Identification of unique B/C recombinant strains of HIV-1 in the southern state of Karnataka, India

We characterized the molecular nature of a large number of primary HIV-1 isolates in the four southern states of India. In addition to confirming a predominance of subtype C infection, for the first time we identified three B/C recombinant viruses in a subset of 115 samples. Unexpectedly, env sequences of two of the three B/C recombinants phylogenetically clustered with subtype B strains of the USA. The determination of the real incidence of the recombinant viruses is of importance.

On the basis of genetic differences, HIV-1 is classified into several subtypes ^[1]. Globally, subtype C strains of HIV-1 cause 56% of infections and are seen in the most populous nations including India, China, sub-Saharan Africa and Brazil ^[2]. It is not known if subtype C viruses are endowed with unique biological properties that make them successful in establishing rapidly growing epidemics. With an estimated 5.1 million infections (National AIDS Control Organization, http://www.naco.nic.in), second only to South Africa, India harbours the largest number of HIV-1 infections in the world ^[3]. Three of the five fastest growing epidemics of India are located in the southern states of Andhra Pradesh, Karnataka and Tamilnadu, where the infection incidence in antenatal clinics was above 1% in 2003 (NACO, India). Studies of viral molecular subtyping in India are few, used small sample numbers and were confined to urban locales ^[4-6]. Using a recently developed subtype-specific polymerase chain reaction (C-PCR) that differentially identifies subtype C viruses, we previously detected the presence of one each of A, B and B/C recombinant viruses ^[7]. To validate our findings further, in the present study, we evaluated a larger number of samples from all the four southern states of India. We demonstrate the identification of two B/C recombinant viruses that are unique in containing env sequences that closely associated with subtype B viruses of north America and Europe.

A total of 352 peripheral blood samples were collected from HIV-seropositive volunteers (195 men, mean age 35.5 years, range 17-60; 124 women, mean age 28.6 years, range 16-60; 22 subjects below 15 years of age; in the rest details were not recorded). Study subjects, representing a heterogeneous community of social and demographic groups, were voluntary participants under the care of several government hospitals, private clinics, and referral centres dedicated to the service of HIV/AIDS, in the southern states of Karnataka, Tamilnadu, Andhra Pradesh and Kerala.

Most of the blood samples were collected over a period of 4 years (2001-2004), after informed consent, in ethylenediamine tetraacetic acid vacutainers (Beckton Dickinson, San Diego, CA, USA) from individuals who were identified to be HIV seropositive by multiple enzyme-linked immunosorbent assays or Western blots. The study was performed under the approval of the institutional biosafety and ethics committees at the participating institutions. Genomic DNA was extracted directly from the peripheral blood and subjected to amplification as described previously ^[7]. The clinical profiles of all the subjects are summarized in Table 1 (supplementary data, online access at http://www.jncasr.ac.in./uday/Supplimantary_data/AIDS/aids.html).

Of a total of 608 primary infections (352 from the present study and 256 from a previous one from our laboratory), 602 strains (99%) were identified to be subtype C in long-term repeat (LTR) by C-PCR. A subset of 115 randomly selected samples from this cohort was further evaluated by heteroduplex mobility assay (HMA) or env sequencing to confirm the C-PCR analysis and also to identify the presence of additional recombinants, if any. The present analysis identified one additional subtype A and two more B/C recombinant viruses. Sequence analysis of the recombinants identified that all three viruses were subtype C in LTR (Fig. 1b) and subtype B in env (Fig. 1c). The three male subjects, from whom the recombinants were identified, hailed from different towns in the state of Karnataka (Shimoga, Mysore and Bangalore) and were not related to one another. None of the subjects used intravenous drugs; however, all three indulged in high-risk sexual behaviour.

Fig. 1

Phylogenetic analysis of the recombinants disclosed that the env sequences of two of the viruses (04/365 and 045/294) were closely related to the standard subtype B strains prevalent in north America and Europe (Fig. 1c). Considering that most of the previously reported B/C recombinants from other parts of the globe contained sequences of Thai B, but not of standard subtype B, and that the individuals had not reportedly traveled abroad, the identification of these B/C recombinants attains importance, suggesting an internal origin of these recombinants and a possible circulation of these recombinants within the population. The env sequence of the third sample (A8/02) clustered with Thai B viruses found in the north-eastern states of India and neighbouring China ^[8,9] and Myanmar ^[10,11]. In a BLAST analysis, all three env sequences differed from one another and from the closest subtype B reference strains by a large number of residues, ruling out the possibility of a laboratory contamination.

The presence of subtype B strains related to north American and European viruses has previously been documented from India in a minority of cases ^[12,13]. Despite the small numbers, these subtype B viruses could be the source of the B/C recombinants we identified in the present study. Importantly, of the total 608 samples, our analysis for recombinants included a subset of only 115 samples, suggesting that there could be more such recombinants in our cohort and in the population. An earlier publication from India reported a single A/C recombinant virus ^[14]. Our report is not only the first to document B/C recombinants of HIV-1 in India, but, to the best of our knowledge, is the first to identify B/C recombinants containing env sequences of subtype B viruses of the USA and Europe.

The data we present here allude to the presence of significant numbers of recombinants circulating in India. Studying the transmission pattern of HIV-1 in a population or a geographical locale is critical for understanding the changing dynamics of subtype incidence and distribution as a function of time. Recombination between different subtypes could significantly influence viral dissemination by increasing genetic diversity and modulating pathogenic properties ^[15]. The emergence of new recombinants could also complicate the development of effective intervention strategies and their implementation. A molecular epidemiological study on larger numbers of samples from different parts of the country is urgently warranted to determine the real incidence of B/C infection and to verify whether a new epidemic of B/C recombinants is emerging in India. It is also important to determine the original source of these recombinants, and whether they possess unique biological properties.

An independent study from India recently identified the presence of B/C recombinants of HIV-1 from the north-eastern regions of the country containing env of Thai B (Tripathy SP et al. AIDS Research and Human Retroviruses, 21, 151-157).

Acknowledgements

The HMA kit and other reagents were received from the National Institutes of Health AIDS Research and Reference Reagent Program and the Centralised Facility for AIDS Reagents, National Institute for Biological Standards and Control, UNAIDS. The Human Brain Tissue Repository for Neurobiological Studies at NIMHANS, Bangalore, is acknowledged for providing samples from the Body Fluid Bank. The authors would like to thank Professor Vinayaka R. Prasad for critically reading the manuscript.

Sponsorship: U.R. acknowledges institutional financial support from JNCASR. N.B.S. and P.K.D. are recipients of a CSIR fellowship from the Government of India. The funding source had no role in the study design, data collection, data analysis, data interpretation, or writing of the report.