Validation of iQ-Check E. coli O157:H7 real-time PCR test kit for detection of Escherichia coli O157:H7 in selected foods.

iQ-Check E. coli O157:H7 (Bio-Rad Laboratories, Hercules, CA) is a real-time PCR kit for detection of E. coli O157:H7 from selected foods.

Gag-specific immune enhancement of lentiviral infection after vaccination with an adenoviral vector in an animal model of AIDS.

The evaluation of vaccine strategies in animal models is essential for the development of a vaccine against HIV. In efficacy trials conducted in non-human primate models of AIDS, vaccines based on adenoviruses compared favourably with other vaccine vectors. To determine whether this strategy could be transposed to another animal model, and by extension, to humans, we have evaluated the efficacy of adenoviral vectors in a natural model of AIDS, infection of the cat by the feline immunodeficiency virus (FIV).

Efficient and specific internal cleavage of a retroviral palindromic DNA sequence by tetrameric HIV-1 integrase.

Background: HIV-1 integrase (IN) catalyses the retroviral integration process, removing two nucleotides from each long terminal repeat and inserting the processed viral DNA into the target DNA. It is widely assumed that the strand transfer step has no sequence specificity. However, recently, it has been reported by several groups that integration sites display a preference for palindromic sequences, suggesting that a symmetry in the target DNA may stabilise the tetrameric organisation of IN in the synaptic complex.

Is oxygen a key factor in the lipodystrophy phenotype?

Background: The lipodystrophic syndrome (LD) is a disorder resulting from selective damage of adipose tissue by antiretroviral drugs included in therapy controlling human-immunodeficiency-virus-1. In the therapy cocktail the nucleoside reverse transcriptase inhibitors (NRTI) contribute to the development of this syndrome. Cellular target of NRTI was identified as the mitochondrial polymerase-gamma and their toxicity described as a mitochondrial DNA (mtDNA) depletion resulting in a mitochondrial cytopathy and involved in fat redistribution.

Inhibition of early steps of HIV-1 replication by SNF5/Ini1.

To replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN.

Evaluation of live feline immunodeficiency virus vaccines with modified antigenic properties.

Live-attenuated viruses have typically been generated from pathogenic viruses by genetic modifications that modified their replicative capacity. The present study investigated whether modification of the antigenic properties of live-attenuated viruses might improve upon the protection that such vaccines afford against lentivirus infection. In a previous study, random amino acid substitutions were introduced into the transmembrane envelope glycoprotein of the feline immunodeficiency virus (FIV), within a highly conserved domain (principal immunodominant domain) bearing immunodominant B-cell epitopes.

Quantification of HFV-integrated DNA in human cells by Alu-LTR real-time PCR.

Integration is described as a key step in viral replication of all retroviruses. A sensitive and quantitative measure of an integrated molecule is a good way to examine the importance of the integration step and to evaluate efficiency of retroviral vectors for gene transfer or anti-integrase drugs. Here, we report a sensitive and quantitative real-time polymerase chain reaction (PCR) technique to measure integrated viral DNA in human cells during a foamy virus (HFV) infection.

Alu-LTR real-time nested PCR assay for quantifying integrated HIV-1 DNA.

An improved Alu-long terminal repeat (LTR) polymerase chain reaction (PCR) assay is described for the quantification of integrated HIV-1 DNA in infected cells. The method includes generation of an infected cell line containing numerous randomly distributed HIV-1 integrated DNA for the construction of the DNA standard and a two-step real-time PCR assay in which the first-round PCR amplifies the DNA sequence between the HIV-1 LTR and the nearest chromosomal Alu element, and the nested PCR specifically amplifies PCR products from the first-round PCR. This assay allows us to quantify proviral DNA with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents) and exhibits a broad range of quantification spanning 5 log10 provirus copies.

CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain.

The protozoan parasite Toxoplasma gondii enters hosts through the intestinal mucosa and colonizes distant tissues such as the brain, where its progeny persists for a lifetime. We investigated the role of CD11c- and CD11b-expressing leukocytes in T. gondii transport during the early step of parasitism from the mouse small intestine and during subsequent parasite localization in the brain.

Oxygen consumption by cultured human cells is impaired by a nucleoside analogue cocktail that inhibits mitochondrial DNA synthesis.

We evaluated oxygen consumption rates in human cells cultured in the presence of a nucleoside analog reverse transcriptase inhibitor (NRTI) cocktail that inhibits mitochondrial DNA synthesis. We treated a proliferating human lymphocyte cell line and a primary culture of human adipose cells with antiretroviral drugs (AZT+ddC+d4T). The effects of these drugs on mitochondrial DNA (mtDNA) levels and oxygen consumption rates were evaluated using semi-quantitative real-time PCR and an on-line monitoring Clark electrode system.

A novel function for spumaretrovirus integrase: an early requirement for integrase-mediated cleavage of 2 LTR circles.

Retroviral integration is central to viral persistence and pathogenesis, cancer as well as host genome evolution. However, it is unclear why integration appears essential for retrovirus production, especially given the abundance and transcriptional potential of non-integrated viral genomes. The involvement of retroviral endonuclease, also called integrase (IN), in replication steps apart from integration has been proposed, but is usually considered to be accessory.

Cell cycle regulation of human immunodeficiency virus type 1 integration in T cells: antagonistic effects of nuclear envelope breakdown and chromatin condensation.

We examined the influence of mitosis on the kinetics of human immunodeficiency virus type 1 integration in T cells. Single-round infection of cells arrested in G1b or allowed to synchronously proceed through division showed that mitosis delays virus integration until 18-24 h postinfection, whereas integration reaches maximum levels by 15 h in G1b-arrested cells. Subcellular fractionation of metaphase-arrested cells indicated that, while nuclear envelope disassembly facilitates docking of viral DNA to chromatin, chromosome condensation directly antagonizes and therefore delays integration.

