Characterization of novel simian immunodeficiency viruses from red-capped mangabeys from Nigeria (SIVrcmNG409 and -NG411).

Two novel simian immunodeficiency virus (SIV) strains from wild-caught red-capped mangabeys (Cercocebus torquatus torquatus) from Nigeria were characterized. Sequence analysis of the fully sequenced SIV strain rcmNG411 (SIVrcmNG411) and gag and pol sequence of SIVrcmNG409 revealed that they were genetically most closely related to the recently characterized SIVrcm from Gabon (SIVrcmGB1). Thus, red-capped mangabeys from distant geographic locations harbor a common lineage of SIV. SIVrcmNG411 carried a vpx gene in addition to vpr, suggesting a common evolutionary ancestor with SIVsm (from sooty mangabeys). However, SIVrcm was only marginally closer to SIVsm in that region than to any of the other lentiviruses. SIVrcm showed the highest similarity in pol with SIVdrl, isolated from a drill, a primate that is phylogenetically distinct from mangabey monkeys, and clustered with other primate lentiviruses (primarily SIVcpz [from chimpanzees] and SIVagmSab [from African green monkeys]) discordantly in different regions of the genome, suggesting a history of recombination. Despite the genetic relationship to SIVcpz in the pol gene, SIVrcmNG411 did not replicate in chimpanzee peripheral blood mononuclear cells (PBMC), although two other viruses unrelated to SIVcpz, SIVmndGB1 (from mandrills) and SIVlhoest (from L'Hoest monkeys), were able to grow in chimpanzee PBMC. The CCR5 24-bp deletion previously described in red-capped mangabeys from Gabon was also observed in Nigerian red-capped mangabeys, and SIVrcmNG411, like SIVrcmGB1, used CCR2B and STRL33 as coreceptors for virus entry. SIVrcm, SIVsm, SIVmndGB1, and all four SIVlhoest isolates but not SIVsun (from sun-tailed monkeys) replicated efficiently in human PBMC, suggesting that the ability to infect the human host can vary within one lineage.

Sequence features downstream of the primer-binding site of HIV type 1 subtype E shared by subtype G and a subset of subtype A.

Two distinct sequence features downstream of the primer-binding site (PBS) were identified in a full-length HIV-1 subtype E clone amplified in this study. Both features are frequently found in HIV-1 subtypes A and G and in more than half of the full-length intersubtype recombinant clones. One of these is the absence of a trinucleotide sequence, which is located 14 nucleotides downstream of the PBS and found only in subtypes B, C, D, F, and H. The other is an insertion of 24 nucleotides immediately downstream of the PBS, which was previously reported as a sequence feature shared by subtypes A, E, and G. The analysis conducted here revealed that this 24-nucleotide insertion contained two sequence motifs duplicated in adjacent regions and was not found in all HIV-1 subtype A clones. Furthermore, our finding suggests that the PBS region of all known full-length subtype E clones, which are A/E intersubtype recombinants, is derived from the group of HIV-1 subtype A, which contains a similar insertion.

HIV-1 subtype C syncytium- and non-syncytium-inducing phenotypes and coreceptor usage among Ethiopian patients with AIDS.

