1 Henry M. Jackson Foundation, 1 Taft Court, Rockville, MD 20850, USA
2 Theoretical Biology and Biophysics (T10), MS K710, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
3 Department of Virology, Swedish Institute for Infectious Disease Control, Karolinska Institute, S-171 82 Solna, Sweden
4 National Public Health Institute, HIV Laboratory, Department of Infectious Diseases, Mannerheimintie 166, FIN-00300 Helsinki, Finland
The phylogenetic complexity of the primate lentiviruses is legendary and our understanding of its scope was dramatically increased recently by the discovery of a new branch of the HIV-1 cluster, the "N" viruses [Simon et al., 1998]. There are now two major branches, "N" and "O", in the phylogenetic tree of HIV-1 sequences in addition to the cluster of sequences that form the "M", or Main group (Figure 1). The HIV-1 M, N, O, and the chimpanzee CPZ sequences cluster some what differently depending on the region compared (Figure~1). This is discussed in detail in conjunction with a new CPZ sequence, CPZ-US, a full length sequence described in [Gao et al., 1999]. (The CPZ-US sequence became available too late for inclusion in this study, but is available in the reference set at our website, http://hiv-web.lanl.gov/ALIGN_99/subtype_alignments.html.) At this point in time, however, only the viruses in the M group have significant public health importance. Genetic diversity within the M group takes the form of phylogenetic clusters which have been named subtypes. There are now at least 8 different subtypes of HIV-1 which circulate to varying extents in populations around the globe. A variety of factors make the genetic structure of HIV-1 particularly fluid both in time and space. This article will provide a description of our current understanding of the major circulating forms in the HIV-1 epidemic, and a subtyping reference set which can be used as a basis for the classification of new sequences.
The number of HIV-1 viruses which have been sequenced in their entirety has increased dramatically in the past few years [Korber et al., 1997], as have the number of tools designed to detect the presence of mosaic genomes [Salminen et al., 1995; Siepel et al., 1995]. It is important to distinguish newly discovered subtypes from recombinants, and to identify recombinant forms of epidemic importance. Now that full-length genomic sequencing is no longer a major obstacle, we propose that a virus isolate should fulfill the following criteria to be considered a subtype: (1) at least two isolates should be sequenced in their entirety, (2) they should resemble each other but no other existing subtype throughout the genome and, (3) they should have been found in at least two epidemiologically unlinked individuals. By these criteria, there are currently 8 subtypes of HIV-1. We are also aware that there are many mosaic genomes of HIV-1, some of which are unique, or restricted to one isolated transmission cluster, and others which are major circulating forms. Recombinant viruses are not as uncommon as previously thought and are especially prevalent in populations where multiple subtypes co-circulate. While possibly interesting for other reasons, the unique recombinant viruses do not play a major role in the global epidemic. In contrast, mosaic viruses which have spread from one location to another and have been associated with new outbreaks of the virus, such as the AE recombinant virus in Southeast Asia, have established a distinct and recognizable genetic lineage. It is proposed that those recombinant viruses be designated "Circulating Recombinant Forms", or CRFs, in distinction to the recombinants which are not known to be in circulation. We propose that a virus isolate should fulfill the following criteria to be considered a CRF: (1) at least two isolates should be sequenced in their entirety, (2) they should resemble each other but no other existing CRF in their subtype structure and (3) they should have been found in at least two epidemiologically unlinked individuals. These forms can be distinguished by associating the CRF with the name of the first full-length viral sequence of that form. By these criteria, there are currently 4 CRFs of HIV-1, the AE virus from Southeast Asia, called "AE(CM240)" [Carr et al., 1996; Gao et al., 1996a; Gao et al., 1996b], the AG recombinant from west and central Africa, called "AG(IbNG)" [Carr et al., 1998], the AGI recombinant from Cyprus and Greece, called "AGI(CY032)" [Gao et al., 1998a; Kostrikis et al., 1995; Nasioulas et al., 1999], and the AB recombinant from Russia, called "AB(Kal153)" This sequence was provided prior to publication by Mika Salminen and is representative of the CRF found in the Kaliningrad IVDU epidemic described in [Liitsola et al., 1998]. The 8 subtypes and 4 major circulating recombinant forms create 12 major branches in the phylogenetic tree representing the lineage of the M group of HIV-1 (Figure 1).
