摘要:

SUMMARY.The mode of replication of the hepatitis C virus (HCV) remains poorly understood. Attempts to produce a tissue culture model containing replicating HCV have been largely unsuccessful. Recent studies on sera from patients chronically infected with HCV have shown that viral particles may be found in high‐or low‐density fractions. High‐density fractions contain non‐infectious virions whilst infectious particles can be derived from low‐density material. Using appropriate infectious fractions we have successfully infected a number of human cell lines allowing studies of HCV replication to be

ISSN:1352-0504    

DOI:10.1111/j.1365-2893.1996.tb00074.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

SUMMARY.Hepatitis C virus (HCV) in highly infectious sera has been shown to be predominantly associated with low‐density lipoproteins. To determine whether the association is specific to low‐density lipoproteins (LDL) or very low‐density lipoproteins (VLDL), we fractionated HCV‐containing plasma by a column chromatographic procedure known to separate these classes. Hepatitis C virus RNA detected by polymerase chain reaction (PCR) was associated primarily with the very low‐density (VLDL) fraction. However, it could not be ruled out that virus‐associated LDL may have eluted with this fraction. Hepatitis C virus virions isolated from sera having sufficient titre for visualization by electron microscopy are generally coated with antiviral antibodies, therefore we utilized the lipid association to isolate antibody‐free virions. Very low‐density lipoproteins were isolated by ultracentrifugal flotation and then treated with deoxycholate to release the virions. These were then isolated in a highly purified form by centrifugation in a sucrose gradient. The 1.10–1.11 gml‐1region of the gradients contained 60–70 nm particles. Particles with similar surface structure but having a diameter of only 30–40 nm constituted about 30% of the total. The latter may represent defective interfering particles. The identity of both small and large particles with HCV virions and associated particles was confirmed by their trapping on grids by an anti‐HCV E2 monoclonal antibody, and by their aggregation by rabbit antiserum to an amino‐terminal peptide of E1. Thus, both E1 and E2 epitopes are displayed on the su

ISSN:1352-0504    

DOI:10.1111/j.1365-2893.1996.tb00075.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

SUMMARY.Antisense oligodeoxynucleotides strategies have been used both to study normal gene function and to block gene expression therapeutically. We have previously shown that a number of antisense oligonu‐cleotides against hepatitis B virus (HBV) mRNA are able to inhibit viral gene expressionin vitro.Here we report the establishment of an animal model producing HBV markers in athymic nude mice and inhibition of HBV gene expression and replication by antisense DNAin vivo.2.2.15 cells (Hep‐G2 cell line transfected with HBV genomes) were injected subcutaneously (s.c.) into athymic BALB/c nude mice at a total cell number of 0.5–1×108per mouse. Transplanted tumours developed about 2 weeks after inoculation. Hepatitis B surface and e antigens (HBsAg and HBeAg), as well as HBV DNA, could be detected in the circulation of tumour‐bearing mice. Hepatitis B surface antigen and hepatitis B core antigen (HBcAg) were demonstrated in tumour cells. After 10 days of tumour growth, antisense phosphorothioate oligonucleo‐tide, complementary to the cap site of the SPII promoter of HBV mRNA, were injected by infiltration into or around the tumour as a daily dose of 20 μg per gram body weight. Treatment for a total of 10 days resulted in an effective inhibition of viral replication and gene expression. These results suggest therapeutic potential for antisense oligomers in the treatment of patients who are chronically infect

ISSN:1352-0504    

DOI:10.1111/j.1365-2893.1996.tb00076.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

