Near full-length genetic analysis of HIV sequences derived from Cyprus: evidence of a highly polyphyletic and evolving infection.

The molecular epidemiology of HIV-1 infection was previously studied in Cyprus but the degree of HIV-1 diversity has remained indefinable. The main objective of the present study is to examine HIV-1 strains isolated from 77 HIV-1-infected individuals representing 38% of the known infected population in Cyprus in the period 1986 to 2006. DNA of the near full-length genome encoding gag, pol, vif, vpr, vpu, tat, rev, env, and 5'-end of nef was amplified by nested PCR/RT-PCR from all HIV-1 seropositives and sequenced using a newly designed assay. Detailed phylogenetic and bootscanning analyses were performed to determine phylogenetic associations and subtype assignments. Phylogenetic analyses of the obtained viral sequences indicated that subtype B was the dominant subtype (61%), followed by subtype A (23.3%), subtype C (5.2%), CRF02_AG (3.9%), and subtype D, CRF01_AE, and CRF04_cpx (1.3% each). Two HIV-1 isolates (2.6%), originating from the Democratic Republic of Congo (DRC), were not classified in any pure (sub)subtype or circulating recombinant form (CRF). Complete phylogenetic and bootscanning analyses revealed that one of these isolates had a new, unique recombinant pattern, comprising segments of subtypes D and G, and is distinct from any other CRFs or URFs reported so far. Detailed analyses of the sequence of the second isolate, which could not be classified, reveal that it is close to subtype K reference sequences but clusters near the root of the clade. At least two epidemiologically unrelated HIV-1 seropositives with an HIV-1 variant similar to this isolate are required to designate this variant as a novel HIV-1 subtype or subsubtype of subtype K. Analogous to results of the earlier epidemiological studies, these data exhibit the extensive heterogeneity of HIV-1 infection in Cyprus, which is being fueled by a continuous entry of new strains from other countries, creating an evolving and polyphyletic infection.