HIV-1 epidemic in India is largely driven by subtype C but
HIV-1 epidemic in India is largely driven by subtype C but other subtypes or recombinants have also been reported from several says of India. (B and C) and the recombinant CRF02_AG strains in North India suggests a rapidly evolving scenario of HIV-1 epidemic in this region with impact on vaccine formulation. Since this is the first report of CRF02_AG envelope from India, it will be important to monitor the spread of this strain and its impact on HIV-1 transmission in India. Introduction HIV-1 displays a tremendous amount of genetic diversity. The binding of the HIV-1 to host cells is usually mediated by envelope glycoprotein. When the HIV-1 envelope protein binds to its primary receptor CD4, it undergoes conformational changes and it then binds to one of the coreceptors (chemokine receptor CCR5, CXCR4 or others) via Rabbit Polyclonal to AML1 its V3 loop. This tri-molecular conversation leads to the viral membrane fusion . HIV-1 envelope is composed of relatively conserved (C1 to C5) and variable regions (V1 to V5). The V3 region elicits neutralizing antibodies and also govern co-receptor usage [1,2]. Replacements in the V3 region with basic amino acids are associated with CXCR4 usage [2,3]. Subtypes A and C usually contain a highly conserved GPGQ amino acid motif, while GPGR is the predominant motif in the V3 loop of subtype B envelopes [4,5]. Mutational patterns in the V3 loop region are likely to be of clinical significance as they can influence their susceptibility to known CCR5 inhibitors. Although all HIV-1 genetic subtypes originated in Africa, it is not fully comprehended how certain subtypes dominate different regions of the world. For e.g. buy Toll-Like Receptor 7 Ligand II subtype B predominates in US and UK but subtype C is usually predominant in India, some parts of Asia and Africa . It is fairly well established that HIV-1 that uses CCR5 chemokine receptor (R5-tropic) is usually transmitted preferentially than the ones that use CXCR4 chemokine receptor . Individuals with a 32 bp deletion in the CCR5 open reading frame (ORF) are largely guarded against HIV-1 contamination [7-9]. Approximately 50% of HIV-1 subtype B infected individuals show buy Toll-Like Receptor 7 Ligand II buy Toll-Like Receptor 7 Ligand II HIV-1 co-receptor switch from CCR5 to CXCR4 which is usually associated with rapid progression of HIV/AIDS . This is observed mainly in US and UK where subtype B predominates. However, in India, where subtype C predominates, the coreceptor switch has not been observed . Replacements of charged amino acids within the V3 region are known to alter the co-receptor usage [2,3,12]. Genetic variations in the subtype C HIV-1 envelope sequences have recently been reported from Southern India with some strains exhibiting multiple co-receptor usage, including CXCR4 chemokine receptor, present predominantly on T-helper lymphocytes [13,14]. It is noteworthy that we recently reported novel B/C LTR  and Vpr B/C/D sequences from North India . Given the large size of India, and with increasing global travel, it is likely that subtypes other than B may also co-circulate, creating an ideal situation for the formation of recombinants. With this in mind, we genetically characterized the HIV-1 envelope sequences from HIV-1 infected individuals from Northern India and report the presence of HIV-1 CRF02_AG for the first time. Methods Genomic DNA isolation and Polymerase chain reaction Genomic DNA was isolated from fresh peripheral blood collected in EDTA using a kit from Qiagen (QIAamp Blood Minikit) as described before by us [8,9]. All requisite ethical clearances were obtained before initiating this study. All the polymerase chain reactions (PCRs) were performed with high fidelity Taq DNA polymerase (Ex-Taq, Takara, Japan) using the following primers: Forward primer: 5′-ATGGGATCAAAGCCTAAAGCCATGTG Reverse primer: 5′-AGTGCTTCCTGCTGCTCCCAAGAACCCAAG Approximately 1.25 Kb DNA fragment corresponding to V1 to V5 region was amplified initially. Thereafter, 700 bp fragment (V3 to V5) was amplified using two internal sets of primers with following sequences: Forward primer: CTGTTAAATGGCAGTCTAGC Reverse primer: CACTTCTCCAATTGTCCCTCA The cycling conditions for amplifying both the fragments were: 35 cycles at 98C for 15 sec, 55C for 30 sec and 72C for 1 min with a final extension at 72C for 10 min. PCR-amplified DNA was cloned into pGem-T expression vector (Promega Biotech. WI, USA) and sequenced in both directions using T7 and SP6-specific primers. The sequence from one representative clone from each sample was used to carry out phylogenetic analysis and sequence comparisons. The final concentration of MgCl2 was 20 mM for both the PCRs. Mother and child samples were processed separately to avoid cross contamination. Patient populace and genetic analysis We carried out genetic analysis of 13 HIV-1 envelope sequences from Northern India. Nine unrelated and 2 mother-child pairs (Pair 1, D & E 57 and Pair 2, D & E 58) were selected randomly from two locations (one.