OBJECTIVE: To assess syncytium-inducing (SI) and non-syncytium-inducing (NSI) frequencies, coreceptor usage and gp120 V3 sequences of HIV-1 isolates from Ethiopian AIDS patients. PATIENTS: Cross-sectional study on 48 hospitalized AIDS patients (CD4 T cells < 200 x 10(6) cell/l) with stage III or IV of the WHO staging system for HIV-1 infection and disease. METHODS: Peripheral blood mononuclear cells (PBMC) from all 48 patients were tested by MT-2 assay to determine SI/NSI phenotypes. Lymphocyte subsets were enumerated using Coulter counting and FACScan analysis. Viral load determination used a nucleic acid sequence-based amplification assay (NASBA). Coreceptor usage of HIV-1 biological clones was measured using U87 CD4/chemokine receptor transfectants and phytohemagglutinin-stimulated PBMC of healthy donors with wild-type CCR5 and homozygous mutation CCR5delta32 (a 32 base-pair deletion in CCR5). Reverse transcriptase polymerase chain reaction sequencing was performed on the third variable region (V3) of the HIV-1 gene gp120. Sequence alignments were done manually; phylogenetic analyses used PHYLIP software packages. RESULTS: SI viruses were detected for 3/48 (6%) AIDS patients only. Lower mean absolute CD4 counts were determined in patients with SI virus compared with NSI (P = 0.04), but no differences in viral load were observed. All patients were found to be infected with HIV-1 subtype C, based on V3 sequencing. NSI biological clones used CCR5 as coreceptor; SI biological clones used CXCR4 and/or CCR5 and/or CCR3. CONCLUSIONS: Ethiopian patients with HIV-1 C-subtype AIDS harbour a remarkably low frequency of SI phenotype viruses. Coreceptor usage of these viruses correlates with their biological phenotypes.

Genetic differences between human immunodeficiency virus type 1 subpopulations in faeces and serum.

To study human immunodeficiency virus type 1 (HIV-1) compartmentalization between intestine and blood, paired faecal and serum samples were collected from 204 HIV-1-infected persons. Direct sequencing of the gp120 V3 region obtained from 33 persons showed that faecal and serum sequences could be nearly homologous (0.3% different) or very dissimilar (11.3% different). Individual clones were obtained and sequenced from the faecal and serum samples of 13 persons. In 6 persons the HIV-1 subpopulations in faeces and serum were similar, whereas in 7 persons, distribution of V3 genotypes showed a marked difference. Genetic characterization of the HIV-1 subpopulations showed less heterogeneity in faecal subpopulations than in serum subpopulations in 5 of the 7 subjects. Furthermore, faecal and serum subpopulations differed predominantly by nonsynonymous nucleotide substitutions (in 6 of 7 persons). Comparison of the HIV-1 subpopulations in faeces and serum of these 7 persons, using resampling techniques, revealed a significant difference between faecal and serum subpopulations at an N-linked glycosylation site, C-terminal of the V3 loop (amino acids 331-333). Sequences from faecal subpopulations of all 7 persons contained a glycosylation site at amino acid position 331-333. Four of these 7 harboured serum variants lacking a glycosylation site at this position. The faecal subpopulations in these 4 persons showed limited nonsynonymous substitutions compared to synonymous substitutions, indicating that purifying selection is operational on these subpopulations.

Analysis of the temporal relationship between human immunodeficiency virus type 1 quasispecies in sequential blood samples and various organs obtained at autopsy.

We studied the temporal relationship between human immunodeficiency type 1 (HIV-1) quasispecies in tissues and in peripheral blood mononuclear cells (PBMC) of infected individuals. Sequential PBMC and tissue samples from various organs obtained at autopsy from three patients who died of AIDS-related complications were available for analysis. Biological HIV-1 clones were isolated from PBMC samples, and cellular tropism and syncytium-inducing (SI) capacity were determined. Genomic DNA was isolated from 1 cm3 of organ tissue, and proviral DNA was amplified by means of PCR and cloned with the PGEM-T vector system. A 185-bp region encompassing the third variable domain of the virus envelope, known to influence HIV-1 biological properties, was sequenced. HIV-1 could be amplified from all PBMC and organ samples, except from liver tissue for two patients. Both SI and non-syncytium-inducing (NSI) genotypes could be detected in the different tissues. Tissue-specific quasispecies were observed in brain, lung, and testis. Lymphoid tissues, such as bone marrow, lymph node, and spleen, harbored several different variants similar to those detected in blood in the last PBMC samples. In general, only tissues in which macrophages are likely to be the main target cell for HIV-1 harbored NSI HIV-1 sequences that clustered separately. Both SI and NSI sequences that clustered with sequences from late-stage PBMC were present in other tissues, which may indicate that the presence of HIV-1 in those tissues is secondary to lymphocyte infiltration rather than to tissue tropism of HIV-1 itself. These data suggest that the viral reservoir may be limited, which will have important implications for the success of HIV-1 eradication.

