Structural and functional state of the bone marrow during its in vitro interaction with ferromagnetic nanoparticles.

Hemopoietic islets, associations of stromal (macrophages, fibroblasts) and blood (including stem) cells, are structural and functional units of the bone marrow. We studied cellular and molecular processes developing following short-term (1 h) contact of hemopoietic islets with ferromagnetic nanoparticles in a multicellular system of the bone marrow in vitro. It was established that nanodispersions of magnetite (Fe(3)O(4), mean particle diameter 18 nm) and iron coated with carbon (Fe(C), particle diameter 5-10 nm) in a dose of 3 mg/liter had a minor effect on processes of necrotic and apoptotic cell death. Nanodispersion of carbon-coated iron (Fe(C)) most mildly stimulated oxidizing processes recorded by intracellular levels of reactive oxygen species. These nanoparticles, in contrast to magnetite, did not reduce the amount of hemopoietic islets in the bone marrow cell suspension.

Infectious simian/human immunodeficiency virus with human immunodeficiency virus type 1 subtype C from an African isolate: rhesus macaque model.

Human immunodeficiency virus type 1 (HIV-1) subtype C is responsible for more than 56% of all infections in the HIV and AIDS pandemic. It is the predominant subtype in the rapidly expanding epidemic in southern Africa. To develop a relevant model that would facilitate studies of transmission, pathogenesis, and vaccine development for this subtype, we generated SHIV(MJ4), a simian/human immunodeficiency virus (SHIV) chimera based on HIV-1 subtype C. SHIV(MJ4) contains the majority of env, the entire second exon of tat, and a partial sequence of the second exon of rev, all derived from a CCR5-tropic, primary isolate envelope clone from southern Africa. SHIV(MJ4) replicated efficiently in human, rhesus, and pig-tailed macaque peripheral blood mononuclear cells (PBMCs) in vitro but not in CEMx174 cells. To assess in vivo infectivity, SHIV(MJ4) was intravenously inoculated into four rhesus macaques (Macaca mulatta). All four animals became infected as determined through virus isolation, PCR analysis, and viral loads of 10(7) to 10(8) copies of viral RNA per ml of plasma during the primary infection phase. We have established a CCR5-tropic SHIV(MJ4)/rhesus macaque model that may be useful in the studies of HIV-1 subtype C immunology and biology and may also facilitate the evaluation of vaccines to control the spread of HIV-1 subtype C in southern Africa and elsewhere.

An efficient test for comparing sequence diversity between two populations.

We address the problem of comparing interindividual genomic sequence diversity between two populations. Although the methods are general, for concreteness we focus on comparing two human immunodeficiency virus (HIV) infected populations. From a viral isolate(s) taken from each individual in a sample of persons from each population, suppose one or multiple measurements are made on the genetic sequence of a coding region of HIV. Given a definition of genetic distance between sequences, the goal is to test if the distribution of interindividual distances differs between populations. If distances between all pairs of sequences within each group are used, then data-dependencies arising from the use of multiple sequences from individuals invalidates the use of a standard two-sample test such as the t-test. Where this problem has been recognized, a typical solution has been to apply a standard test to a reduced dataset comprised of one sequence or a consensus sequence from each patient. Disadvantages of this procedure are that the conclusion of the test depends on the choice of utilized sequences, often an arbitrary decision, and exclusion of replicate sequences from the analysis may needlessly sacrifice statistical power. We present a new test free of these drawbacks, which is based on a statistic that linearly combines all possible standard test statistics calculated from independent sequence subsamples. We describe statistical power advantages of the test and illustrate its use by application to nucleotide sequence distances measured from HIV-1 infected populations in southern Africa (GenBank accession numbers AF110959--AF110981) and North America/Europe. The test makes minimal assumptions, is maximally efficient and objective, and is broadly applicable.

The molecular epidemiology of HIV type 1 of men in Mexico.