SUMMARY.In patients with chronic hepatitis C, the therapeutic effect of interferon (IFN) is influenced by the progression of liver disease. In a previous study, we showed that 2′,5′‐oligoadenylate synthetase activity in the liver homogenate was significantly lower in cirrhotic rats than in controls after injection of murine IFN. To determine the reason for this decrease, we injected IFN into rats with thioacetamide‐induced cirrhosis and used microautoradiography with human lymphoblastoid interferon ([125I]LyIFN). Accumulation of [125I]LyIFN in cirrhotic rat livers was approximately half of that in control rats (2880±900vs5770±600mm2,P<0.01). In the cirrhotic rat livers there were few grains on the hepatocytes, but many on collagen fibres. These results suggest that binding of IFN to its hepatocyte receptors is hindered in the presence of cirrhosis. The decreased amount of IFN reaching hepatocytes may contribute to the poor responses to IFN seen in patients with

ISSN:1352-0504    

DOI:10.1111/j.1365-2893.1996.tb00077.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

SUMMARY.The mechanism of hepatitis C virus (HCV) replication is unknown, although the classification of HCV in the Flaviviridae has led to the postulation that HCV may adopt a replication strategy similar to that of the flaviviruses. To determine if HCV double‐stranded replicative forms, consistent with this strategy, were present in total liver RNA extracted from HCV‐infected individuals, HCV‐specific RNA was detected by reverse transcription followed by polymerase chain reaction (RT‐PCR). Initially, a strand‐specific RT‐PCR resulting from chemical modification of the 3′ end of the RNA was established usingin vitrotranscribed HCV RNA. This procedure allowed the specific detection of positive and negative HCV RNA strands in HCV‐infected liver tissue. The species of HCV RNA was then examined in RNA extracted from liver tissue from naturally infected individuals; total liver RNA was either: (i) fractionated with 2mLiCl (designed to precipitate single‐stranded and partially double‐stranded RNA); or (ii) digested with RNase A in high salt conditions (designed to digest single‐stranded RNA only). Amplification of positive sense HCV RNA from the LiCl‐insoluble fraction, but not from the LiCl‐soluble fraction nor in the RNase A‐digested sample, was consistent with the interpretation that single‐strand, but not double‐stranded HCV RNA, was contained in the liver samples. Thus, it is unclear if a double‐stranded RNA species is formed

ISSN:1352-0504    

DOI:10.1111/j.1365-2893.1996.tb00078.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

SUMMARY.A new vaccine against hepatitis B virus (HBV) infection, produced in mammalian Chinese hamster ovary (CHO) cells, contains the small (s), middle (Pre S2) and large (Pre SI) surface proteins of HBV. Three injections of a 5‐μg or 10‐μg dose were administered intramuscularly (i.m.) at 0, 1 and 6 months to a group of 105 young adults, who were monitored for a period of 6 months after the third injection. Seroconversion rates were 100% after the second injection of the 5‐μg or 10‐μg dose. Geometric mean litres of HBsAb at 1 month after the third injection were 12156 mIU ml‐1and 13 482 mIU ml‐1in those receiving the 5‐μg and 10‐μg dose respectively. The vaccine was well tolerated with no significant adverse events. These preliminary results suggest that the Pre S‐s recombinant vaccine, produced in mammalian cells, is highly immunogenic, leading to 100% seroconversion in the population tested after injection of

ISSN:1352-0504    

DOI:10.1111/j.1365-2893.1996.tb00079.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

SUMMARY.Haemophilic patients have a high prevalence of hepatitis C virus (HCV) infection because of the use of unsterilized clotting factor concentrates. Six major genotypes of HCV have been distinguished so far, with epidemiological evidence suggesting that genotypes 1–3 are common in the indigenous UK and US populations. The aim of this study was to analyse the changes in viral load and composition of the HCV quasispecies in haemophilic patients receiving therapy with interferon‐α (IFN‐α) using the four major methods currently available for HCV genotyping. The most consistent genotype results were obtained using restriction fragment‐length polymorphism (RFLP) analysis when compared with the DNA sequence analysis, and showed that the dominant genotype can change in patients with mixed genotype infections treated with IFN‐α. This study indicates the difficulties in studying this group of patients with mixed HCV genotype infections, and that frequent sampling is necessary, together with viral load measurement to monitor response to I

ISSN:1352-0504    

DOI:10.1111/j.1365-2893.1996.tb00080.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY