HMA. The structural distortions of the DNA double helix caused by mismatch nucleotides (resulting from base substitution mutations) and unpaired nucleotides or gaps (resulting from insertions or deletions within otherwise annealed regions) reduce the electrophoretic mobility of the DNA [Figure 2]. The reference C2 and C3 strains have approximately 10% nucleotide mismatches.  The distinctions between the intraclade subtype, i.e., C2, C3 and C4 and among the interclade subtypes were made based on the heteroduplex DNA mobilities. Any deletion or insertion in V1 to V5 region or recombinants can be detected by using HMA. The heteroduplex mobility ratio was calculated from the mobility distance of the heteroduplexes and homoduplexes from the well bottom. Various intraclade subtypes such as C1 and C2; C2 and C4 showing the same mobility distance ratio were considered to be homotypic recombinants. Similarly, the same mobility ratio shown by two interclade subtypes was considered to be due to the heterotypic recombinants. HMA is a simple, reliable, inexpensive, reproducible and a quick method of subtyping of HIV-1.
Almost 100% correlation was found between the HMA data and the sequencing data of C2-V3-C3 region of gp120 of HIV-1 subtypes in an earlier study. 
A total of 177 HIV-1 seropositive samples collected from the Department of Microbiology, of a teaching hospital in Pune, were analyzed. The predominant mode of transmission was heterosexual ( n = 162). There were five cases due to vertical transmission and one due to transfusion of blood and blood products. Clinical details of the cases studied revealed that the majority of patients ( n = 106) were symptomatic [Figure 3]. The majority of the cases were in the age group 31-40 yrs ( n = 74). The predominant sample size was from Maharashtra and Gujarat (western India). The data presented in [Table 2] and [Table 3] show their subtype distribution as follows: 170 (96%) were HIV-1 subtype C with predominance of C3 as evident from the fact that 158 out of 177 cases (89.2%) were of subtype C3, 11 were subtype C2 (6.2%), one was C4 (0.5%) and four (2.2%) were subtype B. In three samples [1.6%], the subtype distribution was homotypic mixed, i.e., one was subtype C1 and C2 recombinant and the other two were C2 and C4 recombinant, respectively. No definitive assignment of subtype was possible in these two samples by HMA and these perhaps could be circulating recombinant forms (CRFs) of HIV-1. Subtype B sequences obtained were from the IVDU cases from the northeastern state of India and were related to subtype B2 (Thailand sequences). 
Subtype C was found to be the predominant strain present in almost every region of India and in many densely populated countries of the developing world, including East and South Africa, China, Brazil and transmitted primarily by heterosexual contact.  However, due to greater interaction among different geographic regions, the geographic patterns are becoming blurred. This might lead to increased possibility of super infections by different subtypes, which in turn will present the virus with greater opportunities to evolve into newer genetic variants by recombination. Gadkari et al  studied the transmission of genetically diverse strains of HIV-1 in 46 patients with STD in Pune, India by HMA. Of these, 44 (96%) were HIV-1 subtype C and one each of subtypes A and B. Further analysis revealed that 66% of the C subtype sample had maximum homology to the C3 Indian reference strain, while 34% were most homologous to the C2- Zambian strain. Sahni et al ,  in 2002, studied the genomic diversity of HIV-1 in India by HMA. In their study, out of a total of 125 samples analyzed, 98 (78.4%) were subtype C, 11(8.8%) were subtype B, three (2.4%) were subtype A and two (1.6%) were subtype E. Further genotype distribution of subtype C was: 68% subtype C3, 8% subtype C2 and 2.4% subtype C4. Initial studies identified subtype C in few samples from western India. , Jameel et al  analyzed the HIV-1 samples from Punjab and found the presence of multiple HIV-1 subtype variants, with subtype C being the predominant one. Tripathy et al  studied the HIV-1 isolates from northern and western India and they found the predominance of subtype C in these areas. Gupta and Sahni et al  carried out HMA and sequencing of gag and env and found that subtype C predominates in India, along with the presence of subtypes A, B, E and recombinants. A study on gag gene sequences and C2-V3-C3 sequences of env region of gp 120 of HIV-1 has also reported the presence of multiple subtypes. ,, Mandal et al  analyzed 52 samples from commercial sex workers from Kolkata (eastern India) and found 90% to be of subtype C.
A study using dried blood spots for PCR conducted on limited cases from southern India  reported the presence of subtype C along with the rare subtype A. All these studies show the presence of multiple subtypes in India, with subtype C being reported as the most prevalent subtype of HIV-1.
The results obtained in the present study are consistent with the other molecular epidemiological studies on the analysis of HIV-1 subtypes from the different geographical regions of India. The predominance of subtype C in India may be due to complex, highly efficient viral transmission in the population due to specific virological and host factor interaction. V3 loop sequence of Indian subtype C isolates has considerable conserved amino acid sequences. This may have profound implications as regard to the biological behavior, evolution and phenotypic character of the virus in determining the immunopathogenesis of the disease process. One or more factors might have caused this characteristic conservation of subtype C strains circulating in India. Predominant heterosexual transmission of the subtype C strain in the population of this subcontinent may also influence the evolution of these strains in India. ,
| Conclusion|| |
The study reveals the presence of multiple subtypes of HIV-1 in India, the predominant strain being HIV-1 subtype C. The global scenario indicates that subtype C is the most prevalent subtype of HIV-1. Any future vaccine strategy to be adopted for this country must take into account the variability of HIV-1 in India and the candidate vaccine to be developed should be based on Indian subtype C3, this being the predominant circulating subtype in India. However, further large-scale molecular epidemiological studies are required to be conducted to study the biological behavior and evolution of the virus. HMA is a powerful tool for determining the molecular epidemiology of the Indian HIV epidemic.
| Acknowledgments|| |
We are grateful to the Director, National AIDS Control Organization, (NACO), Ministry of Health and Family Welfare, Govt. of India for financially supporting this study. We are extremely thankful to Dr. N. K. Ganguly, Director General of the Indian Council of Medical Research (ICMR) for the periodic review and valuable comments that helped in the completion of this project.
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A K Sahni
Department of Pathology, Army Hospital (R and R), Delhi Cantt., New Delhi - 110 010
Source of Support: None, Conflict of Interest: None
[Figure 1a and b], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]
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