The 12 major genetic forms of the HIV-1 M group are listed, with selected full-length sequences to use as references, in Table 1. The subtypes are A, B, C, D, F, G, H, J, and the four CRFs are: AE(CM240), AG(IbNG), AGI(CY032) and AB(KAL153). New to this edition of the database are the first full-length sequences from subtypes F, G, H and J, as well as more new isolates from subtypes A, C and D. In addition, there are new full-length sequences from the CRFs AGI(CY032), AB(Kal153) and AG(IbNG).
Subtype A, the most prevalent subtype in Africa, has recombined with many other subtypes in a myriad of permutations and combinations. So far, however, only four of those recombinations, to our knowledge, have yielded viruses which have spread to a significant extent. The first is a recombination with subtype E, forming the AE virus prevalent in Southeast Asia [Carr et al., 1996; Gao et al., 1996a; Gao et al., 1996b]. The parental E virus has never been found. The virus contains a subtype E env and LTR, but most if not all of the remainder of the virus derives from subtype A. The second and third A recombinants which have spread extensively are ones which have recombined with subtype G. The first of these viruses is called "AG(IbNG)". The first isolate of this CRF which was fully sequenced was from Ibadan, Nigeria and was named "IbNG" [Howard et al., 1994; Howard et al., 1996; Gao et al., 1996b]. Other viruses with the same structure have been fully sequenced from Djibouti and Ivory Coast [Carr et al., 1998; Carr et al., 1999] and there are many partial sequences from west or west central Africa, all of which cluster with IbNG [Ellenberger et al., 1999; Takehisa et al., 1998; McCutchan et al., 1999]. The AG(IbNG) virus is mosaic in pol and LTR, but since both gag and env derive largely from subtype A these viruses were initially classified as subtype A [Howard et al., 1996]. In fact, in both gag and env they form a significant subcluster within the A subtype and can be recognized even using partial sequencing of familiar regions. The third major CRF is AGI(CY032), a recombinant between subtypes A and G and possibly another previously unknown subtype, I. Like the parental E virus, the parental, "pure" I virus is not known. Two of the three viruses of this type have been found in epidemiologically unlinked individuals in Cyprus and Greece [Gao et al., 1998; Nasioulas et al., 1999]. The last A recombinant to be identified is the AB(Kal153) virus from the city of Kaliningrad in Russia. Some of this recombinant is from subtype A but most of the env region is subtype B. This recombinant has been responsible for an explosive epidemic among drug users in the city of Kaliningrad [Liitsola et al., 1998].
The genetic structure of the first full length AG(IbNG) and subtype G viruses has been recently published [Carr et al., 1998]. In protease, in the accessory gene region, and at the very end of env, an unusual phylogenetic relationship exists between subtypes A, G, AE(CM240) and AG(IbNG). In a genetic sense, they are neither as close, nor as distant, as in other regions of the genome, where it is simple to identify the region as belonging to a given subtype. In these regions they show an intermediate relationship. While this phenomenon is observed with the A, G, E and IbNG cluster, it is not observed with the other subtypes in the same regions. It is therefore not due to a general weakness in the information content of the region or to the analytic approach. Some have suggested that the G viruses are actually recombinant with subtype A in these regions (Gao et al., 1998), and while this is a possibility, others are unable to convincingly demonstrate a recombinant nature for the G viruses [Carr et al., 1998]. At the moment the issue is not completely resolved.
A variety of intersubtype recombinants combining segments of A and C, A and D, B and F, and others have been described or are known in yet-to-be-published studies. Each of these unique forms could potentially spread epidemically, and as new recombinants are studied it is increasingly important to compare them in detail to the full spectrum of known recombinant forms. The initial events leading to the emergence of recombinants may be better understood in future years.
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