Showing papers by "Jens Bukh published in 1997"

Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee

Abstract: We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5′ and 3′ termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase–PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA transcripts. However, we found no evidence for HCV replication. Sequence analysis of these and 16 additional full-length clones revealed that seven clones were defective for polyprotein synthesis, and the remaining nine clones had 6–28 amino acid mutations in the predicted polyprotein compared with the consensus sequence of H77. Next, we constructed a consensus chimera from four of the full-length cDNA clones with just two ligation steps. Injection of RNA transcripts from this consensus clone into the liver of a chimpanzee resulted in viral replication. The sequence of the virus recovered from the chimpanzee was identical to that of the injected RNA transcripts. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of HCV.

The quasispecies of hepatitis C virus and the host immune response

Abstract: Infection with hepatitis C virus (HCV) represents an important public health problem worldwide, because it is a major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) [110]. Approximately 1% of the world population is infected with HCV, although geographic variation exists and further epidemiological studies are needed to draw a conclusive epidemiological map of HCV infection. The most striking and alarming feature of HCV infection is its high rate of progression to chronicity. Chronic hepatitis C develops in more than 80% of the acutely infected individuals and about 20-35% of them develop cirrhosis during the course of the disease [1, 2, 19, 25]. Currently, HCV-related end-stage liver disease is the leading indication of orthotopic liver transplantation worldwide [141]. Prospective studies of post-transfusion non-A, non-B hepatitis have been fundamental in unravelling the natural history of HCV infection [ 122]. Despite its inevitable progression, the course of chronic hepatitis C is usually indolent and subclinical. The average time to the clinical presentation of chronic hepatitis is about 10 years, to cirrhosis 20 years and to the development of HCC 30 years [64, 133]. In some patients, however, the course of chronic HCV infection may be rapidly progressive, leading to liver-related death within 5 years after infection ([1] and H.J. Alter, unpublished data). The overall rate of liver-related mortality varied markedly in different prospective studies, with a prevalence ranging from 1.6 to 15% [122]. A controlled clinical study conducted by Seeff et al. [111] in elderly subjects who underwent open-heart surgery failed to demonstrate an increase in the mortality rate after a mean follow-up of 18 years for patients who had developed post-transfusion hepatitis C compared to HCV-uninfected control subjects. By contrast, Tong et al. [133] showed a mortal-

How Escherichia coli can bias the results of molecular cloning: Preferential selection of defective genomes of hepatitis C virus during the cloning procedure

Abstract: Cloned PCR products containing hepatitis C virus (HCV) genomic fragments have been used for analyses of HCV genomic heterogeneity and protein expression. These studies assume that the clones derived are representative of the entire virus population and that subsets are not inadvertently selected. The aim of the present study was to express HCV structural proteins. However, we found that there was a strong cloning selection for defective genomes and that most clones generated initially were incapable of expressing the HCV proteins. The HCV structural region (C-E1-E2-p7) was directly amplified by long reverse transcription–PCR from the plasma of an HCV-infected patient or from a control plasmid containing a viable full-length cDNA of HCV derived from the same patient but cloned in a different vector. The PCR products were cloned into a mammalian expression vector, amplified in Escherichia coli, and tested for their ability to produce HCV structural proteins. Twenty randomly picked clones derived from the HCV-infected patient all contained nucleotide mutations leading to absence or truncation of the expected HCV products. Of 25 clones derived from the control plasmid, only 8% were fully functional for polyprotein synthesis. The insertion of extra nucleotides in the region just upstream of the start codon of the HCV insert led to a statistically significant increase in the number of fully functional clones derived from the patient (42%) and from the control plasmid (72–92%). Nonrandom selection of clones during the cloning procedure has enormous implications for the study of viral heterogeneity, because it can produce a false spectrum of genomic diversity. It can also be an impediment to the construction of infectious viral clones.

Evaluation of commercially available and in-house reverse transcription-PCR assays for detection of hepatitis G virus or GB virus C.

Abstract: Serum samples from 96 Spanish hemodialysis patients, as well as serial dilutions of RNA extracted from a reference strain of hepatitis G virus (HGV), were tested for HGV or GB virus C (GBV-C) RNA. Two different reverse transcription (RT)-PCR-based methods of detection were compared for the ability to detect RNA extracted from the samples: an RT-nested PCR assay with primers derived from the 5' noncoding region (5'NC) or nonstructural region 3 (NS3) sequences and a commercially available RT-PCR assay with primers derived from the 5'NC or NS5A sequences. When RT-nested PCR was performed on 10-fold serial dilutions of RNA from the HGV reference strain, the last positive dilution was 10(-7) to 10(-8). With the commercial RT-PCR assay, the last positive dilution was 10(-6) to 10(-7). When equal amounts of RNA extracted from serum samples from 96 hemodialysis patients were tested for HGV or GBV-C RNA, 25 patients (26%) were positive by the RT-nested PCR. However, only 21 (84%) of these 25 positive patients were positive for HGV or GBV-C by the commercial RT-PCR assay. Analysis of the 5'NC and NS3 sequences amplified by RT-nested PCR demonstrated that all but two positive patients had unique HGV or GBV-C sequences. In summary, RT-nested PCR and a commercially available RT-PCR assay for HGV or GBV-C gave concordant results for 96% of the patients tested.