Genotypic characteristics of human immunodeficiency virus type 1 (HIV-1) in Mexico were investigated in a multicenter study that involved centers in five geographic regions of the country. Study samples (n = 65) collected from male patients in 1998-1999 were sequenced within the C2-V5 region of the gp120 env gene. Phylogenetic analysis revealed that subtype B predominates in Mexico. The level of interpatient nucleotide diversity (mean value of 8.9%) was congruent with multiple introductions of the virus and the "aging" epidemic in Mexico. One-third of samples (30.8% of cases) showed polymorphism within the crown of the V3 loop demonstrating non-GPGR motifs. Two new motifs in the V3 loop crown - HPGG and GPEG - were observed. The evolution of the AIDS epidemic in Mexico should be closely monitored since non-B HIV-1 subtypes might be introduced. The nucleotide sequences were deposited in the GenBank under accession numbers AF200855-AF200869, AF200871-AF200892, and AF200894-AF200921.

Molecular cloning and biological characterization of full-length HIV-1 subtype C from Botswana.

Human immunodeficiency virus type 1 (HIV-1) subtype C is now responsible for more than half of all HIV-1 infections in the global epidemic and for the high levels of HIV-1 prevalence in southern Africa. To facilitate studies of the biological nature and the underlying molecular determinants of this virus, we constructed eight full-length proviral clones from two asymptomatic and three AIDS patients infected with HIV-1 subtype C from Botswana. Analysis of viral lysates showed that Gag, Pol, and Env structural proteins were present in the virions. In four clones, the analysis suggested inefficient envelope glycoprotein processing. Nucleotide sequence analysis of the eight clones did not reveal frameshifts, deletions, premature truncations, or translational stop codons in any structural, regulatory, or accessory genes. None of the subtype C clones were replication competent in donor peripheral blood mononuclear cells (PBMCs), macrophages, Jurkat(tat) cells, or U87. CD4.CCR5 cells. However, infection by two clones could be rescued by complementation with a functional subtype C envelope clone, resulting in a productive infection of PBMCs, macrophages, and U87. CD4.CCR5 cells.

HIV type 1 A/J recombinant with a pronounced pol gene mosaicism.

A nearly full-length genome sequence of a novel HIV-1 A/J recombinant with a complex structure of the pol gene has been analyzed. This virus was isolated in 1998 from a 35-year-old female from Botswana. The virus demonstrated a dual pattern for CXCR4/CCR5 coreceptor utilization. Using short-term enrichment of the donor's PBMCs, the 98BW21 isolate was long-range amplified, cloned, and sequenced. The sequence of the clone 98BW21.17 spanned 9103 bp from the PBS site to the U5 region of the 3' LTR. The phylogenetic relationship of the 98BW21.17 clone to HIV-1 sequences represented by M, N, and O groups and A-K subtypes of the M group was examined across the entire viral genome. The 98BW21.17 clone demonstrated a unique phylogenetic topology clustering within subtype A or subtype J reference sequences. However, the subtype origin of two regions within the pol gene (p51 RT and integrase) could not be identified. Recombination patterns of the 98BW21.17 clone were different from known AGJ/AGIJ-type viruses such as isolates BFP90 and 95ML84. This study demonstrated the existence and replication competence of a new dual-tropic X4/R5 recombinant form of HIV-1 on the subtype J backbone. The nucleotide sequence of the 98BW21.17 clone was submitted to GenBank under accession number AF192135.

Elevated tumor necrosis factor-alpha activation of human immunodeficiency virus type 1 subtype C in Southern Africa is associated with an NF-kappaB enhancer gain-of-function.

