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. The virus was shown to be closely related to the gibbon ape leukaemia virus (GALV) with 87% similarity when the sequence was compared with the published genome of the Seato strain of GALV. At the level of the long terminal repeat where comparison was possible with other strains, the closest relationship was found with the San Francisco strain of GALV and with the simian sarcoma virus. These results suggest that the isolate should be considered as a strain of GALV.

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. Accordingly, viral culture was used to clarify skin infectivity and the nested polymerase chain reaction (PCR) was used to assess the reliability of skin PCR testing. METHODS: Viral culture and nested PCR performed with gag and pol specific primers were investigated in cadaveric skin and blood from 12 HIV-1 infected patients. Samples were collected repeatedly between one and five days in seven patients. In most cases, analyses were performed on triplicate skin samples: fresh (n = 26); cryopreserved in 5% dimethylsulphoxide (n = 21), or cryopreserved in 30% glycerol (n = 26). RESULTS: HIV was isolated in two of 26 cultures of fresh skin specimens (8%), seven of 47 cryopreserved skin specimens (15%), and eight of 26 blood specimens (31%). The nested PCR detected HIV-1 in all skin samples (n = 73), regardless of the postmortem interval or cryopreservation. In blood, a positive signal was found in eight of 12 patients but two of them had discordant results on successive samples. CONCLUSIONS: These data suggest that nested PCR on postmortem skin samples can detect HIV more reliably than on blood. They also demonstrate the potential viral infectivity of fresh or stored skin postmortem samples in HIV infected patients. They underscore the need for caution during the handling of skin tissue from HIV infected cadavers and confirm the potential risk related to accidental allografting of HIV contaminated skin.

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 1.16 g/cm3. The RNA purified from the culture medium was not detected by reference DNA probes for either HIV-1 and -2 or HTLV-I and -II. In an indirect immunofluorescence assay, sera from patients seropositive for these human retroviruses failed to recognize any antigen in the cells producing only the C-type particles. Using the same technique, plasma samples from 100 blood donors also gave negative results. The presence of this retrovirus could be the result of previous laboratory contamination but the possibility that it is indeed a human virus has to be considered.

Isolation of HIV-1 from seropositive people living in Cotonou, Benin.

Benin is located in West Africa and is situated between HIV-2 and HIV-1-endemic zones. The first cases of HIV-1 infection in Benin were reported in 1987. Since then, AIDS cases have been diagnosed there and the number of known HIV-seropositive people has rapidly increased. Blood samples were collected from 14 seropositive and 11 seronegative patients living in the main city, Cotonou, and their peripheral blood mononuclear cells were cultured. In seven of the seropositive cases, a retrovirus was detected by measurement of Mg2(+)-dependent reverse transcriptase activity and electron microscopy. HIV-1 antigen assay and genomic analysis indicated that the isolated viruses belong to the first serotype. In each positive case, an HIV-1 DNA probe hybridized to the RNA extracted from the virus and six isolates were found positive by the polymerase chain reaction using HIV-1-specific primers.

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. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these preparations revealed one distinct band with an apparent molecular mass of approximately 39 kilodaltons. Antiserum was prepared by immunizing a rabbit with the serogroup A flagellin, which had been eluted from the gel. In immunoblotting, this antiserum cross-reacted with the flagellin of the other strains. When the cells were deflagellated by a short sonication, the cross-reactions observed by slide agglutination with A, G, and K antisera were suppressed. Similarly, shearing of flagella allowed specific slide agglutination of the 12 subgroups of serogroup A.

Rat hybridoma antibodies against canine parvovirus.

Hybrid cells have been produced by fusion between rat myeloma cells IR983F and spleen cells from a LOU rat immunized with canine enteritis parvovirus. Four stable cell lines were selected each secreting a monoclonal antibody directed against this virus. By indirect immunofluorescence, the antigenic determinants recognized by these antibodies were found to be present not only in canine parvovirus infected cells but also in cells infected by the related parvoviruses of mink enteritis and feline panleukopenia. Two of the monoclonal antibodies inhibit hemagglutination by the canine virus and neutralize its infectivity. Hybrid cells were transplanted into histocompatible animals, and large amounts of the antibodies were obtained.

Canine hemorrhagic enteritis: detection of viral particles by electron microscopy.

At necropsy, several dogs which died showing symptoms of hemorrhagic diarrhea, had significant lesions of the mucosa that were found especially in the duodenum and upper part of the small bowel. Study of ultrathin sections from the diseased mucosa revealed particles resembling parvoviruses in altered nuclei of cells of the intestinal crypts. Electron microscopic examination of intestinal contents by negative staining has shown the presence of many viral particles which have a diameter of 24 nm and whose profile is consistent with an icosahedral shape. These virions float at a density of 1.43 g/cm3 in cesium chloride and agglutinate rhesus monkey and swine red blood cells at 4 degrees C. A possible etiological role is discussed. This virus is compared with the minute virus of canines and the Feline Panleukopenia virus.