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T Leitner

Publications and source records attributed to T Leitner.

At least 19 recordsLinked to original sources
PubMed2005

The X4 phenotype of HIV type 1 evolves from R5 in two children of mothers, carrying X4, and is not linked to transmission.

Previously, we found that emergence of the X4 viral phenotype in HIV-1-infected children was related to the presence of X4 in their mothers (C.H. Casper et al., J Infect Dis 2002; 186:914-921). Here, we investigated the origin of the X4 phenotype in the child, analyzing two mother-child pairs (Ma-Ca, Mb-Cb) where the mothers carried X4 and their children developed X4 after an initial presence of R5. We used nested polymerase chain reaction of the env V3 region to generate 203 HIV-1 clones for sequencing (Ma, n = 44; Ca, n = 73; Mb, n = 61; Cb, n = 25) from DNA of peripheral blood mononuclear cell (PBMC) lysates, altogether 167 clones, or from cDNA of plasma RNA, 36 clones. PBMC and plasma isolate sequences from each time point enabled us to assign the probable phenotype to clone sequences in a phylogenetic tree. The transmission and evolution were reconstructed using the maximum likelihood method. In mother-child pair Ma-Ca, one maternal R5 isolate clustered with the child's R5 sequences, at the earliest time when R5 was isolated in the child, confirming this as a likely source of the transmitted R5 phenotype. At age 3, an X4 population was present in the child that had evolved from the child's own R5-associated population, clearly distinct from the maternal X4 sequences. The second mother-child pair (Mb-Cb) displayed a similar pattern. Amino acid substitution patterns corroborated the conclusions from the phylogenetic tree. Thus, in both children, the X4 virus developed from their own R5 population, and was not caused by transmission of X4.

Amino Acid Sequence↗
PubMed2002

Coreceptor change appears after immune deficiency is established in children infected with different HIV-1 subtypes.

Change of HIV-1 coreceptor use has been connected to progression of disease in children infected with HIV-1, presumably subtype B. It has not been possible to discern whether the appearance of new viral phenotypes precedes disease development or comes as a consequence of it. We studied the evolution of coreceptor use in HIV-1 isolates from 24 vertically infected children. Their clinical, virological, and immunological status was recorded and the env V3 subtype was determined by DNA sequencing. Coreceptor use was tested on human cell lines, expressing CD4 together with CCR5, CXCR4, and other chemokine receptors. The children carried five different env subtypes (nine A, five B, four C, three D, and one G) and one circulating recombinant form, CRF01_AE (n = 2). Of the 143 isolates, 86 originated from peripheral blood mononuclear cells (PBMCs) and 57 originated from plasma, received at 90 time points. In 52 of 54 paired plasma and PBMC isolates the coreceptor use was concordant. All 74 isolates obtained at 41 time points during the first year of life used CCR5. A change from use of CCR5 to use of CXCR4 occurred in four children infected with subtype A, D, or CRF01_AE after they had reached 1.5 to 5.8 years of age. There was a significant association with decreased CD4+ cell levels and severity of disease but, interestingly, the coreceptor change appeared months or even years after the beginning of the immunological deterioration. Thus CXCR4-using virus may emerge as a possible consequence of immune deficiency. The results provide new insights into AIDS development in children.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Monitoring resistance to human immunodeficiency virus type 1 protease inhibitors by pyrosequencing.

The emergence of drug-resistant viral variants is the inevitable consequence of incomplete suppression of human immunodeficiency virus type 1 (HIV-1) replication during treatment with antiretroviral drugs. Sequencing to determine the resistance profiles of these variants has become increasingly important in the clinical management of HIV-1 patients, both in the initial design of a therapeutic plan and in selecting a salvage regimen. Here we have developed a pyrosequencing assay for the rapid characterization of resistance to HIV-1 protease inhibitors (PIs). Twelve pyrosequencing primers were designed and were evaluated on the MN strain and on viral DNA from peripheral blood mononuclear cells from eight untreated HIV-1-infected individuals. The method had a limit of detection of 20 to 25% for minor sequence variants. Pattern recognition (i.e., comparing actual sequence data with expected wild-type and mutant sequence patterns) simplified the identification of minor sequence variants. This real-time pyrosequencing method was applied in a longitudinal study monitoring the development of PI resistance in plasma samples obtained from four patients over a 2 1/2-year period. Pyrosequencing identified eight primary PI resistance mutations as well as several secondary mutations. This sequencing approach allows parallel analysis of 96 reactions in 1 h, facilitating the monitoring of drug resistance in eight patients simultaneously and, in combination with viral load analysis, should be a useful tool in the future to monitor HIV-1 during therapy.

