Identification of subtype C human immunodeficiency virus type 1 by subtype-specific PCR and its use in the characterization of viruses circulating in the southern parts of India

Abstract

Human immunodeficiency virus type 1 (HIV-1) subtype C viruses are associated with nearly half of worldwide HIV-1 infections and are most predominant in India and the southern and eastern parts of Africa. Earlier reports from India identified the preponderance of subtype C and a small proportion of subtype A viruses. Subsequent reports identifying multiple subtypes suggest new introductions and/or their detection due to extended screening. The southern parts of India constitute emerging areas of the epidemic, but it is not known whether HIV-1 infection in these areas is associated with subtype C viruses or is due to the potential new introduction of non-subtype C viruses. Here, we describe the development of a specific and sensitive PCR-based strategy to identify subtype C-viruses (C-PCR). The strategy is based on amplifying a region encompassing a long terminal repeat and gag in the first round, followed by two sets of nested primers; one amplifies multiple subtypes, while the other is specific to subtype C. The common HIV and subtype C-specific fragments are distinguishable by length differences in agarose gels and by the difference in the numbers of NF-kappaB sites encoded in the subtype C-specific fragment. We implemented this method to screen 256 HIV-1-infected individuals from 35 towns and cities in four states in the south and a city in the east. With the exception of single samples of subtypes A and B and a B/C recombinant, we found all to be infected with subtype C viruses, and the subtype assignments were confirmed in a subset by using heteroduplex mobility assays and phylogenetic analysis of sequences. We propose the use of C-PCR to facilitate rapid molecular epidemiologic characterization to aid vaccine and therapeutic strategies.

FIG.1.

C-PCR strategy, genomic position, and variability in sequences corresponding to subtype C-specific primers. (Top) LTR/gag region corresponding to primers used in this study and their relative positions (not drawn to scale). The upstream subtype C-specific primer, N415F, was anchored on the upstream stimulatory factor site in the LTR, while the downstream primer was anchored on the NF-κB site. (Bottom) Levels of variability in sequences corresponding to these primers among various subtypes. For each subtype, using the number of sequences indicated, the proportion of different nucleotides at each position was calculated and plotted as a percentage. Subtype C sequences from India (C_IN) are shown separately. The numbers below the subtypes indicate the average uncorrected nucleotide distances between the primer and the corresponding subtype. The asterisks indicate the four positions that accounted for much of the variability within subtype C sequences. International Union of Pure and Applied Chemistry codes were used for positions with redundant nucleotides.

FIG. 2.

Specificity and sensitivity of C-PCR. (A) The Specificity of C-PCR was assessed using molecular clones derived from different subtypes of HIV-1 in the following order (from left): A (p92UG037.1), A (p90CF402.1.8), B (pBH10), B (pYU2), C₂ (pMJ4), C₃ (pINDIE), C₄ (p92BR025.8), D (p94UG114.1), D (p94UG114.1.6), D (p84ZR085.1), E (p90CF402.1), F (p93BR020.1), G (p92NG003.1), G (p92NG083.2), and H (p90CF056.1). Lane M, DNA size standard; −, control lane. The extra NF-κB site present in the subtype C₄ molecular clone can be seen to lead to increased size of the subtype C-specific fragment. (B) Sensitivity of C-PCR. The subtype C molecular clone pINDIE was used at the copy numbers shown above the lanes. Optimization of uniplex PCR conditions and their subsequent use in multiplex PCR with no loss of sensitivity are seen. −, control lane.

FIG. 3.

Subtyping clinical samples with C-PCR, HMA, and sequencing. (A) Illustration of typical gel profiles of subtype C sequences obtained using C-PCR. Each lane represents a different clinical sample. −, control reaction mixture that contained DNA from an individual not infected with HIV-1. (B) HMA analysis of a subset of samples identified as subtype C by C-PCR. These representative samples demonstrate the typical HMA profiles of subtype C viruses. The labeling above the lanes indicates the subtype standards used. Although additional subtype standards were used in the analysis, only profiles with subtype A and C standards are shown. −, control lanes that did not include subtype standards. (C) A non-C virus identified as subtype B by HMA. C-PCR failed to amplify the LTR fragment from this sample. (D) A single recombinant virus identified in this study was subtype C in the LTR and subtype B in env. (E) Insertion of additional sequences resembling NF-κB motif is common in subtype C strains and results in bands with lower mobility than expected (Fig. 1A). The insertion of a 15-bp sequence between two authentic NF-κB motifs of one such clinical sample has been confirmed by sequencing the LTR. An NF-κB-like motif is highlighted.

FIG. 4.

Phylograms depicting the relationship between env and LTR sequences obtained in this study (solid circles) and sequences obtained from the HIV database. Single non-subtype C env sequences obtained from the database are identified by the final digits of their accession numbers. Subtype C sequences from eight other countries were included in the analysis and are identified by open triangles along with their two-letter country codes and the accession numbers. The open circles identify subtype C sequences from India available in the database. The phylograms were obtained from gap-stripped alignments and edited. The log likelihood (lnL) scores for the phylograms and the scale bars corresponding to substitutions per codon are indicated.

FIG. 5.

Geographic spread in the assessment of subtype prevalence in India. The four states in the south and the one state in the east of India from which the clinical samples for this study were collected are shaded. The urban centers of the states are indicated by stars, and the towns and cities are indicated by triangles. Subtype C viruses were found in all of these places, and the three places where non-subtype C viruses or recombinants were detected are circled.