Development of an env gp41-based heteroduplex mobility assay for rapid human immunodeficiency virus type 1 subtyping

Abstract

The gp120 region of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene exhibits a high level of genetic heterogeneity across the group M subtypes. The heteroduplex mobility assay (HMA) has successfully been used to assign subtype classifications, but C2V5 primers often fail to amplify African strains. We developed an env gp41-based HMA for which the target sequence is amplified with highly conserved gp41 primers, known to efficiently amplify nucleic acids from HIV-1 group M, N, and O viruses. By using gp41 from a new panel of reference strains, the subtype assignments made by our modified HMA were concordant with those obtained by sequencing and phylogenetic analysis of 34 field strains from 10 countries representing subtypes A to G. Testing of field strains from Nigeria further demonstrated the utility of this modified assay. Of 28 samples, all could be amplified with gp41 primers but only 17 (60.7%) could be amplified with the standard C2V5 primers. Therefore, gp41-based HMA can be a useful tool for the rapid monitoring of prevalent subtypes in countries with divergent strains of circulating HIV-1.

FIG. 1

HIV-1 subtyping by gp41-based HMA. The subtypes of HIV-1 strains IC2239, UG31, TH135, TH24, 98ZA142, BR063, and 97GLB24 identified are marked with the prefixes A, B, C, D, E, F, and G, respectively. The designations A1, A2, B1, B2, B3, C1, C2, D1, D2, E1, E2, F1, F2, G1 and G2 indicate the subtype reference strains used in the formation of the heteroduplex; C, control.

FIG. 2

Phylogenetic classification of env gp41 HIV-1 sequences (arrowheads) from representative group M subtype A to G strains obtained worldwide (GenBank accession numbers AF43895 to AF343910). Sequences denoted with the abbreviations LB, UG, TZ, TH, CN, ZA, IL, BR, IC, and SG were from Lebanon, Uganda, Tanzania, Thailand, China, South Africa, Israel, Brazil, Ivory Coast, and Singapore, respectively. The numbers before those abbreviations indicate the year of specimen collection. Values on the branches represent the percentages of 100 bootstrap replicates. HIV-1 subtypes and groups are marked with the prefixes A, B, C, D, E, F, G, H, and J and the prefixes N and O, respectively. The scale bar indicates the evolutionary distance of 0.10 nucleotides per position in the sequence. Vertical distances are for clarity only.