Acquired Immunodeficiency Syndrome↗
PubMed1999

The molecular clock of HIV-1 unveiled through analysis of a known transmission history.

Detailed knowledge about the rate and mode of the genetic variation is vital for understanding how HIV-1 induces disease and develops resistance as well as for studies on the molecular epidemiology and origin of the virus. To unveil the molecular clock of HIV-1 we analyzed a unique set of viruses from a known transmission history with separation times between samples of up to 25 years. The env V3 and p17gag regions of the genome were sequenced, and genetic distances were estimated by using the true tree and a nucleotide substitution model based on a general reversible Markov process with a gamma distribution to account for differences in substitution rates among sites. Linear regression analysis showed that separation times were significantly correlated with synonymous as well as nonsynonymous nucleotide distances in both V3 and p17, giving strong support for the existence of a molecular clock. The estimated rate of nucleotide substitution was 6.7 +/- 2.1 x 10(-3) substitutions/site per year in V3 and 2.7 +/- 0.5 x 10(-3) in p17. Importantly, the regression analyses showed that there was a significant genetic distance at zero divergence times. This pretransmission interval exists because the ramifications in the phylogenetic trees do not correspond to time of transmission, but rather to the coalescence time of the most recent common ancestor of the viruses carried by the transmitter and the recipient. Simulation experiments showed that neither the V3 nor the p17 clocks were overdispersed, which indicates that the introduction of nucleotide substitutions can be described adequately by a simple stochastic Poisson process.

Disease Transmission, Infectious↗
PubMed1998

Stochastic processes strongly influence HIV-1 evolution during suboptimal protease-inhibitor therapy.

It has long been assumed that HIV-1 evolution is best described by deterministic evolutionary models because of the large population size. Recently, however, it was suggested that the effective population size (Ne) may be rather small, thereby allowing chance to influence evolution, a situation best described by a stochastic evolutionary model. To gain experimental evidence supporting one of the evolutionary models, we investigated whether the development of resistance to the protease inhibitor ritonavir affected the evolution of the env gene. Sequential serum samples from five patients treated with ritonavir were used for analysis of the protease gene and the V3 domain of the env gene. Multiple reverse transcription-PCR products were cloned, sequenced, and used to construct phylogenetic trees and to calculate the genetic variation and Ne. Genotypic resistance to ritonavir developed in all five patients, but each patient displayed a unique combination of mutations, indicating a stochastic element in the development of ritonavir resistance. Furthermore, development of resistance induced clear bottleneck effects in the env gene. The mean intrasample genetic variation, which ranged from 1.2% to 5.7% before treatment, decreased significantly (P < 0.025) during treatment. In agreement with these findings, Ne was estimated to be very small (500-15,000) compared with the total HIV-1 RNA copy number. This study combines three independent observations, strong population bottlenecking, small Ne, and selection of different combinations of protease-resistance mutations, all of which indicate that HIV-1 evolution is best described by a stochastic evolutionary model.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Correlation between HIV sequence evolution, specific immune response and clinical outcome in vertically infected infants.

