Analysis of the near full length genomes of HIV-1 subtypes B, F and BF recombinant from a cohort of 14 patients in São Paulo, Brazil

Abstract

The human immune deficiency virus (HIV) exhibits strikingly tremendous amount of genetic variability. Such feature is critically important for the virus to adapt to environmental changes by escaping the host immune system and by escaping candidate vaccine. Therefore, understanding of such diversity is fundamental for the design of successful drugs or vaccine, which is urgently needed to bring the HIV/AIDS epidemic under control. In this study, we investigated the magnitude of diversity of the HIV-1 near full-length genomes from patients previously assigned as infected with non-recombinant HIV-1 subtypes B and F1 variants based on small portion of viral genome. HIV-1 proviral DNA was extracted from 14 samples previously classified in our laboratory as six subtypes B and eight subtypes F on the basis of small amplicon sequencing. Reamplifications of DNA from these samples were carried out by an overlapping PCR followed by direct sequencing. The data were phylogenetically inferred. Sequence analysis revealed that two out of six partially identified subtype B and six out of eight partially identified subtype F were in fact BF recombinants throughout their full genomes. Two pairs BF recombinants had identical genomic recombination structure and distinct from the Argentinean CRF 12_BF strains, probably represents a novel circulating recombinant forms in Brazil. Our data provided new genetic material of some of the HIV-1 subtypes currently circulating in the country and points to the widespread of BF recombinants which are expected to change the epidemic nature by approaching the level of subtype B in Brazil.

Introduction

Human immunodeficiency virus-1 (HIV-1) is the causative agent of AIDS and can persist in individuals for years before causing disease. The HIV-1 genome is approximately 9.5 kb and displays significant sequence variations as a result of constant mutation and evolutionary pressure. Based on these genetic variations and pattern observed in phylogenetic reconstruction, researchers have classified the virus into groups, subtypes and sub-subtypes (Robertson et al., 2000). Currently, three groups (M, main; O, outlier; N, neither) have so far been recognized. HIV-1 group M viruses are responsible for the current global epidemic and are further classified into nine (A–D, F–H, J and K) subtypes, approximately equidistant, although subtypes B and D seem to be more closely related. Moreover, early sequencing studies have provided evidence of interstrand crossovers between genomes of different HIV subtypes (Sabino et al., 1994, Robertson et al., 1995). Such interclade recombinant strains are consistently reported from regions where two or more clades are predominant. Recombinant strains from unlinked epidemiological sources that exhibit identical patterns of mosaicism have been classified separately as circulating recombinant forms (CRFs) (Carr et al., 1998). There are currently 16 defined CRFs, namely CRF01–CRF16 and are epidemiologically important as subtypes. HIV-1 subtypes and CRFs show considerably different patterns of distribution in different geographical regions.

In South America, HIV epidemic is concentrated among people at increased risk of infection like injecting drug users, and men who have sex with men. The most prevalent HIV genetic subtypes are subtypes B, BF recombinants and F (Hierholzer et al., 2002). CRF12_BF and its related recombinant forms are widely circulating in Argentina (Carr et al., 2001, Thomson et al., 2002).

Brazil is the Latin American country that has been hardest hit by the HIV epidemic and has the second highest number of HIV-1 cases in the Americas, after the USA with an estimated number of 610,000 HIV-1/AIDS cases at the end of 2001 (Global AIDS program, 2004). HIV-1 subtype B is a major genetic form circulating in the country, however, existence of small proportion of other subtypes such as F, C, B/C and B/F have been consistently reported (Sabino et al., 1994, Cornelissen et al., 1996, Bongertz et al., 2000, Caride et al., 2000, Brindeiro et al., 1999). Typing strategies of these strains are based on sequencing of one or multiple short fragments of the viral genome, which may not accurately define the true HIV-1 genetic structure. Efforts to search for CRFs in Brazil in two recent studies did not find a common ancestry of Brazilian recombinants or their relationship to CRF12_BF (Thomson et al., 2004, Sa Filho et al., 2005). The current study was undertaken to characterize HIV-1 near full-length genomes from patients previously assigned as infected with non-recombinant HIV-1 subtypes B and F1 variants based on small portion of viral genome.