The human immunodeficiency virus type 1 (HIV-1) epidemic within southern Africa is predominantly associated with the HIV-1C subtype. Functional analysis of the enhancer region within the long terminal repeat (LTR) indicates that HIV-1C isolates have >/=3 NF-kappaB binding sites, unlike other subtypes, which have only 1 or 2 sites. A correlation was shown between NF-kappaB enhancer configuration and responsiveness to the proinflammatory cytokine tumor necrosis factor (TNF)-alpha within the context of naturally occurring subtype LTRs, subtype-specific NF-kappaB enhancer regions cloned upstream of an isogenic HXB2 core promoter or a heterologous SV40 minimal promoter, and full-genome subtype clones. In all cases, TNF-alpha activation was correlated with the subtype configuration of the NF-kappaB enhancer. Whether the naturally occurring gain-of-function in the NF-kappaB enhancer of HIV-1C observed in this study can provide a selective advantage for the virus in vivo remains to be determined and warrants further study.

Molecular cloning and phylogenetic analysis of human immunodeficiency virus type 1 subtype C: a set of 23 full-length clones from Botswana.

To better understand the virological aspect of the expanding AIDS epidemic in southern Africa, a set of 23 near-full-length clones of human immunodeficiency virus type 1 (HIV-1) representing eight AIDS patients from Botswana were sequenced and analyzed phylogenetically. All study viruses from Botswana belonged to HIV-1 subtype C. The interpatient diversity of the clones from Botswana was higher than among full-length isolates of subtype B or among a set of full-length HIV-1 genomes of subtype C from India (mean value of 9. 1% versus 6.5 and 4.3%, respectively; P < 0.0001 for both comparisons). Similar results were observed in all genes across the entire viral genome. We suggest that the high level of HIV-1 diversity might be a typical feature of the subtype C epidemic in southern Africa. The reason or reasons for this diversity are unclear, but may include an altered replication efficiency of HIV-1 subtype C and/or the multiple introduction of different subtype C viruses.

Molecular epidemiology of an HIV-1 subtype A subcluster among injection drug users in the Southern Ukraine.

Phylogenetic characterization of primary isolates from 17 HIV-1-infected individuals within a recent epidemic in the city of Odessa, Ukraine was conducted. The isolates were drawn from two time periods, 1993 and 1996. The 1996 isolates coincided with the first apparent expansion of HIV-1 among injection drug users (IDU). Multi-locus phylogenetic analysis indicated that HIV-1 gag, env, tat, and long terminal repeat (LTR) sequences all conformed to the HIV-1 classification of a subcluster within subtype A. There was no evidence for intersubtype recombinants among these isolates. A number of potential signature sequences, particularly within env, were identified in these two time periods, possibly suggesting a selective pressure on viral evolution among IDU. Results of this study are consistent with a recent introduction and subsequent independent evolution of an HIV-1 subtype A subcluster among IDU in the Southern Ukraine. This study demonstrates a congruence of multi-locus phylogenetic analysis, and suggests that non-B genetic subtypes, such as HIV-1 subtype A, may become relevant to the study of IDU transmission in the future.

Divergent transcriptional regulation among expanding human immunodeficiency virus type 1 subtypes.

The current AIDS pandemic represents the uneven spread of multiple genetically related subtypes (A to J) of human immunodeficiency virus type 1 (HIV-1). Notably, HIV-1 E in southeast Asia and HIV-1 C in sub-Saharan Africa are expanding faster and are likely of greater global significance than the HIV-1 B subtype prevalent in the United States and Europe. While many studies have focused on genetic variation among structural genes, we chose to conduct a comparative analysis of the long terminal repeats of HIV-1 E and HIV-1 C isolates and report subtype-specific differences in enhancer copy numbers and sequences, as well as divergent activation in response to the cellular transcriptional activators Rel-p65 and NFATc and viral Tat. This study is the first to identify functional distinctions in promoter architecture between HIV-1 subtypes and raises the possibility that regulatory divergence among the subtypes of HIV-1 has occurred. Divergent transcriptional regulation may explain some of the epidemiologically observed differences in transmission and pathogenesis and underscores the need for further comparative analysis of HIV-1 regulation.