Quantitation of human immunodeficiency virus type 2 DNA in peripheral blood mononuclear cells by using a quantitative-competitive PCR assay.

A new quantitative-competitive PCR-based human immunodeficiency virus type 2 (HIV-2) proviral DNA assay (QC-PCR) was developed and used to determine the proviral load in HIV-2-infected individuals. Proviral load varied considerably, with means of 1,831 copies per 10(6) peripheral blood mononuclear cells for asymptomatic subjects (n = 19) and 2,587 for AIDS patients (n = 2). HIV-2 viral and proviral loads also varied significantly over time in asymptomatic patients. These data suggest that a high level of virus replication occurs throughout the asymptomatic phase of HIV-2 infection.

Virological and molecular demonstration of human immunodeficiency virus type 2 vertical transmission.

To demonstrate that human immunodeficiency virus type 2 (HIV-2) mother-to-child transmission exists, HIV-2 isolates were obtained from both an asymptomatic mother (HIV-2 strain ARM), and her child (HIV-2 strain SAR), who had a diagnosis of AIDS. To determine their biological phenotype, primary isolates were used to infect various primary mononuclear cells and cell lines. HIV-2 ARM replicates in primary cells and Jurkat-tat, while HIV-2 SAR infects these cells plus SupT1, which led us to classify HIV-2 ARM as a slow/low virus and HIV-2 SAR as having an intermediate (slow/low-3) phenotype. Molecular analysis of the env region corresponding to gp125 was performed. Viral DNA was cloned, sequenced, and used to construct phylogenetic trees. The DNA sequence analysis demonstrated an overall nucleotide diversity of 7.6%. The results present evidence that the child's strain is more virulent than the mother's strain, which is in agreement with the immunodeficiency of the child. The phylogenetic trees that were constructed demonstrate that the two isolates cluster together, being closer to each other than to any other isolate described until now.

Amplification of full-length HIV-2 envelope genes.

In contrast to HIV-1, no studies have been published on the genetic and functional analysis of the envelope gene of primary NSI isolates of HIV-2. However several studies on HIV-1 have shown that NSI strains are the most frequently transmitted strains and probably the most important strains in the pathogenesis of HIV infection. Furthermore, it has been shown that the genetic and biological characteristics of primary isolates of HIV-1 differ widely from those of T-cell-line adapted isolates. Two different polymerase chain reaction (PCR) methods, nested-polymerase chain reaction and overlapp-extension amplification, were used to amplify the envelope genes from a primary non-syncytium-inducing HIV-2 isolate, HIV-2ALI, and from the T-cell line adapted syncytium-inducing isolate, HIV-2ROD. These methods could amplify the complete envelope gene from both viruses. Nested-polymerase chain reaction method was highly sensitive, enabling the amplification of one proviral copy of HIV-2ALI in 10000 peripheral blood mononuclear cells. The use of the methods described herein may help to expand our knowledge on the genetic diversity of HIV-2 as well as on the structure and function of the envelope glycoproteins of primary HIV-2 isolates.

Detection of HIV-1 DNA by polymerase chain reaction incorporation of digoxigenin-11-dUTP and hybridization to immobilized probes.

We have developed a new non-isotopic polymerase chain reaction (PCR) based method for the detection of the human immunodeficiency virus type 1 (HIV-1) DNA in clinical samples. In this method a two step PCR is used to amplify and label HIV-1 DNA segments by incorporation of digoxigenin-11-dUTP (dig-dUTP). Digoxigenin labelled amplified products are hybridized to membrane immobilized complementary DNA probes. Hybridization is detected non-radioactively by incubating the filters with antidigoxigenin antibody conjugated with alkaline phosphatase followed by a standard phosphatase assay. With this method the detection limit was between 1 and 10 HIV-1 DNA copies in a background of 1 microgram of human genomic DNA. Furthermore, we were able to detect HIV-1 DNA in 41 out of 41 HIV-1 antibody positive individuals while 10 out of 10 HIV-1 seronegative individuals gave consistently negative results. Our data indicate that this simple non-isotopic technique is sensitive and specific for the detection of HIV-1 DNA in clinical samples and can constitute a good alternative to other non-isotopic methods described to date.

Detection of HIV1 proviral DNA by PCR and hybridization with digoxigenin labelled probes.

One of the main obstacles for the introduction of PCR method to identify HIV1 proviral DNA in routine diagnostic laboratories is the use of radiolabelled oligodeoxynucleotide probes. Nonradioactive labelled probes have several advantages over radioactive labelling: they are stable for over 1 year, they can be produced easily in large amounts and they are safe. Polymerase chain reaction is an efficient and simple method to produce vector free inserts to use as probes. In this paper we describe a procedure for labelling DNA probes with digoxigenin-11-dUTP using the polymerase chain reaction. This non-radioactive labelling system was applied to detect HIV proviral sequences, amplified in vitro by PCR, from peripheral blood mononuclear cells DNA of infected subjects. We found identical sensitivities and specificities for probes synthesized with the non-radioactive and radioactive labelling procedures. The digoxigenin-11-dUTP can be efficiently incorporated during amplification of a DNA fragment using the polymerase chain reaction. This labelling and detection method proved to be specific, sensible and simple enough to be used in routine diagnostic laboratories for the detection of HIV1 infected individuals.