Human immunodeficiency virus type 1 env sequences from Calcutta in eastern India: identification of features that distinguish subtype C sequences in India from other subtype C sequences

Abstract

India is experiencing a rapid spread of human immunodeficiency virus type 1 (HIV-1), primarily through heterosexual transmission of subtype C viruses. To delineate the molecular features of HIV-1 circulating in India, we sequenced the V3-V4 region of viral env from 21 individuals attending an HIV clinic in Calcutta, the most populous city in the eastern part of the country, and analyzed these and the other Indian sequences in the HIV database. Twenty individuals were infected with viruses having a subtype C env, and one had viruses with a subtype A env. Analyses of 192 subtype C sequences that included one sequence for each subject from this study and from the HIV database revealed that almost all sequences from India, along with a small number from other countries, form a phylogenetically distinct lineage within subtype C, which we designate C(IN). Overall, C(IN) lineage sequences were more closely related to each other (level of diversity, 10.2%) than to subtype C sequences from Botswana, Burundi, South Africa, Tanzania, and Zimbabwe (range, 15.3 to 20.7%). Of the three positions identified as signature amino acid substitution sites for C(IN) sequences (K340E, K350A, and G429E), 56% of the C(IN) sequences contained all three amino acids while 87% of the sequences contained at least two of these substitutions. Among the non-C(IN) sequences, all three amino acids were present in 2%, while 22% contained two or more of these amino acids. These results suggest that much of the current Indian epidemic is descended from a single introduction into the country. Identification of conserved signature amino acid positions could assist epidemiologic tracking and has implications for the development of a vaccine against subtype C HIV-1 in India.

FIG. 1

Deduced amino acid sequences of partial HIV-1 env sequences obtained from the 21 subjects in this study. Sequences from 20 subjects harboring subtype C were aligned with the subtype C consensus sequence. Subtype A sequences from subject 12 were aligned with a consensus sequence derived from the four sequences sampled from this individual. IN99C and IN99A in the names indicate the year of sampling and subtype assignment. Numbers in parentheses indicate the number of sequences with identical amino acid sequences. The regions corresponding to V3 and V4 in the envelope protein are highlighted. The nine amino acid positions identified to be particularly discriminatory for subtype C_IN sequences (Table 2) are indicated (¶). In addition, the amino acids at positions 51, 61, and 156 (corresponding to positions 340, 350, and 429, respectively) that were conserved in more than 70% of the C_IN sequences are underlined. Within the alignment, dots indicate identity with the consensus sequence, dashes indicate deletions, and asterisks indicate stop codons.

FIG. 2

DNA distances between subtype C sequences sampled from various countries. The inset shows the mean DNA distances for comparison of sequences sampled within and between each of the six countries where eight or more sequences were available for comparison. The solid red line in the plot depicts the distribution of DNA distances when sequences sampled within India were compared to each other. Other lines illustrate the distribution of pairwise DNA distances when sequences from India were compared to sequences from each of the other countries.

FIG. 3

Phylogenetic relationships among subtype C HIV-1 env sequences sampled from different countries. Neighbor-joining analysis using 192 sequences encoding V3-V4 region was implemented using the TVM+I+G evolutionary model as described in the text.

FIG. 4

Maximum likelihood (TVM+G evolutionary model) phylogram of all C_IN lineage sequences along with other sequences sampled from India and reference sequences for other subtypes. C_IN lineage sequences identified in Fig. 3 are shown within the gray box. C_IN lineage sequences clustered into two lineages, one containing only sequences from India (except one from Israel in an unpublished study; ILNO10.X94393) and another containing a small number of sequences from African countries. Sequences from India are in bold, and those isolated in this study are underlined. Sequence identifiers show the two-letter ISO 3166 country codes (http://www.din.de/gremien/nas/nabd/iso3166ma/codlstp1/en_listp1.html) and the year of isolation, when available. The log likelihood score for the phylogram was −5654.97269.