Genetic characterization of the nef gene from human immunodeficiency virus type 1 group M strains representing genetic subtypes A, B, C, E, F, G, and H.

Most efforts to characterize sequence variation of HIV isolates has been directed toward the structural envelope gene. Few studies have evaluated the sequence variability of auxiliary genes such as nef. In this study 41 new HIV-1 strains, representing the majority of the described envelope subtypes of HIV-1 (A to H), were genetically characterized in the nef region. Phylogenetic analysis showed that 34 strains could be classified in the same subtype in nef and env, and 7 (19%) of the 41 new viruses were recombinants. For two of the seven strains, recombination occurred upstream of the nef gene, whereas for five of the seven strains recombination occurred within the nef gene with a crossover close to the 5' end of the LTR (long terminal repeat). The low intersubtype distance between subtype B and D in the nef gene confirms previous observations in the pol, env, and gag genes, which suggest a common ancestor for these subtypes. The majority of all the previously described functional domains in the nef gene were relatively conserved among the different subtypes, with only minor differences being observed. The myristoylation signal among the different subtypes, with only minor differences being observed. The myristoylation signal was less conserved for subtype C, with one or more amino acid changes being observed at positions 3, 4, and 5. The highly conserved acidic region (positions 62 to 65), critical for the enhancement of viral synthesis with an increased virus growth rate, was less conserved among the subtype G strains from our study. At least three epitopic regions of the nef gene have been defined and each can be recognized by CTLs under a variety of HLA restrictions; all were also relatively well conserved between the different genetic subtypes. Despite the relatively important genetic variation in nef sequences obtained among the different genetic subtypes, functional domains and CTL epitopes were relatively well conserved. In vitro and/or in vivo studies are necessary to study the relevance of the observed differences.

Presence of diverse human immunodeficiency virus type 1 viral variants in Cameroon.

Phylogenetic analysis of the gp41 region of 123 HIV-1-seropositive specimens from Cameroon showed that 89 were subtype A (71% of these sequences were IbNg-like), 12 (10%) were subtype D, 11 (9%) were subtype G, 5 (4%; closely related to subtype F2) were subtype F, 1 was subtype H, 2 (1.6%) remained unclassifiable, while 3 were group O. Further analysis of the two unclassifiable specimens in gag(p24), pol(prot), and env (C2V3 or gp41) showed that one (98CM19) was a complex mosaic between subtype A in p24 and subtype J prot, and unclassifiable in env (C2V3 or gp41). The second, 98CM63, clustered distinctly from all known subtypes in p24, prot, C2V3, or gp41. 98CM63 clustered with a specimen from Cyprus and these two geographically and epidemiologically unlinked specimens, with their distinct clustering pattern, may represent a new subcluster of subtype A. In conclusion, these findings confirm the high HIV-1 genetic variability and further suggest the continuous appearance of new viral strains in this population.

Characterization of a highly replicative intergroup M/O human immunodeficiency virus type 1 recombinant isolated from a Cameroonian patient.

A Cameroonian patient with antibodies reacting simultaneously to human immunodeficiency virus type 1 (HIV-1) group O- and group M-specific V3-loop peptides was identified. In order to confirm that this patient was coinfected with both viruses, PCRs with O- and M-specific discriminating primers corresponding to different regions of the genome were carried out with both primary lymphocyte DNA and the corresponding viral strains isolated from three consecutive patient samples. The PCR data suggested that this patient is coinfected with a group M virus and a recombinant M/O virus. Indeed, only type M gag sequences could be amplified, while for the env region, both type M and O sequences were amplified, from plasma or from DNA extracted from primary lymphocytes. Sequence analysis of a complete recombinant genome isolated from the second sample (97CA-MP645 virus isolate) revealed two intergroup breakpoints, one in the vpr gene and the second in the long terminal repeat region around the TATA box. Comparison of the type M sequences shared by the group M and the recombinant M/O viruses showed that these sequences were closely related, with only 3% genetic distance, suggesting that the M virus was one of the parental viruses. In this report we describe for the first time a recombination event in vivo between viruses belonging to two different groups, leading to a replicative virus. Recombination between strains with such distant lineages (65% overall homology) may contribute substantially to the emergence of new HIV-1 variants. We documented that this virus replicates well and became predominant in vitro. At this time, group O viruses represent a minority of the strains responsible for the HIV-1 pandemic. If such recombinant intergroup viruses gained better fitness, inducing changes in their biological properties compared to the parental group O virus, the prevalences of group O sequences could increase rapidly. This will have important implications for diagnosis of HIV-1 infections by serological and molecular tests, as well as for antiviral treatment.

