[Construction of nested set of unidirectional deletion of recombinant plasmid DNA for sequencing].

In order to rapidly sequence a batch of large DNA fragments, we developed a method for the construction of nested set of unidirectional deletions. A nested set of unidirectional deletions of the large DNA fragment of a c-type retrovirus gene was successfully constructed by the adoption of this method. The recombinant plasmid DNA was excised by BamHI and SphI at on end of the target DNA to create 5' and 3' overhanging ends.

Variant human immunodeficiency virus type 1 proteases and response to combination therapy including a protease inhibitor.

The objective of this observational study was to assess the genetic variability in the human immunodeficiency virus (HIV) protease gene from HIV type 1 (HIV-1)-positive (clade B), protease inhibitor-naïve patients and to evaluate its association with the subsequent effectiveness of a protease inhibitor-containing triple-drug regimen. The protease gene was sequenced from plasma-derived virus from 116 protease inhibitor-naïve patients. The virological response to a triple-drug regimen containing indinavir, ritonavir, or saquinavir was evaluated every 3 months for as long as 2 years (n = 40).

Comparison of DNA sequencing and a line probe assay for detection of human immunodeficiency virus type 1 drug resistance mutations in patients failing highly active antiretroviral therapy.

The resistance of human immunodeficiency virus type 1 (HIV-1) to drugs is a major cause of antiretroviral treatment failure. We have compared direct sequencing to a line probe assay (LiPA) for the detection of drug resistance-related mutations in 197 clinical samples, and we have investigated the sequential appearance of mutations under drug pressure. For 26 patients with virological failure despite the use of two nucleoside analogues and one protease inhibitor (indinavir [n = 6], ritonavir [n = 10], and saquinavir [n = 10]), genotypic resistance assays were carried out retrospectively every 3 months for up to 2 years by using direct sequencing (TruGene; Visible Genetics) and a LiPA for detection of mutations in the reverse transcriptase (INNO-LiPA HIV-1 RT; Innogenetics) and the protease (INNO-LiPA HIV Protease, prototype version; Innogenetics) genes.

Molecular analysis of GB virus C/hepatitis G virus in HIV-1-positive intravenous drug users in Belgium.

Objective: A high prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) RNA and E2 antibodies is observable in human immunodeficiency virus type 1 (HIV-1)-infected individuals in Belgium, including intravenous drug users (IDUs), in whom the highest prevalence is observed. A molecular analysis of GBV-C/HGV could give indications on the origin of this infection in IDUs.

Methods: We directly sequenced 7 GBV-C/HGV isolates from this IDU population and performed a phylogenetic analysis comparing the results to known GBV-C/HGV sequences.

Influence of CD4+ lymphocyte counts on GB virus C/hepatitis G virus carriership in HIV-positive individuals.

The duration of the GB virus C or hepatitis G virus (GBV-C/HGV) carriership varies according to the patient group studied. The immune competence of the host may be important. GBV-C/ HGV was studied in human immunodeficiency virus (HIV)-infected persons and an attempt was made to correlate the presence of viral RNA or E2 antibodies with CD4+ lymphocyte counts.

Acute parvovirus B19 infection associated with fulminant hepatitis of favourable prognosis in young children.

Background: The cause of fulminant hepatitis (FH) in children is unexplained in up to 50% of cases. We report parvovirus B19 as an agent associated with FH in children and compare clinical characteristics of these patients with those of age-matched patients with FH of other origin.

Methods: 45 patients presented with FH.

Characterization of a C-type retrovirus isolated from an HIV infected cell line: complete nucleotide sequence.

As previously reported, a C-type retrovirus, referred to as retrovirus X was isolated from HIV infected cells. In order to further characterize this virus, the proviral DNA was cloned and sequenced. The organization of the genome (8379 bp) appeared to be typical of the mammalian type C retroviruses.

Evidence for persistence of human parvovirus B19 DNA in bone marrow.

A nested polymerase chain reaction assay (nPCR) was used to investigate the potential of human parvovirus B19 DNA to persist in blood or bone marrow samples obtained either from blood donors or cadaveric bone donors or from patients presenting with clinical signs of parvovirus B19 infection. The presence of parvovirus B19 specific antibody in blood was tested by enzyme immunoassay (EIA). B19 virus genome was not detected in any blood sample of 115 blood donors, of whom 92 (80%) had anti-B19 IgG antibody only as an indication of past infection.

HIV-1 detection by nested PCR and viral culture in fresh or cryopreserved postmortem skin: potential implications for skin handling and allografting.

Aims: To date, the risk relating to the handling or allografting of human immunodeficiency virus type 1 (HIV-1) infected postmortem skin remains hypothetical. While blood screening for HIV antibodies is still the key safety procedure to detect HIV infected cadavers, false negative results are a concern. Conversely, false positive results may hamper the collection of skin allografts.

