Rapid detection of GB virus C RNA by reverse transcription-polymerase chain reaction (RT-PCR) using primers derived from the 5nontranslated region.
Erker JC. Desai SM. Mushahwar IK.
Virus Discovery Group, Experimental Biology Research, Abbott Laboratories, North Chicago, IL 60064, USA. email@example.com
A simple reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of GB virus C (GBV-C) RNA in serum or plasma is described. In this method, total nucleic acid, extracted from a small volume of human plasma, is reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in PCR employing GBV-C specific primers designed to highly conserved regions of the 5'nontranslated region (NTR). For additional sensitivity, a second round of nested amplification is performed. Reactions are analyzed on an agarose gel and samples showing an ethidium bromide stained band of the appropriate size in the first and second amplification, or in the second amplification only, are designated to be positive. This protocol allows for the rapid and sensitive detection of GBV-C infection in human plasma or serum.
Genotyping of hepatitis E virus in clinical specimens by restriction endonuclease analysis.
Gouvea V. Hoke CH Jr. Innis BL.
Department of Virus Diseases, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA. firstname.lastname@example.org
The genomic variability of hepatitis E virus (HEV) was examined by restriction endonuclease analysis (REA) of four genomic cDNA copies comprising a 499 bp segment of the putative polymerase gene, a 264 bp segment of the helicase gene, and two, 680 bp and 448 bp, segments of the capsid gene. Analysis of the deduced restriction sites of all 27 HEV sequences currently available in the GenBank, and digestion of reverse-transcribed and nested PCR amplified segments obtained from six Nepali isolates were used to devise and test a REA genotyping assay. The assay allowed easy discrimination between the Mexico and Asian genotypes, and the classification of the Asian genotypes into three, or perhaps four subgenotypes. In addition, endonucleases identifiers of individual isolate or clusters of isolates were found. This assay permits rapid identification of a large number of HEV isolates directly from clinical specimens for studies on the molecular epidemiology and evolution of HEV.
Detection of HCV and GBV-C/HGV infection by multiplex PCR in plasma samples of transfused subjects.
Caudai C. Padula MG. Bettini V. Valensin PE.
Department of Molecular Biology, Microbiology Section, University of Siena, Italy.
A multiplex polymerase chain reaction (PCR) was applied to clinical samples for simultaneous detection of hepatitis C virus (HCV) and GBV-C/HGV genome. With both RNA viruses, the amplification was performed with primers of the 5' UTR region starting from the single viral RNA reverse transcripted (cDNA) with random hexanucleotide primer mix. GBV-C/HGV RNA was detected in plasma sample of seven out of 50 transfused patients (14%). The multiplex PCR demonstrated a sensitivity up to 7.8 x 10(2) copies/ml respectively for GBV-C/HGV and HCV RNA in plasma samples of 5/50 patients with GBV-C/HGV/HCV co-infection and in patients with HCV (27/50) or GBV-C/HGV infection alone (2/50).
The MxA protein levels in whole blood lysates of patients with various viral infections.
Chieux V. Hober D. Harvey J. Lion G. Lucidarme D. Forzy G. Duhamel M. Cousin J. Ducoulombier H. Wattre P.
Laboratoire de Virologie, Institut Gernez Rieux, Centre Hospitalier et Universitaire, Lille, France.
Interferon alpha (IFNalpha), a type I interferon, can be considered as a viral infection marker because this cytokine is induced during many viral infections. However, it is quite difficult to detect IFNalpha in sera. Investigations are interested in various intra-cellular IFNalpha-induced proteins as viral infection markers. However the activity of these enzymes increased not only in response to type I IFNs but also to type II IFN. MxA protein can be detected in the cytoplasm of IFNalpha/beta-treated cells, whereas other cytokines, including IFNgamma, are poor inducers. Using an immunochemiluminescent assay, we studied MxA protein in whole blood of 34 patients with various viral infections. The whole blood was drawn into sterile vacuum tubes containing heparin or EDTA. MxA values were relatively similar in heparin-treated samples and EDTA-treated samples, with differences not exceeding 1 ng/ml. The levels of MxA protein were compared in whole blood obtained by using two different lysis procedures. A correlation was found between the MxA levels obtained by using procedure I and procedure II, but higher amounts of MxA protein were found with procedure II. The second procedure is rapid and more convenient than the other and it is carried out in one step which reduce technical problems. High levels of MxA protein were found in peripheral blood cells of patients with acute viral infections (Rotavirus, Adenovirus, RSV, CMV), but MxA protein was not elevated in bacterial infections. The MxA levels were also studied in peripheral blood of 32 HCV positive patients. MxA protein was not found in most of IFNalpha-untreated patients, even those with high viral load. In contrast, high levels of MxA protein were found in IFNalpha-treated patients. MxA quantitation can be considered as a specific marker of acute viral infections, and could be useful in the management of treatment with IFNalpha.
Biophysical characterization of GB virus C from human plasma.
Melvin SL. Dawson GJ. Carrick RJ. Schlauder GG. Heynen CA. Mushahwar IK.
Virus Discovery Group, Experimental Biology Research, Abbott Diagnostic Division, Abbott Laboratories, North Chicago, IL 60064, USA.
Viral characterization studies were carried out on GB virus C (GBV-C) RNA positive plasma from normal human donors and from donors co-infected with GBV-C and hepatitis C virus (HCV). GBV-C RNA was detected by reverse-transcriptase polymerase chain reaction (RT-PCR) and probe hybridization in a single tube assay. Sequential filtration of GBV-C positive plasma indicated that GBV-C RNA is associated with a particle 50-100 nm in diameter. The peak of GBV-C RNA in sucrose gradients was observed at a buoyant density of 1.05-1.13 g/ml. GBV-C RNA titer was reduced following treatment with chloroform or with five detergents indicating that GBV-C has a lipid-containing envelope. Sucrose density gradients and self-forming cesium chloride gradients of detergent-treated GBV-C showed a shift in the RNA peak to heavier buoyant density only when RNase inhibitor (RNasin) and high detergent concentrations were present. The treated material was non-filterable and the RNA had a density of > 1.5 gm/ml.