GENETIC DIVERSITY AND DRUG RESISTANCE PATTERNS OF HIV-1 ISOLATES FROM DELTA, ANAMBRA AND IMO STATES, NIGERIA

Abstract

Human Immunodeficiency Virus (HIV) is characterized by high genetic diversity which affects diagnosis, transmission, disease progression, vaccine design, co-receptor usage, response to antiretroviral therapy and development of drug resistance. Previous studies in Northern and Southwestern parts of Nigeria indicate the presence of multiple HIV strains, with different dominant strains and increasing resistance to some antiretroviral drugs. There is however limited information on strains circulating in the Southeastern part of the country. This study was undertaken to determine the diversity of circulating HIV-1 strains, their co-receptor usage potential and resistance patterns to Protease Inhibitors (PIs) in Delta, Anambra and Imo States. Blood samples were collected from 246 consenting HIV-1 infected individuals attending antiretroviral treatment centers in Delta, Anambra and Imo States. Genomic DNA was extracted from the samples using phenol/chloroform extraction procedure, and P24 gag, protease and C2-V3 envgenes amplified by nested PCR using specific primers for each of the genes. Thirty isolates with amplified products in all 3 genomic regions were sequenced using Sanger’s method. The nucleotide sequences were assembled, edited, blasted in genbank and phylogenetically analysed using ClustalW and Neighbour Joining method based on their nucleotide and amino acid sequences. Co-receptor usage and resistance patterns were determined using Geno2Pheno method and Stanford algorithm respectively. Data were analysed using descriptive statistics and Chi-square at α0.05. A total of 17 gag, 28 protease and 14 env sequences were obtained. Phylogenetic analysis showed that 17.7, 29.4 and 41.2% of the gag sequences were subtypes A, G and CRF02_AG respectively(p=0.00001) while 35.7 and 57.1% of protease sequences were identified as subtypes G and CRF02_AG respectively (p=0.00001). Also, 11.8 of gag and 7.1% of protease sequences were unclassifiable (U). Based on the env C2-V3 sequences, 7.1, 35.7, 50.0 and 7.1% were identified as subtypes A, G, CRF02_AG and J, respectively (p=0.00001). Mosaic structure was found among 71.4 and 26.7% of isolates in which 3 and 2 genomic regions respectively were successfully sequenced. The V3 loop of the 14 env sequences displayed total amino acid diversity of 82.9%. The CXCR4-tropic and CCR5-tropic viruses accounted for 42.9 and 57.1%, respectively (p=0.047715) among the study population. Maraviroc associated resistant mutations occurred in 12.5% of the CCR5-tropic viruses. From the 28 protease sequences, amino acid substitution occurred in 41.4% out of the 99 amino acid positions. Major PI resistance-associated mutations were found at two protease sites (M46L and V82L) in 3 (10.7%) of the sequences. Minor PI mutations identified include; L10V/I (25.0%), K20I (100%) and N88D (3.6%). Other polymorphisms identified include; I13V/A, E35Q, M36I/L, N37D/S/E/H, R57K/G, L63T/P/S/Q, C67E/S, H69K/R, K70R, V82I and L89M in the range of 28.6% to 100% among the different subtypes. The predominant HIV-1 subtypes circulating in Delta, Anambra and Imo States, Nigeria are CRF02_AG and G. Identification of mosaic strains including unclassifiable sequences highlights the complexity of HIV epidemic in Nigeria. Significant proportion of the isolates uses CXCR4 co-receptor with the potential of undermining success of Maraviroc in treatment.