Hepatitis C virus genotypes in hemodialyzed patients: a multicentric study.

The few studies concerning HCV genotypes in hemodialyzed patients concerned small groups of patients, issued from single units. Using the RFLP typing method in the 5' non-coding region (5' NCR), we performed the genotyping of HCV strains of 80 patients issued from 14 Belgian dialysis units. Forty-seven of the 80 patients were also tested by Inno-Lipa II (Innogenetics, Gent, Belgium).

Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene.

HIV-1 and related viruses require co-receptors, in addition to CD4, to infect target cells. The chemokine receptor CCR-5 (ref.1) was recently demonstrated to be a co-receptor for macrophage-tropic (M-tropic) HIV-1 strains, and the orphan receptor LESTR (also called fusin) allows infection by strains adapted for growth in transformed T-cell lines (T-tropic strains).

An anti-HIV-1 gag protein rat monoclonal antibody library.

A set of 18 rat monoclonal antibodies (MAbs) directed against human immunodeficiency virus type 1 (HIV-1) gag proteins was derived from 4 independent fusion protocols. The epitopes recognized were delineated using a random fragment expression library representing the whole HIV-1IIIB genome. This panel of rat MAbs was used to analyze the antigenicities of the HIV-1 CAp24 major core protein and the HIV-1 NCp7 nucleocapsid protein.

HIV Infection in Children Born Before and After Immigration to Belgium.

Methods: The records of 119 children, either infected with HIV or born to HIV-infected mothers were reviewed. This group was mainly represented by children whose families came to Belgium from Central Africa. Seven children were infected through blood or blood products transfusion, whereas the remaining 112 were born to infected mothers.

Isolation of a C-type retrovirus from an HIV infected cell line.

A study by electron microscopy of HUT 78 cells infected with the ARV-2 strain of HIV-1 revealed, in addition to virions having the characteristic morphology of a lentivirus, the presence of numerous C-type particles, suggesting that the analysed specimen might in fact contain two different viruses. In order to further investigate this observation, the cell culture supernatant was filtered and mixed at serial dilutions with uninfected HUT 78 cells. In this way, it was possible to obtain cells producing only virions with C-type morphology and a Mn++ dependent reverse transcriptase activity which in a sucrose gradient was found to peak at a buoyant density of 1.

Study on rat-rat hybridoma technique and production of rat monoclonal antibodies against HIV and HBsAg.

The rat-rat hybridoma technique has some definite advantages in the three systems (mouse, rat or human system) currently utilized for monoclonal antibody production. The study on rat-rat hybridoma technique and its application to the productions of monoclonal antibodies against human immunodeficiency virus (HIV) and hepatitis B virus surface antigen (HBsAg) are described as examples in this paper.

Characterization of flagella of Clostridium difficile and their role in serogrouping reactions.

Slide agglutination with rabbit antisera allows the differentiation of 10 serogroups of Clostridium difficile, namely, A, B, C, D, F, G, H, I, K, and X. Each serogroup displays a specific protein profile in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, except for A, which displays 12 different protein profiles (A1 to A12). In the present work, electron microscopy revealed the presence of uniformly distributed flagella in the reference strains of serogroups G and K and in all strains representative of the 12 subgroups within serogroup purified by differential centrifugation.

Disseminated BCG in HIV infection.

A boy, born to a mother with AIDS related complex, was immunised with BCG on the 10th day of life. At the age of 4 months he presented with a local enlarged lymph node, fever, hypotonia, and diarrhoea. Mycobacterium bovis, BCG strain, was grown from the lymph node and cerebrospinal fluid; this proved dissemination.

Anti-HIV antibodies in the CSF of AIDS patients: a serological and immunoblotting study.

CSF and serum samples from 16 AIDS patients were tested for the presence of anti-HIV antibodies either by classical serological methods or by an immunoblot technique based on agarose gel isoelectric focusing and transfer of the specific IgG antibodies onto HIV antigens-loaded nitrocellulose sheets. This method enabled the demonstration of an intrathecal synthesis of anti-HIV oligoclonal IgG antibodies, often superimposed on diffuse polyclonal production, in 14 patients. The two negative cases were devoid of neurological signs or symptoms.

Characterization of a nonhemagglutinating mutant of canine parvovirus.

A nonhemagglutinating mutant of a 1978 isolate of canine parvovirus (CPV) was derived after repeated passages in the NLFK feline kidney cell line. The mutant CPV was antigenically indistinguishable from wild-type virus when tested with 82 monoclonal antibodies, and it replicated in cat and dog cell lines in culture. Sequences of the VP-1 and VP-2 genes revealed two nucleotide and two predicted amino acid sequence differences at 77 and 88 genome map units in the mutant compared to hemagglutinating viruses.