HIV-1 subtype and viral tropism determination for evaluating antiretroviral therapy options: an analysis of archived Kenyan blood samples

Abstract

Background: Infection with HIV-1 is characterized by genetic diversity such that specific viral subtypes are predominant in specific geographical areas. The genetic variation in HIV-1 pol and env genes is responsible for rapid development of resistance to current drugs. This variation has influenced disease progression among the infected and necessitated the search for alternative drugs with novel targets. Though successfully used in developed countries, these novel drugs are still limited in resource-poor countries. The aim of this study was to determine HIV-1 subtypes, recombination, dual infections and viral tropism of HIV-1 among Kenyan patients prior to widespread use of antiretroviral drugs.

Methods: Remnant blood samples from consenting sexually transmitted infection (STI) patients in Nairobi were collected between February and May 2001 and stored. Polymerase chain reaction and cloning of portions of HIV-1 gag, pol and env genes was carried out followed by automated DNA sequencing.

Results: Twenty HIV-1 positive samples (from 11 females and 9 males) were analyzed. The average age of males (32.5 years) and females (26.5 years) was significantly different (p value < 0.0001). Phylogenetic analysis revealed that 90% (18/20) were concordant HIV-1 subtypes: 12 were subtype A1; 2, A2; 3, D and 1, C. Two samples (10%) were discordant showing different subtypes in the three regions. Of 19 samples checked for co-receptor usage, 14 (73.7%) were chemokine co-receptor 5 (CCR5) variants while three (15.8%) were CXCR4 variants. Two had dual/mixed co-receptor use with X4 variants being minor population.

Conclusion: HIV-1 subtype A accounted for majority of the infections. Though perceived to be a high risk population, the prevalence of recombination in this sample was low with no dual infections detected. Genotypic co-receptor analysis showed that most patients harbored viruses that are predicted to use CCR5.

Figure 1

Phylogenetic tree of HIV-1 gag gene. Clones generated from each patient sample were aligned and compared with reference sequences obtained from the Los Alamos HIV database. Phylogenetic relationships were constructed by neighbor-joining method and rooted with SIVcpzGAB. The bootstrap values (of 1000 replicates) above 70% are indicated next to the node. Brackets on the right indicate the subtype clusters.

Figure 2

Phylogenetic tree of HIV-1 pol gene. Cloned patient samples were aligned and compared with reference sequences obtained from the Los Alamos HIV database. Phylogenetic relationships were constructed by neighbor-joining method and rooted with SIVcpzGAB. The bootstrap values (of 1000 replicates) above 70% are indicated next to the node. Brackets on the right indicate the subtype clusters.

Figure 3

Phylogenetic tree of HIV-1 env-C2V3 region. Clones generated from each patient sample were aligned and compared with reference sequences obtained from the Los Alamos HIV database. Phylogenetic relationships were constructed by neighbor-joining method and rooted with SIVcpzGAB. The bootstrap values (of 1000 replicates) above 70% are indicated next to the node. Brackets on the right indicate the subtype clusters.