HIV type 1 subtype C in Addis Ababa, Ethiopia.

PIP: HIV-1 variants in different geographic regions have been phylogenetically classified into the genetic subtypes A-I and O on the basis of sequence differences in the V3 regions of their gp120 envelope genes. The existence of all HIV-1 subtypes except subtype I has been confirmed in Africa. This paper describes the distribution of HIV-1 subtypes in Ethiopia. The first Ethiopian AIDS case was reported in 1986 and the AIDS epidemic has now become a rapidly growing problem in Addis Ababa, the capital city. HIV-1 seroprevalence in the city is estimated to be 10-27% among pregnant women, 47-59% among prostitutes, and 7% among blood donors. Preliminary sequence data on a limited number of samples indicated the presence of subtype C in Addis Ababa in 1988. 94 sera collected from prostitutes, pregnant women, and blood donors during 1989-95 were analyzed to assess the distribution of HIV-1 subtypes in Addis Ababa. HIV-1 subtype C was identified in 93 of 94 cases. One case of subtype A virus was identified. Subtype C was also highly abundant also before the 1995 sera collection. Finally, the authors discuss how the Ethiopian subtype C sequences differ slightly from the consensus C sequence.^ieng

Evidence for HIV type 1 strains of U.S. intravenous drug users as founders of AIDS epidemic among intravenous drug users in northern Europe.

To establish an epidemiological link between HIV-1 epidemics in U.S. and European homosexual men and intravenous drug users (IVDUs) we analyzed the HIV-1 gp120 V3 sequences in both risk groups. Signature pattern analysis revealed that the V3 sequences of viruses from IVDUs in Northern Europe are distinguishable from those of homosexual men on the basis of one amino acid and two synonymous nucleotide substitutions, which the most conserved was a synonymous nucleotide substitution in the second glycine codon at the tip of the gp120 V3 loop (GGC). This substitution was seen in 17 of 20 (85%) viruses of IVDUs in Northern Europe, in none of 41 homosexual men in either Europe or the United States, and in 5 of 11 (45%) U.S. IVDUs sequences analyzed. Subsequent phylogenetic and multivariate principal coordinate (PCOORD) analyses showed that 16 of 20 (80%) of the Northern European IVDU sequences clustered together with the 5 U.S. IVDU sequences carrying the GGC substitution and away from the sequences of homosexual men from either Europe or the United States. Taken together with the higher level of heterogeneity of U.S. IVDU sequences compared to the Dutch IVDU sequences taken at the same time, these data present suggestive evidence for a U.S. instead of a European origin of the AIDS epidemic among Northern European IVDUs.

Consistent risk group-associated differences in human immunodeficiency virus type 1 vpr, vpu and V3 sequences despite independent evolution.

Human immunodeficiency virus 1 type vpr, vpu and V3 sequences from 15 homosexual men and 19 intravenous drug users in the Amsterdam Cohort studies were analysed. Previously, we reported that V3 domains of viruses from drug users are distinguishable from those of homosexual men on the basis of two silent mutations. Phylogenetic analysis of vpr, vpu and V3 shows that differences in all three regions correlate with risk group. Two positions in both vpr and vpu were found to differ significantly between the risk groups. The distinguishing positions were confirmed for sequences from 11 Scottish and four German samples. The three regions show relatively independent evolution patterns; they resulted in different phylogenies, the only stable clustering being that based on the risk group distinction. Pairwise differences between sequences of the genes were moderately correlated (around 0.30). Surprisingly, when only silent changes are counted, the correlations dropped almost to zero, indicating that the evolution towards independence was more advanced in the silent than in the non-silent positions. This suggests that selection at the amino acid level is not the primary driving force for the independent evolutionary behaviour of the genes. Recombination, combined with restrictions on certain amino acids because of epistatic interactions between the genes, could be an alternative explanation of this phenomenon.