OBJECTIVE: To evaluate sequence evolution in relation to different rates of disease progression in infants infected with HIV-1. DESIGN: Variability in the gp120 V3 region was analysed in HIV-1-infected children with different clinical courses, slow progression (n = 2) versus progressive disease (n = 3). METHODS: Cloning and sequencing of virus-derived DNA from uncultured peripheral blood mononuclear cells was performed at two to three timepoints from birth and up to the fifth year of life. Sequence variability was estimated by calculating the genetic distance and the proportion and ratio of synonymous and non-synonymous nucleotide substitutions over time. RESULTS: Genetic distances were significantly shorter in children with fast progression to disease, a predominance of synonymous nucleotide substitutions also being detected at later timepoints. Conversely, a preferential accumulation of non-synonymous nucleotide substitutions was apparent in children with slow disease progression. Furthermore, a positive correlation between a decreased ratio of synonymous/non-synonymous nucleotide substitutions and the ability of children's sera to react with synthetic peptides representing the autologous virus sequence was determined. CONCLUSION: Data suggest that an antigenically more diverse virus population emerges in infected children with slower progression to disease as a result of a stronger immune pressure.

Amino Acid Sequence↗
PubMed1997

HIV-2 genetic variation and DNA load in asymptomatic carriers and AIDS cases in Guinea-Bissau.

The purpose of this study was to document which genetic subtypes of HIV-2 are present in Guinea-Bissau and to investigate whether asymptomatic HIV-2 carriers and AIDS patients carry distinct genetic variants. A secondary aim was to correlate proviral DNA load to clinical and immunologic status of the patients. Thirty-eight asymptomatic HIV-2 carriers and 11 AIDS patients from Bissau, Guinea-Bissau were included in a cross-sectional study in which HIV-2 env V3 sequences, HIV-2 DNA load, and CD4-positive (CD4+) lymphocyte counts were determined. Phylogenetic analyses showed that all investigated subjects carried subtype A HIV-2 variants and that the sequences from AIDS patients and asymptomatic carriers did not form distinct subclusters in the tree. As expected, patients with AIDS had significantly higher median HIV-2 DNA load than did asymptomatic carriers (4.6 vs. 2.0 log10 HIV-2 DNA copies/10(6) CD4+ lymphocytes). Our study indicates that the HIV-2 epidemic in Guinea-Bissau is almost exclusively caused by subtype A HIV-2 variants and that the HIV-2 infections among the asymptomatic carriers and AIDS cases included in the study do not have distinct epidemiologic histories.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Most HIV-1 genetic subtypes have entered Sweden.

OBJECTIVE: The aim of this study was to document which genetic subtypes of HIV-1 have entered Sweden and to study transmission patterns of these virus variants. PATIENTS: All HIV-1 infected individuals at Danderyds Hospital, Stockholm, Sweden, who were suspected of carrying a virus of African origin were prospectively included in the study. The study subjects originated from 15 different African countries. METHODS: The V3 domain of the HIV-1 envelope was directly sequenced from uncultured peripheral blood mononuclear cells from 75 individuals included in the study. Phylogenetic analyses were used to determine genetic subtype and to study transmission patterns. RESULTS: The virus strains carried by the study subjects belonged to six established subtypes of HIV-1 (27A, 4B, 18C, 18D, 2G, 2H). Two individuals from Zaire carried a subtype, which had not been classified previously, provisionally named subtype 1. Eleven transmissions of non-subtype B strains in Sweden were documented. CONCLUSIONS: This study shows that most genetic HIV-1 subtypes have entered Sweden despite the relatively low prevalence of HIV infection in the country. Thus, the complete dominance of subtype-B infections which was seen during the early phase of the HIV-1 epidemic in Europe and the US has been broken in Sweden.

HIV Infections↗
PubMed1997

The role of virologic and immunologic factors in mother-to-child transmission of HIV-1.

PROBLEM: More than 90% of human immunodeficiency virus type 1 (HIV-1) infection in children is acquired by mother-to-child transmission. However, infection of the child occurs in between 14 and 35% of cases. METHOD OF STUDY: To understand the mechanisms involved in HIV-1 transmission, we have investigated the antigenic, molecular, and phenotypic characteristics of the virus harbored in infected mothers and their children. RESULTS: A clear correlation was observed between the transmission of the virus and the isolation of viral variants with a rapidly replicating and syncytium-inducing phenotype from the mother. Furthermore, non-transmitting mothers were able to neutralize several primary isolates more frequently than transmitting mothers. The comparison of the viral phenotype and genotype of mother-child pairs showed that the transmitted virus did not have common features, suggesting that transmission is usually not a selective process. CONCLUSIONS: This study suggests that transmission is governed by an interaction of both viral and immunological factors. The results obtained indicate that different strategies can be applied for the prevention of transmission.

