HIV-1 Subtype Distribution Determined by Phylogenetic Analysis of pol Gene Sequences and Automated Subtyping Tools among HIV-1 Isolates from the Aegian Region of Turkey

Human immunodeficiency virus (HIV) exhibiting remarkable genetic variability, includes two genotypes namely HIV-1 (group M, N, O and P) and HIV-2 (group A-H). HIV-1 group M, which is mainly the cause of the AIDS pandemic, is divided into nine pure subtypes, more than 45 circulating recombinant forms (CRF) and numerous unique recombinant forms (URF). According to the documents of Turkish Government of Health, among a total of 6802 HIV-positive cases, 1096 of them were defined as AIDS as of June 2013 in Turkey. Although subtype B is the predominant subtype, recent studies indicate higher proportion of CRFs similar to their increasing role in the HIV pandemic. The aim of this study was to determine the subtype distribution of HIV-1 strains isolated from 70 patients (61 male, 9 female; age range: 16-73 yrs, mean age: 39.6 yrs) who presented to our institution between April 2008-June 2013. HIV-1 strains were subtyped by phylogenetic analysis of the pol gene region and commonly used automated subtyping tools namely, Stanford HIV db v6.2.0 and Rega v3.0. Pol sequences retrieved from the Los Alamos database and from GeneBank, were trimmed from full-length genomes. Phylogenetic analysis of the 1302 base pair of the pol gene region was performed using Mega v5.2 software. The sequences were aligned using Muscle and phylogenetic distances between sequences were estimated by using Kimura two-parameter model (transition/transversion ratio: 2.0). Tree topology was obtained using neighbour-joining method and bootstrap value was set at 1000. Sixty-one (87.1%) patients were antiretroviral treatment (ART)-naive and nine were on different ART regimens. The subtypes of the isolates according to phylogenetic analysis were found as follows; 31(44.2%) subtype B, 24 (34.2%) CRF42_BF, 6 (8.5%) B/CRF02_AG recombinants, 5 (7.1%) sub-subtype A1, 1 (1.4%) sub-subtype F1, 1 (%1.4) CRF 25_cpx, 1 (1.4%) CRF02_AG and 1 (1.4%) CRF01_AE. Rega v3.0 subtyping tool produced five discrepant results (4 B/CRF02-AG and 1 CRF42_BF) compared to phylogenetic analysis. Stanford HIVdb v6.2.0 had eight results (3 CRF42_BF, 2 subtype B, 2 sub-subtype A1, 1 CRF25_cpx) that were not concordant with phylogenetic analysis. Stanford HIVdb v6.2.0 was able to subtype all B/CRF02_AG recombinant strains. B/CRF02_AG recombinants which were seen among homosexual men in France were for the first time isolated in Turkey from five men (2 homosexual, 2 bisexual, 1 heterosexual) and one heterosexual woman. CRF42_BF had not been found in Turkey previously and it has not been a common type isolated in neighboring countries either. Full genome sequencing could be helpful to further analysis of those isolates. Our results support the latest studies from Turkey reporting increase in the proportion of CRF-related infections. This is not an unusual finding when geographical location of Turkey is considered. Nevertheless, more comprehensive data regarding molecular epidemiology and subtype distribution of HIV-1 isolates in Turkey are needed