Evidence for gene expression by unintegrated human immunodeficiency virus type 1 DNA species.

The integrated form of human immunodeficiency virus type 1 (HIV-1) DNA is classically considered to be the sole template for viral gene expression. However, several studies have suggested that unintegrated viral DNA species could also support transcription. To determine the contribution of the different species of HIV-1 DNA to viral expression, we first monitored intracellular levels of various HIV-1 DNA and RNA species in a single-round infection assay.

Early detection of a two-long-terminal-repeat junction molecule in the cytoplasm of recombinant murine leukemia virus-infected cells.

We showed that a U5-U3 junction was reproducibly detected by a PCR assay as early as 1 to 2 h postinfection with a DNase-treated murine leukemia virus (MLV)-containing supernatant in aphidicolin-arrested NIH 3T3 cells, as well as in nonarrested cells. Such detection is azidothymidine sensitive and corresponded to neosynthesized products of the reverse transcriptase. This observation was confirmed in two additional human cell lines, TE671 and ARPE-19.

[Evolution and virulence of primate lentiviruses].

While the AIDS epidemic caused by human immunodeficiency viruses (HIV) has resulted in the death of over 20 million people worldwide, simian immunodeficiency virus (SIV) infection, found in numerous African primate species, does not induce disease symptoms. The factors accounting for this difference between humans and natural host of SIV remain poorly understood. The entangled nature of the host/virus relationship could be the answer, rather than independent virus or host factors.

Analysis of early human immunodeficiency virus type 1 DNA synthesis by use of a new sensitive assay for quantifying integrated provirus.

A novel Alu-long terminal repeat (LTR)-based real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type 1 (HIV-1) DNA in infected cells with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents). Parallel assays for total HIV-1 DNA and two-LTR HIV-1 DNA circles allowed the synthesis and fate of the different HIV-1 DNA species to be monitored upon a single round of viral replication.

Quantitation of blood lymphocyte mitochondrial DNA for the monitoring of antiretroviral drug-induced mitochondrial DNA depletion.

Objective: To investigate the impact of antiretroviral treatment on the mitochondrial DNA (mtDNA) content of peripheral blood mononuclear cells (PBMCs) from HIV-1-infected patients.

Design: As absolute mtDNA copy numbers widely differ between individuals, we performed a longitudinal analysis where the patient's first historical specimen was obtained as a baseline reference for relative comparison with subsequent samples from that patient.

Methods: mtDNA and nuclear DNA quantitation per cell (beta-globin gene copies) were both measured by real-time polymerase chain reaction analysis of whole DNA extracts of 361 serial live-cryopreserved PBMCs collected in former trials and clinical follow-ups from 60 individuals with established or recently acquired HIV-1 infections before and during administration of various antiviral combination therapies.

Biphasic DNA synthesis in spumaviruses.

Spumaviruses are complex retroviruses whose replication cycle resembles that of hepadnaviruses, especially by a late-occurring reverse transcription step. The possible existence of an early reverse transcription as observed in other retroviruses was not documented. Using real-time quantitative PCR, we addressed directly the kinetics of DNA synthesis during spumavirus infection.

Longitudinal monitoring of 2-long terminal repeat circles in peripheral blood mononuclear cells from patients with chronic HIV-1 infection.

Objective: To evaluate the potential use of 2-long terminal repeats (LTR) HIV circular DNA quantification for the monitoring of ongoing virus replication in treated HIV-1-infected patients.

Design And Methods: In a longitudinal setting, where the natural course of HIV-1 infection was in most cases disrupted by continuous or discontinuous antiviral therapy, 2-LTR circles of HIV-1 DNA were quantified in serial peripheral blood mononuclear cell samples, selected in retrospect from 16 patients with chronic HIV-1 infection, using quantitative real-time PCR. We compared variations of 2-LTR circle level with concomitant variations in plasma viral RNA level and with the frequency of productively infected cells and chromosome associated proviral DNA copy numbers in patient's peripheral blood mononuclear cells.

Live, attenuated simian immunodeficiency virus SIVmac-M4, with point mutations in the Env transmembrane protein intracytoplasmic domain, provides partial protection from mucosal challenge with pathogenic SIVmac251.

Attenuated molecular clones of simian immunodeficiency virus (SIVmac) are important tools for studying the correlates of protective immunity to lentivirus infection in nonhuman primates. The most highly attenuated SIVmac mutants fail to induce disease but also fail to induce immune responses capable of protecting macaques from challenge with pathogenic virus. We recently described a novel attenuated virus, SIVmac-M4, containing multiple mutations in the transmembrane protein (TM) intracytoplasmic domain.

Lymphoid activation: a confounding factor in AIDS vaccine development?

In a previous vaccination trial, inoculation of env gene DNA failed to elicit a detectable antibody response, yet accelerated virus dissemination in most immunized cats following challenge with feline immunodeficiency virus. This result raised the possibility that cell-mediated immune responses had given rise to immune-mediated enhancement of infection. Since high-level replication of immunodeficiency viruses in lymphocytes requires cellular activation, antigen-specific responses or non-specific polyclonal activation may have increased the frequency of optimal target cells.

B-cell epitopes of the envelope glycoprotein of caprine arthritis-encephalitis virus and antibody response in infected goats.

Goats infected with caprine arthritis-encephalitis virus (CAEV) develop high titres of antibodies to Env. Not only is no consistent neutralizing response found but anti-Env antibodies have even been associated with disease in infected goats. To identify the continuous antigenic determinants involved in this atypical anti-Env response, we mapped CAEV-CO Env by screening an epitope expression library with infected goat sera.