Evidence for limited within-person evolution of the V3 domain of the HIV-1 envelope in the amsterdam population.

OBJECTIVE: To study the development of the V3 region of the HIV-1 envelope over time, both within subjects and population-wide. METHODS: Direct V3 sequences were obtained from viral RNA from seroconversion samples of 138 individuals [32 intravenous drug users (IVDU), 106 homosexual men], as well as from 5-year follow-up samples of 45 of these individuals (11 IVDU, 34 homosexual men). RESULTS: The population-wide variation of the V3 region in both the seroconversion samples and the 5-year samples steadily increased over consecutive years and were of similar magnitude in each calendar year. The variation in the IVDU group was slightly lower (presumably lagging behind) than in the homosexual group, but also increased over time. The consensus sequence, representing the centre of the swarm of variants, remained almost stationary in 10 years of evolution. The V3 sequences from virions in serum collected 5 years after seroconversion still resembled those from the seroconversion sample, either in overall similarity or in specific (signature) amino acids. Seroconversion and late sequences from a donor-recipient pair were also very similar. CONCLUSIONS: The variation in V3 sequences from seroconversion samples is as large as that in 5-year follow-up samples from the same calendar year, suggesting that there is no strong selection for a particular V3 genotype at transmission. The HIV-1 subtype B quasispecies in a naive population appears to evolve through unbiased expansion around a stationary consensus sequence. Despite its large variability, the V3 region retains many of its individual characteristics after 5 years of infection. This indicates that the sampling moment (relative to the seroconversion data) will not greatly influence the results of phylogenetic analyses.

Intrahost human immunodeficiency virus type 1 evolution is related to length of the immunocompetent period.

The antigenic diversity threshold theory predicts that antigenic sites of human immunodeficiency virus type 1, such as the V3 region of the external glycoprotein gp120, evolve more rapidly during the symptom-free period in individuals progressing to AIDS than in those who remain asymptomatic for a long time. To test this hypothesis, genomic RNA sequences were obtained from the sera of 44 individuals at seroconversion and 5 years later. The mean number of nonsynonymous nucleotide substitutions in the V3 region of the viruses circulating in 31 nonprogressors (1.1 x 10(-2) +/- 0.1 x 10(-2) per site per year) was higher than the corresponding value for 13 progressors (0.66 x 10(-2) +/- 0.1 x 10(-2) per site per year) (P < 0.01), while no difference between the mean numbers of synonymous substitutions in the two groups was seen (0.37 x 10(-2) +/- 0.1 x 10(-2) and 0.51 x 10(-2) +/- 0.2 x 10(-2) per site per year for nonprogressors and progressors, respectively; P > 0.1). The mean ratios of synonymous nucleotide p distance to nonsynonymous p distance were 0.35 for nonprogressors and 0.62 for progressors. The number of nonsynonymous substitutions was not associated with virus load or virus phenotype, which are established predictors of disease progression, but correlated strongly with the duration of the immunocompetent period (r2 = 0.41; P = 0.001). This indicates that there is no causative relationship between intrahost evolution and CD4+ cell decline. Our data suggest that intrahost evolution in human immunodeficiency virus type 1 infection is driven by selective forces, the strength of which is related to the duration of the immunocompetent period.

Nuclear counterparts of the cytoplasmic mitochondrial 12S rRNA gene: a problem of ancient DNA and molecular phylogenies.