Antigenic Variation↗
PubMed1997

Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis.

Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore, the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster. The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine. Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C. trachomatis being the more recently evolved species within the genus Chlamydia.

Animals↗
PubMed1997

Tempo and mode of nucleotide substitutions in gag and env gene fragments in human immunodeficiency virus type 1 populations with a known transmission history.

The complex evolutionary process of human immunodeficiency virus type 1 (HIV-1) is marked by a high level of genetic variation. It has been shown that the HIV-1 genome is characterized by variable and more constant regions, unequal nucleotide frequencies, and preference for G-to-A substitutions. However, this knowledge has largely been neglected in phylogenetic analyses of HIV-1 nucleotide sequences, even though these analyses are applied to a number of important biological questions. The purpose of this study was to identify a realistic model of HIV-1 evolution and to statistically test if the application of such a model significantly improves the accuracy of phylogenetic analyses. A unique and recently reported HIV-1 transmission cluster consisting of nine infected individuals, for whom the direction and time for each transmission were exactly known, formed the basis for the analyses which were performed under a general model of nucleotide substitution using population sequences from the env V3 and p17gag regions of the HIV-1 genome. Examination of seven different substitution models by maximum-likelihood methods revealed that the fit of the general reversible (REV) model was significantly better than that of simpler models, indicating that it is important to account for the asymmetric substitution pattern of HIV-1 and that the nucleotide substitution rate varied significantly across sites. The shape parameter alpha, which describes the variation across sites by a gamma distribution, was estimated to be 0.38 and 0.25 for env V3 and p17gag, respectively. In env V3, the estimated average transition/transversion rate ratio was 1.42. Thus, the REV model with variable rates across sites (described by a gamma distribution) provides the best description of HIV-1 evolution, whereas simple models are unrealistic and inaccurate. It is likely that the accuracy of phylogenetic studies of HIV-1 and many other viruses would improve substantially by the use of more realistic nucleotide substitution models. This is especially true when attempts are made to estimate the age of distant viral ancestors from contemporary viral sequences.

Base Sequence↗
PubMed1997

Sequence analysis of domestic dog mitochondrial DNA for forensic use.

A method has been developed for the direct sequencing of hypervariable region 1 (HV1) of domestic dog (Canis familiaris) and wolf (Canis lupus) mitochondrial DNA (mtDNA) using single hairs as template. The method uses a robotic work-station and an automated sequencer to allow for robust routine analysis. A population data base was created in order to investigate the forensic and population-genetic informativeness of domestic dog HV1. Sequence variation, partitioning of dog breeds among sequence variants and phylogenetic relations between the variants were determined. Samples from 102 domestic dogs of 52 different breeds and two captive wolves were analyzed. Nineteen dog-sequence variants were found and the frequencies of the variants ranged from 1 to 21%. The calculated discrimination power of the region, i.e., the exclusion capacity, implied that nine out of ten disputed individuals can be excluded by this analysis. The sequence variants were found to cluster into four phylogenetic groups.

Animals↗
PubMed1996

Molecular epidemiology and MT-2 cell tropism of Russian HIV type 1 variant.

