GENETIC DIVERSITY AND CORECEPTOR USAGE OF HIV-1 STRAINS AMONG INFECTED INDIVIDUALS PRESENTING AT A VOLUNTARY COUNSELLING AND TESTING CENTRE IN IBADAN, NIGERIA

Abstract

Molecular analyses of Human Immunodeficiency Virus (HIV) have shown great genetic diversity among strains of the virus. The diversity has implications on efficiency of its transmission, pathogenesis, diagnosis, vaccine design, coreceptor usage, response to anti-retroviral therapy and development of drug resistance. The HIV epidemic in Nigeria is characterised by the presence of multiple strains of the virus and increasing viral resistance to some anti-retroviral drugs. This study was carried out to determine the circulating HIV-1 strains and their coreceptor usage patterns at a voluntary counseling and testing centre in Nigeria. Blood samples were collected from 85 consenting HIV-1 infected (40 seroconcondant couples and partners of 5 serodiscordant couples) anti-retroviral therapy-naïve patients. They include 42 males and 43 females with median age of 37 years (range 18-58 years) who presented at a voluntary counselling and testing centre in the University College Hospital, Ibadan. Genomic DNA was extracted from whole blood using a commercial DNA extraction kit. The env C2-V3 region of HIV-1 from the genomic DNA extract was amplified by nested PCR using primers WT1 and WT2, and KK40 and KK30 for the first and second rounds, respectively. Amplified DNA was directly sequenced in both forward and reverse directions, edited and analysed for phylogenetic relationships by ClustalW and Maximum Likelihood methods in a specialised software based on their nucleotide and amino acid sequences. The target region was successfully sequenced from 64 of the 85 patients comprising 23 of the 40 seroconcordant couples, 13 single partners of the remaining 17 seroconcordant couples and the 5 serodiscordant couples. Overall, 3.1%, 53.1%, 28.1%, 14.1% and 1.6% of the HIV-1 env C2-V3 nucleotide sequences were identified as subtype A, G, CRF02_AG, CRF06_cpx, and CRF35_AD, respectively, with variation in subtype distribution. Eighteen of the 23 HIV-1 positive seroconcordant couples were infected with the same HIV-1 strain while the other five HIV-1 seroconcordant couples were infected with different HIV-1 strains indicating independent sources of infection of each spouse. The V3 loop region of the viruses had amino acid sequence conservation in the base positions 1-8 and 26-35 and tip regions and sequence variability, including mutations and deletions at positions 9-25. Most (76.5%) of the sequences had the GPGQ crown motif while the GPGQ/L/R/K substitution was observed in 18.8%. The number of N-linked glycosylation sites ranged from 0 to 4 per env C2-V3 amino acid sequence with only 37.5% of the sequences having all four N-linked glycosylation sites. The CXCR4-tropic viruses known to be associated with the presence of multiple amino acid mutations were predominant (68.8%) among the studied population. Ten percent of the CCR5-tropic viruses showed Maraviroc associated resistant mutations. There was evidence of circulation of HIV-1 CRF35_AD in Nigeria, and multiple circulation of HIV-1 subtypes, with the predominance of HIV-1G. The predominance of CXCR4-tropic viruses and presence of primary resistance to Maraviroc in 10% of CCR5-tropic viruses suggest the need for further investigation prior to introduction of coreceptor inhibitors like Maraviroc for management of HIV infection in Nigeria. Keywords: HIV-1 strains, Genetic diversity, C2-V3 region, Coreceptor usage Word count: 492