Monkey mummy bones and teeth originating from the North Saqqara Baboon Galleries (Egypt), soft tissue from a mummified baboon in a museum collection, and nineteenth/twentieth-century skin fragments from mangabeys were used for DNA extraction and PCR amplification of part of the mitochondrial 12S rRNA gene. Sequences aligning with the 12S rRNA gene were recovered but were only distantly related to contemporary monkey mitochondrial 12S rRNA sequences. However, many of these sequences were identical or closely related to human nuclear DNA sequences resembling mitochondrial 12S rRNA (isolated from a cell line depleted in mitochondria) and therefore have to be considered contamination. Subsequently in a separate study we were able to recover genuine mitochondrial 12S rRNA sequences from many extant species of nonhuman Old World primates and sequences closely resembling the human nuclear integrations. Analysis of all sequences by the neighbor-joining (NJ) method indicated that mitochondrial DNA sequences and their nuclear counterparts can be divided into two distinct clusters. One cluster contained all temporary cytoplasmic mitochondrial DNA sequences and approximately half of the monkey nuclear mitochondriallike sequences. A second cluster contained most human nuclear sequences and the other half of monkey nuclear sequences with a separate branch leading to human and gorilla mitochondrial and nuclear sequences. Sequences recovered from ancient materials were equally divided between the two clusters. These results constitute a warning for when working with ancient DNA or performing phylogenetic analysis using mitochondrial DNA as a target sequence: Nuclear counterparts of mitochondrial genes may lead to faulty interpretation of results.

Similarity in env and gag genes between genomic RNAs of human immunodeficiency virus type 1 (HIV-1) from mother and infant is unrelated to time of HIV-1 RNA positivity in the child.

Variation in the env (V3 region) and gag (p17 region) genes of genomic RNA of human immunodeficiency virus type 1 was studied in three mother-child pairs. One infant was human immunodeficiency virus type 1 RNA positive at birth (pair 114), one became positive 6 weeks after birth (pair 127), and one became positive 30 months after birth (pair 564). The first two children were born to seropositive mothers, and the last child was infected by breast-feeding following seroconversion of the mother after delivery. In both V3 and p17gag, intrasample variability was much higher in the maternal samples, including the first seropositive sample of the seroconverted mother, than in the infants' samples. Variability was less in p17gag than in V3, except in the postnatally infected child. In all three cases, infection of the child was established by variants representing a minority of the cell-free virus population in the maternal samples. For the two infants born to seropositive mothers, V3 sequences were more similar to the sequence populations of maternal samples collected during pregnancy than to those of samples collected at delivery or thereafter. However, in pair 114 a V3 variant identical to the child's virus was also detected in the sample collected at delivery. In contrast to the V3 region, p17gag sequences of maternal samples of the first trimester of pregnancy and at delivery had comparable resemblance to the child's sequences in pair 114, while in pair 127, similarity to sequences of the sample collected at delivery was higher than that to sequences of the sample from early in pregnancy. In the last pair, V3 and p17gag sequences from a maternal sample collected 18 months prior to the first RNA-positive sample of the child resembled the infant's sequences as much as the sample collected close to the presumed time of infection. Taken together, the evolutionary characteristics for genomic RNA env and gag genes did not point to a particular time of mother-to-child transmission.

Phylogeny of African monkeys based upon mitochondrial 12S rRNA sequences.

The suborder Anthropoidea of the primates has traditionally been divided in three superfamilies: the Hominoidea (apes and humans) and the Cercopithecoidea (Old World monkeys), together comprising the infraorder Catarrhini, and the Ceboidea (New World monkeys) belonging to the infraorder Platyrrhini. We have sequenced an approximately 390-base-pair part of the mitochondrial 12S rRNA gene for 26 species of the major groups of African monkeys and apes and constructed an extensive phylogeny based upon DNA evidence. Not only is this phylogeny of great importance in classification of African guenons, but it also suggests rearrangements in traditional monkey taxonomy and evolution. Baboons and mandrills were found to be not directly related, while we could confirm that the known four superspecies of mangabeys do not form a monophyletic group, but should be separated into two genera, one clustering with baboons and the other with mandrills. Patas monkeys are clearly related to members of the genus Cercopithecus despite their divergence in build and habitat, while the talapoin falls outside the Cercopithecus clade (including the patas monkey).

Differences in human immunodeficiency virus type 1 V3 sequences from patients with and without AIDS dementia complex.