Twenty-two HIV-1-infected Russian individuals were studied to gain better insight on the genetic and biological characteristics of HIV-1 variants present in Russia. The V3 domain of the HIV-1 envelope was directly sequenced from cultured peripheral blood mononuclear cells (PBMCs). Phylogenetic analyses were used to determine the HIV-1 genetic subtype and to study transmission patterns. Virus isolates were obtained from PBMCs and the biological phenotype was determined by coculture with MT-2, CEM, and Jurkat-tat cells. Twelve homo- and bisexual men carried subtype B variants, whereas 6 heterosexually infected individuals carried subtype F virus. A heterosexual man infected in the Ivory Coast and a nosocomially infected young female carried subtype A and G virus, respectively. Interestingly, phylogenetic analyses suggested that subtype B may have entered Russia on at least four occasions and, even more surprisingly, that the relatively rare subtype F has entered Russia on at least three occasions. Two suspected transmission clusters of subtype F were supported by the phylogenetic analyses, whereas one suspected subtype B transmission cluster was not. Positively charged amino acids in positions 311 and 325 of V3 loop have been shown to be important determinants for the MT-2-positive biological phenotype of virus isolates. Interestingly, we found that the direct PBMC sequences from individuals with MT-2-positive isolates displayed specific neutral, instead of positively charged, amino acids, in these positions. This indicates that it may be more difficult to predict the biological phenotype of HIV-1 using sequences from uncultured PBMCs than from virus isolates.

Amino Acid Sequence↗
PubMed1996

Accurate reconstruction of a known HIV-1 transmission history by phylogenetic tree analysis.

Phylogenetic analyses are increasingly used in attempts to clarify transmission patterns of human immunodeficiency virus type 1 (HIV-1), but there is a continuing discussion about their validity because convergent evolution and transmission of minor HIV variants may obscure epidemiological patterns. Here we have studied a unique HIV-1 transmission cluster consisting of nine infected individuals, for whom the time and direction of each virus transmission was exactly known. Most of the transmissions occurred between 1981 and 1983, and a total of 13 blood samples were obtained approximately 2-12 years later. The p17 gag and env V3 regions of the HIV-1 genome were directly sequenced from uncultured lymphocytes. A true phylogenetic tree was constructed based on the knowledge about when the transmissions had occurred and when the samples were obtained. This complex, known HIV-1 transmission history was compared with reconstructed molecular trees, which were calculated from the DNA sequences by several commonly used phylogenetic inference methods [Fitch-Margoliash, neighbor-joining, minimum-evolution, maximum-likelihood, maximum-parsimony, unweighted pair group method using arithmetic averages (UPGMA), and a Fitch-Margoliash method assuming a molecular clock (KITSCH)]. A majority of the reconstructed trees were good estimates of the true phylogeny; 12 of 13 taxa were correctly positioned in the most accurate trees. The choice of gene fragment was found to be more important than the choice of phylogenetic method and substitution model. However, methods that are sensitive to unequal rates of change performed more poorly (such as UPGMA and KITSCH, which assume a constant molecular clock). The rapidly evolving V3 fragment gave better reconstructions than p17, but a combined data set of both p17 and V3 performed best. The accuracy of the phylogenetic methods justifies their use in HIV-1 research and argues against convergent evolution and selective transmission of certain virus variants.

Amino Acid Sequence↗
PubMed1996

Analysis of ENV V3 sequences from HIV-1-infected brain indicates restrained virus expression throughout the disease.

The isolation of human immunodeficiency virus type 1 (HIV-1) from the cerebrospinal fluid (CSF) of asymptomatic virus carriers suggests that the viral infection spreading to the brain occurs early during infection. The aim of the present study was to investigate whether HIV-1 infection of the brain parenchyma also occurs during the early phase of infection. We also wished to compare the degree of replication of the virus in the brain at different clinical stages associated with HIV-1 infection. With the use of polymerase chain reaction (PCR), the viral genomes present in seven of eight brain specimens obtained from two asymptomatic HIV-1 carriers and six AIDS patients were amplified. Thereafter, the number of viral copies present in each brain specimen was quantified, the third variable region (V3) of the gp 120 glycoprotein was sequenced and these results compared with the histopathological findings in the tissue. The HIV-1 DNA genome was amplified from seven of the eight brain tissues, including the specimens obtained from the two asymptomatic carriers. An increased number of viral copies in the brain was found in association with histopathological findings of HIV-1 encephalitis. The analysis of the V3 sequences, however, revealed the presence of a homogeneous virus population in the brain at every clinical stage of the disease. These results suggest that, although entry of the virus in the parenchyma may occur early during infection, HIV-1 replication in the brain is constrained until the terminal phase of AIDS encephalitis.

Adult↗