Efficiencies of four versions of the AMPLICOR HIV-1 MONITOR test for quantification of different subtypes of human immunodeficiency virus type 1.

Three versions of a commercial human immunodeficiency virus (HIV) type 1 (HIV-1) load test (the AMPLICOR HIV-1 MONITOR Test versions 1. 0, 1.0+, and 1.5; Roche Diagnostics, Branchburg, N.J.) were evaluated for their ability to detect and quantify HIV-1 RNA of different genetic subtypes. Plasma samples from 96 patients infected with various subtypes of HIV-1 (55 patients infected with subtype A, 9 with subtype B, 21 with subtype C, 2 with subtype D, 7 with subtype E, and 2 with subtype G) and cultured virus from 29 HIV-1 reference strains (3 of subtype A, 6 of subtype B, 5 of subtype C, 3 of subtype D, 8 of subtype E, 3 of subtype F, and 1 of subtype G) were tested. Detection of subtypes A and E was significantly improved with versions 1.0+ and 1.5 compared to that with version 1. 0, whereas detection of subtypes B, C, D, and G was equivalent with the three versions. Versions 1.0, 1.0+, and 1.5 detected 65, 98, and 100% of the subtype A-infected samples from patients, respectively, and 71, 100, and 100% of the subtype E-infected samples from patients, respectively. Version 1.5 yielded a significant increase in viral load for samples infected with subtypes A and E (greater than 1 log10 HIV RNA copies/ml). For samples infected with subtype B, C, and D and tested with version 1.5, only a slight increase in viral load was observed (<0.5 log10). We also evaluated a prototype automated version of the test that uses the same PCR primers as version 1.5. The results with the prototype automated test were highly correlated with those of the version 1.5 test for all subtypes, but were lower overall. The AMPLICOR HIV-1 MONITOR Test, version 1.5, yielded accurate measurement of the HIV load for all HIV-1 subtypes tested, which should allow the test to be used to assess disease prognosis and response to antiretroviral treatment in patients infected with a group M HIV-1 subtype.

Most env and gag subtype A HIV-1 viruses circulating in West and West Central Africa are similar to the prototype AG recombinant virus IBNG.

The genetic subtype was identified in gag and env of 219 HIV-1-positive samples collected in different African countries, 44 from Senegal, 55 from Cameroon, 82 from Gabon, and 38 from Djibouti. In total, 20 (9.1%) samples had discordant subtypes between gag and env, 6 of 44 (13.9%) in Senegal, 4 of 55 (7.2%) in Cameroon, 1 of 38 (2.6%) in Djibouti, and 10 of 82 (12.1%) in Gabon. Subtypes A and G were predominantly involved in the recombination events. Phylogenetic tree analysis of gag showed that an important number of the A sequences form a distinct subcluster with the AG-IBNG prototype strain (a complex A/G mosaic virus): 27 of 32 (84.3%) in Senegal, 12 of 17 (70.6%) in Nigeria, 24 of 39 (61.5%) in Cameroon, and 38 of 70 (54.3%) in Gabon. Full-length genome analysis of 3 and additional sequences in pol for 10 such strains confirmed that they have a similar complex A/G mosaic genomic structure. These data suggest that in West Africa, most probably between 60% and 84% of the subtype A viruses are recombinant AG-IBNG viruses. This finding has potential implications on future vaccine, diagnostic, and treatment strategies. The actual and future role of these viruses in the global pandemic must be monitored in all new molecular epidemiologic studies, a discrimination between subtype A and AG-IBNG-like viruses is necessary.