Paired serum and cerebrospinal fluid (CSF) samples from 10 AIDS patients with and 10 without AIDS dementia complex (ADC) were studied, in an attempt to uncover ADC-associated variation in V3 sequences. Sequences were obtained from four of the patients with and eight of those without ADC. Comparison of the sequences using a resampling technique revealed a significant ADC-associated difference occurring at several amino acid positions. Results from serum and CSF sequences were comparable. These differences may indicate that the virus found in ADC and that in non-ADC patients have different biological properties. Comparison of serum versus CSF sequences within samples from both ADC and non-ADC patients, using the same resampling technique, revealed no clear distinctions. In some patients, the sequence populations in serum and CSF were completely distinct, while in others, there was no difference in distribution. These patterns were not associated with ADC.

Conserved V3 loop sequences and transmission of human immunodeficiency virus type 1.

The third variable region (V3) of the surface glycoprotein (gp120) of envelope sequence subtype B, type 1 human immunodeficiency virus (HIV-1B), is highly variable among T cell line-adapted viruses and syncytium-inducing HIV-1-B isolates. Here we analyze the V3 region sequences from 93 individuals close to the time of seroconversion and show that the cysteine-bridged V3 loop, which also encompasses a major neutralizing determinant, is highly conserved, whereas sequences immediately surrounding the loop are similarly divergent in all HIV-1-B strains. Viruses with this conserved V3 loop have been reported to be more resistant to antibody-mediated neutralization than T cell-adapted viruses with divergent V3 sequences. We hypothesize, therefore, that on transmission from a donor to a recipient, virions inherently more resistant to neutralization by donor antibodies have a greater chance of initiating infection than those more sensitive to neutralization. This might explain the conservation of V3 early in infection and has implications for the design of HIV vaccines.

Identification of a unique group of human papillomavirus type 16 sequence variants among clinical isolates from Barbados.

The naturally occurring sequence variation of human papillomavirus type 16 (HPV-16) was analysed by direct sequence analysis of the PCR products of the long control region (LCR), the E5 and E7 open reading frames (ORFs), a segment of the L2 ORF overlapping the early viral poly(A) signal and a small segment of the L1 ORF or clinical isolates from Barbados and The Netherlands. Despite the widely different geographical and ethnic origin of the two groups of specimens, sequence analysis revealed relatively few mutational differences. Analysis of the LCR and the E5 ORF appeared to be the minimum requirement for the correct positioning of these variants in the HPV-16 phylogenetic tree. Most of the Barbadian variants appeared to be located at a unique position in the HPV-16 phylogenetic tree, at the internal branch close to the point where the European and Asian branches diverge. In contrast, most of the Dutch samples were located on the European branch.

Antibody responses to HIV-1 envelope and gag epitopes in HIV-1 seroconverters with rapid versus slow disease progression.

We studied the relationship between the rate of disease progression after HIV-1 seroconversion and the level of IgG antibody response to HIV-1 envelope and core epitopes. This was done by comparing a group of fast-progressing individuals and a group of slow-progressing individuals for serum IgG titers to peptides from the gp120-V3 neutralization domain, to a peptide from the immunodominant gp41 epitope (residues 590 to 607), and to recombinant gp120 and p24. The two groups displayed a large overlap in titers to the envelope epitopes, which precluded their differentiation at most time points after seroconversion. Low responsiveness to envelope antigens was not only found in a few fast-progressors but also in one individual who remained asymptomatic for at least 92 months after seroconversion. The only significant differences between the groups were found in the first months after seroconversion when the responses to the V3 domain and the gp41 epitope were more vigorous in the group of fast-progressors. Furthermore, on evaluating ratios of anti-V3 antibody titers to anti-gp120 antibody titers we found no indication that fast disease progression was associated with a restriction in antibody response to the V3 epitope. We did confirm the finding that fast disease progression is associated with low levels of p24-directed antibodies, both early after seroconversion and at later stages. These data demonstrate that levels of IgG antibodies to envelope epitopes are poor predictors of rapid disease progression and suggest that the role of V3-directed neutralizing antibodies in preventing subversion of the immune system is not decisive in natural HIV-1 infection.