Independent introduction of transmissible F/D recombinant HIV-1 from Africa into Belgium and The Netherlands.

Most HIV-1 subtype F viruses described so far have been isolated from individuals originating in South America, Romania, or Central Africa. Previous studies have shown that subtype F viruses from these three areas can be distinguished by phylogenetic tree analysis of various parts of the HIV genome. Subtype F strains circulating in Central Africa and classified as subgroup F2 and F3 have relatively large nucleotide distances from strains of subgroup F1, which includes some African strains, along with strains from Romania and South America. Subtype F strains have now appeared in Europe. In this study, we analyzed the complete gag gene and a large fragment of the pol gene of seven strains of African origin that represent the three F subgroups. At least five of the seven strains appear to be intersubtype recombinants. Of four strains circulating in Belgium and the Netherlands, three were F/D mosaics and the fourth harboured a G(gag)/GH(pol)/F3(env) recombinant structure. Two of the three F/D mosaics showed identical breakpoints and were independently introduced in Belgium and the Netherlands. At least two of the mosaics were further transmitted. The remaining three strains of the seven we studied were isolated from individuals in Cameroon. Two included large or smaller F1 fragments in gag and pol. The third strain was subtype D along the entire gag and pol fragment. A parental African subtype F that showed no evidence for recombination was not found.

Genetic evidence of multiple transmissions of HIV type 1 subtype F within Romania from adult blood donors to children.

We studied the phylogeny of HIV-1 subtype F viruses from children and adults in Romania in order to (1) clarify whether the Romanian subtype F epidemic was caused by one or several virus introductions and (2) gain insight into the route of spread of the HIV-1 subtype F virus among children and adults in Romania. env (V3), gag (p17/half p24), and pol (prot/half RT) sequences were obtained from three districts in Romania: Tirgu Mures (n = 9, children), Craiova (n = 15, children), and Bucharest (n = 13, adults). Of 37 HIV V3 sequences from Romania, 35 belonged to the genetic subtype F in the neighbor-joining tree, whereas 2 sequences from adults clustered with subtypes A and C. Within the subtype F cluster, no bootstrap-supported subclusters were observed according to geographic area in Romania. Two of the adult V3 sequences that clustered with the children were obtained from individuals who tested HIV seropositive in 1989 and 1990, showing that the subtype F virus was present among adults when the HIV epidemic began among children in Romania. The HIV-1 subtype F viruses obtained from children showed a mean pairwise V3 nucleotide distance of 7.9% and maximum distances of between 18 and 19%; both are higher than previously described. The mean V3 distances (overall, synonymous, and nonsynonymous) were significantly higher for adults than for children. One V3 sequence from the Democratic Republic of Congo clustered within the Romanian sequences, suggesting that the subtype F virus in Romania may originate from this area. Our data also suggest that HIV-1 subtype F was present among Romanian adults before it appeared in 1989 among institutionalized children. The juvenile population was most likely infected with the HIV-1 subtype F virus on more than one occasion, presumably through HIV-contaminated blood (products) obtained from adults.

Spread of distinct human immunodeficiency virus type 1 AG recombinant lineages in Africa.

To identify new subtype G human immunodeficiency virus type 1 (HIV-1) strains and AG recombinant forms, we collected 28 serum samples from immigrants to the Netherlands from 12 countries throughout Africa. Based on the gag sequences 22 isolates were identified as subtype A or G. Phylogenetic analysis of discontinuous regions of the gag (726 nt), pol (1176 nt) and env (276 nt) genes revealed 13 AG recombinants with the mosaic structure A(gag)/G(pol)/A(env), three with A(gag)/G(pol)/G(env) and one other with A(gag) /G(pol)/G(env), in addition to 'pure' subtypes A(gag)/A(pol)/A(env) (n=1) and G(gag)/G(pol)/G(env) (n=4). To analyse the crossover points in more detail, a new RT-PCR was developed resulting in a large contiguous sequence of 2600 nt from the gag region to half the pol region. All the 13 A(gag)/G(pol)/A(env) recombinants appeared to belong to the circulating recombinant form (CRF) AG (IbNG). The three A(gag)/G(pol) /G(env) recombinants differed from the CRF AG (IbNG) subtype, suggesting the identification of a new CRF subtype. The recovery of AG recombinants from African countries a thousand miles apart indicates the active spread of new recombinants.

pol gene diversity of five human immunodeficiency virus type 1 subtypes: evidence for naturally occurring mutations that contribute to drug resistance, limited recombination patterns, and common ancestry for subtypes B and D.

Naturally occurring mutations in the polymerase gene of human immunodeficiency virus type 1 (HIV-1) have important implications for therapy and the outcome of clinical studies. Using 42 virus isolates obtained from the UNAIDS sample collection, we analyzed the protease (99 amino acids [aa]) and the first 297 aa of reverse transcriptase (RT) coding regions. Based on the V3 sequence analysis, the collection includes subtype A (n = 5), subtype B (n = 12), subtype C (n = 1), subtype D (n = 11), and subtype E (n = 13) viruses. Of the 42 protease genes, 37 contained naturally occurring mutations at positions in the gene that contribute to resistance to protease inhibitors (indinavir, saquinavir, ritonavir, and nelfinavir) in clade B isolates. The phenotypic effect of these substitutions in non-B isolates is unclear. The The 5'half RT coding region of the 42 isolates was found to be less variable, although 19 of the 42 RT sequences contained amino acid substitutions known to contribute to nucleoside and/or nonnucleoside drug resistance. Since the virus isolates were obtained in 1992, it is unlikely that the infected subjects received protease inhibitors, but we found evidence that one subject acquired a zidovudine (AZT)-resistant HIV-1 strain from a contact who had received AZT. Phylogenetic analysis identified five subtype pol clusters: A, B, C, D, and A'. Comparison of env and pol sequences of the same viruses showed no more recombination events than were already identified on the basis of gag/env comparison (M. Cornelissen, G. Kampinga, F. Zorgdrager, J. Goudsmit, and the UNAIDS Network for HIV Isolation and Characterization, J. Virol. 70:8209-8212, 1996). In one of the known recombinants, a crossover site between subtypes A and C could be identified, and in another, a crossover site could not be identified due to lack of a reference subtype F pol sequence. We analyzed the ds/da ratio of gag, pol, and env sequences of 35 isolates, excluding the recombinants. Our analysis showed that gag and pol are subjected to purifying selection with an average ds/da ratio above 1, independent of the subtype and in contrast with V3 (ds/da approximately 1). Based on the low ds/da ratio of the intergroup analysis of A/E and B/D gag and pol sequences, we analyzed the evolutionary relation between subtypes B and D in more detail by constructing separate phylogenetic trees for synonymous and nonsynonymous substitutions. Our analysis suggests a common ancestry for subtypes B and D that is distinct from that of subtypes A and E.