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ABSTRACTThe new severe acute respiratory syndrome (SARS) coronavirus (CoV), described in February 2003, infected a total of 8,439 people. A total of 812 people died due to respiratory insufficiency. Close contact with symptomatic patients appeared to be the main route of transmission. However, potential transmission by blood transfusion could not be definitely excluded. Two real-time SARS-specific PCR assays were assessed for their sensitivities, agreement of test results, and intra-assay variabilities. Both assays rely on reverse transcription and amplification of extracted RNA. Dilutions of gamma-irradiated cell culture supernatants of SARS CoV-infected Vero E6 cells were prepared to determine the precisions, linear ranges, and accuracies of the assays. The linear range for the Artus RealArt HPA-Coronavirus assay (Artus assay) was 1 × 102to 1 × 107copies/ml, and that for the Roche LightCycler SARS CoV Quantification kit (Roche assay) was 1 × 104to 2 × 108copies/ml. The detection limit of the Roche assay was 3,982.1 copies/ml, whereas that of the Artus assay was 37.8 copies/ml. Detection limits were calculated with a standard preparation that was recommended for use by the World Health Organization. However, quantification of CoV in this preparation may be imprecise. In summary, both assays are suitable for quantitative measurement of SARS CoV at the high concentrations expected in sputum samples. The Artus assay is also suitable for detection of SARS CoV at the low concentrations found in serum samples.

The sequence variability within the core‐ and non‐structural 5 (NS5)‐region of the hepatitis C virus (HCV) was investigated in tumor and non‐tumor tissue of 8 patients with HCV‐associated hepatocellular carcinoma (HCC). Analyses of multiple clones containing polymerase chain reaction‐amplified HCV sequences revealed a significantly higher variability within the core‐region of tumor tissue isolates than of isolates from non‐tumor tissue. Mutant sequence diversity ranged from silent mutations, as well as amino acid substitutions, appearance of in frame stop codons and deletions leading to frame‐shifts. In contrast, the variability of the NS5‐region sequences between isolates from tumor and non‐tumor tissue was not significantly different. These observations might have important implications on the pathology of HCV, especially its potential tumorigenicity. J. Med. Virol. 63:128–134, 2001. © 2001 Wiley‐Liss, Inc.

An association between a specific mutational pattern within the nonstructural (NS)3 region of GB virus‐C/hepatitis G virus (GBV‐C/HGV) genome and fulminant hepatic failure has been suggested recently. The mutational pattern consists of 3–6 nucleotide mutations of which one is leading to an amino acid exchange. In the present study, patients with GBV‐C/HGV mono‐infection (n = 24) or GBV‐C/HGV and HCV co‐infection (n = 20) were investigated prospectively. In 6/44 patients (14%) the mutational pattern within GBV‐C/HGV NS3 previously associated with fulminant hepatic failure was identified by direct sequence analysis of the NS3 region. All 44 patients were asymptomatic clinically and had normal liver functions at initial presentation and after a median follow‐up of 2.2 years. In 22/24 patients with GBV‐C/HGV mono‐infection and all patients with GBV‐C/HGV and HCV co‐infection GBV‐C/HGV RNA remained detectable at the end of the study period, whereas two patients infected with GBV‐C/HGV alone became negative for GBV‐C/HGV RNA and developed GBV‐C/HGV anti‐E2 antibodies indicating recovery from GBV‐C/HGV infection. Aminotransferase levels remained elevated or became normal independent of the persistence of serum GBV‐C/HGV RNA. The median rate of nucleotide substitutions in GBV‐C/HGV mono‐infected and HCV co‐infected patients was 3.4 × 10−3and 3.2 × 10−3per site per year, respectively. In conclusion, the prevalence of the mutational pattern within NS3 region of GBV‐C/HGV associated previously with fulminant hepatic failure is about 14% and not associated specifically with severe liver disease. Over a median follow‐up of 2.2 years less than 5% of patients cleared spontaneously GBV‐C/HGV and no correlation between viraemia and elevated liver enzymes was observed. J. Med. Virol. 62:191–198, 2000. © 2000 Wiley‐Liss, Inc.

An association between a specific mutational pattern within the nonstructural (NS)3 region of GB virus-C/hepatitis G virus (GBV-C/HGV) genome and fulminant hepatic failure has been suggested recently. The mutational pattern consists of 3–6 nucleotide mutations of which one is leading to an amino acid exchange. In the present study, patients with GBV-C/HGV mono-infection (n = 24) or GBV-C/HGV and HCV co-infection (n = 20) were investigated prospectively. In 6/44 patients (14%) the mutational pattern within GBV-C/HGV NS3 previously associated with fulminant hepatic failure was identified by direct sequence analysis of the NS3 region. All 44 patients were asymptomatic clinically and had normal liver functions at initial presentation and after a median follow-up of 2.2 years. In 22/24 patients with GBV-C/HGV mono-infection and all patients with GBV-C/HGV and HCV co-infection GBV-C/HGV RNA remained detectable at the end of the study period, whereas two patients infected with GBV-C/HGV alone became negative for GBV-C/HGV RNA and developed GBV-C/HGV anti-E2 antibodies indicating recovery from GBV-C/HGV infection. Aminotransferase levels remained elevated or became normal independent of the persistence of serum GBV-C/HGV RNA. The median rate of nucleotide substitutions in GBV-C/HGV mono-infected and HCV co-infected patients was 3.4 × 10⁻³ and 3.2 × 10⁻³ per site per year, respectively. In conclusion, the prevalence of the mutational pattern within NS3 region of GBV-C/HGV associated previously with fulminant hepatic failure is about 14% and not associated specifically with severe liver disease. Over a median follow-up of 2.2 years less than 5% of patients cleared spontaneously GBV-C/HGV and no correlation between viraemia and elevated liver enzymes was observed. J. Med. Virol. 62:191–198, 2000. © 2000 Wiley-Liss, Inc.

Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2"-O-allyl modified hammer­head ribozyme was designed to cleave specifically the mutated form of the Ki-ras mRNA (GUU motif in codon 12). The activity was monitored by RT–PCR on Ki-ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2"-deoxy-uridine incorporation on cell prolifer­ation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene.

In patients infected with the hepatitis C virus (HCV), a heterogeneous population of viruses, so‐called quasispecies exists in vivo. The hypervariable regions (HVR) within the second envelope gene (HCV‐E2) show particularly highly intratypic variability and are considered to be the target of neutralizing antibodies. The aims of the study were to optimize a genotype‐independent primer set for amplification of HVR‐1 and to establish a sensitive SSCP analysis for rapid and non‐isotopic detection of predominant serum HCV quasispecies. Using the optimized SSCP technique, changes of quasispecies composition were investigated in five chronically infected patients with HCV before and during interferon‐α treatment. HCV genotyping was performed by sequence and phylogenetic analysis. In addition, serial viremia and serum alanine aminotransferase (ALT) levels were determined. The SSCP analysis was performed at two time points before and during interferon‐α therapy, respectively. Four patients showed an alteration of the SSCP pattern during the first three months of interferon‐α therapy, whereas in one patient the SSCP pattern changed before therapy and remained stable during treatment with interferon‐α. The present approach for non‐isotopic analysis of single strand conformation polymorphism provides a direct, rapid, and sensitive technique for detection of the heterogeneous population of HCV quasispecies of different genotypes. Using this test procedure, investigations of large cohorts of patients with chronic hepatitis C can be undertaken. J. Med. Virol. 53:245–251, 1997.© 1997 Wiley‐Liss, Inc.

In human pancreatic carcinoma (PCa) mutations in the p53 tumor suppressor gene are present in up to 50 of cases. Conformational change and cellular accumulation, together with subsequent release of mutant and normal p53 protein from transformed cells, may initiate a B-cell response with generation of circulating autoantibodies to p53 protein (anti-p53). In the present study we analyzed the sera of 85 consecutive patients with acute pancreatitis (N19). chronic pancreatitis (N33), and PCa (N33) to evaluate the specificity of autoantibodies to p53 protein as a serological marker for PCa. Detection of anti-p53 was performed using an enzyme-linked immunosorbent assay system with immobilized recombinant wild-type p53 protein. Autoantibodies to p53 were detectable in 1 of 19 patients with acute (5.3) and in 4 of 33 patients with chronic pancreatitis (12.1). All anti-p53-positive patients with acute or chronic pancreatitis were carefully examined and no underlying malignant disease was found. During follow-up (range, 281-647 days; mean, 472 days) none of these patients showed any evidence for subsequent development of PCa or any other malignant disease. In patients with PCa, anti-p53 was detected in 6 of 33 cases, resulting in a sensitivity of 18.2 with a specificity of 90.4. In contrast to anti-p53, detection of serum carbohydrate antigen (CA 19–9) resulted in a sensitivity and specificity of 69.7 and 71.2 (CA 19–9, >37 U/ml) and 51.5 and 96.2 (CA 19–9, >1OO U/ml) for the detection of PCa, respectively. Taken together, the sensitivity of anti-p53 formation was low in patients with PCa (18.2). Furthermore, the detection of anti-p53 was not specific for malignancy, indicating that severe inflammatory processes may also induce anti-p53 formation.

Nucleic acid testing was introduced at our blood transfusion service in order to reduce the diagnostic preseroconversion window for transfusion relevant viruses. Up to 96 donor samples were pooled overnight by two Tecan Genesis RSP 150 pipettors. Pools were centrifuged at 48.000g for one hour to enrich viruses. Viral nucleic acids were extracted from centrifugation pellets using modified Qiagen viral RNA kit. HIV and HBV sequences were amplified by in house TaqMan PCR's with patented primers and probes for HBV. HCV PCR was performed by Roche Amplicor V2.0. “Nadis” software was developed for pooling, resolving positive pools and data communication with main frame computer. PCR testing was introduced in January 1997 for HCV HBV and HIV‐1 for all plasma products and for labile components at the 21“ of April 1997. Throughput increased from 2000 to 4000 samples per day. PCR testing is done in parallel to serological testing and products are released eight hours after pooling is completed. Until January 2000, 1.078.940 donations have been tested in 13.274 pools. A total of seven PCR only positives were identified (3 HCV, 3 HBV, 1 HIV). yield of PCR testing for transfusion relevant viruses confirms theoretical estimates on the residual risk of antibody screening.