Detection of genomic and intermediate replicative strands of hepatitis C virus in liver tissue by in situ hybridization.

Aria, K T Nouri; Sallie, Richard; Sangar, D. V.; Alexander, Graeme; Smith, Heather M.; Byrne, Janet C.; Portmann, Bernard; Eddleston, A. L. W. F.; Williams, Roger L.

doi:10.1172/jci116449

Cited by 99 publications

(55 citation statements)

References 36 publications

(14 reference statements)

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“…The sinusoidal cells contain three functionally different cells including endothelial, stellate, and Kupffer cells. A similar observation has been reported previously, 22 however, the effect of HCV replication in sinusoidal cells on liver damage is unknown.…”

Section: Discussionsupporting

confidence: 87%

Dynamics of Hepatitis C Virus Replication in Human Liver

Chang

Williams

Mittler

et al. 2003

The American Journal of Pathology

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Hepatitis C virus (HCV) replication at the cellular level is not fully understood. This study describes an optimized system for quantifying replication of HCV in hepatocytes and in liver tissues. A digital image analysis method was developed to quantify signal intensities of HCV genomic and replicative-intermediate1 Symptomatic differences among infected individuals have generated considerable interest in elucidating the pathogenic mechanisms of HCV disease.Previous studies have described the levels of HCV genomes in patient sera in cohorts either on therapeutic drug regimens, untreated, or undergoing liver transplantation. As a whole, these studies have failed to find a consistent relationship between serum titers and the degree of hepatic injury.2-4 The ratios of HCV genome RNA in liver and serum compartments are significantly different among chronically infected patients.2,5-8 These reports suggest that hepatic injury, serum titers, and intrahepatic viral replication do not display a linear relationship during chronic hepatitis C infection.HCV is a positive strand RNA virus that is classified as a Hepacivirus within the Flaviviridae family.9 By analogy with other members of the Flaviviridae, it is assumed that HCV replication requires production of negative strand replicative-intermediate RNA, from which progeny genomic strand RNA is transcribed. The presence of negative strand RNA in cells indicates ongoing HCV replication and synthesis of progeny genomic RNA in these cells. Previously, we and others have used strand-specific in situ hybridization to localize positive strand and negative strand HCV RNAs in infected human liver tissue. 10 The percentage of hepatocytes positive for genomic RNA ranged from 4.8 to 87.6%, 10 -12 whereas those positive for replicative-intermediate RNA ranged from 4 to 25%. 10,13 On visualization, the distribution and abundance of genomic RNA appeared to be different from that of replicative-intermediate RNA, suggesting compartmentalization or regulation of replication may occur.To further characterize HCV replication in liver tissue, we developed an image analysis method to measure quantitative amounts of genomic and replicative-intermediate RNAs in infected liver tissues, and to examine the

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Section: Discussionsupporting

confidence: 87%

Dynamics of Hepatitis C Virus Replication in Human Liver

Chang

Williams

Mittler

et al. 2003

The American Journal of Pathology

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“…It is possible that GGT elevation is a result of HCV infection of biliary epithelial cells and/or HCV-induced bile duct damage. 28,29 Furthermore, the association between elevated GGT and insulin resistance 30 -32 may explain a reduced virological response to IFN-␣. 33 Patients with persistently normal baseline ALT levels showed a different distribution of ALT patterns during treatment than patients with elevated baseline ALT levels.…”

Section: Discussionmentioning

confidence: 99%

Viral kinetics during antiviral therapy in patients with chronic hepatitis C and persistently normal ALT levels

et al. 2004

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The aim of the present study was to compare viral kinetics between patients with chronic hepatitis C and persistently normal alanine aminotransferase (ALT) levels and those with elevated ALT levels. Kinetic parameters were derived from nonlinear, least square fitting of serum hepatitis C virus RNA quantifications collected from patients with chronic hepatitis C and persistently normal (n ‫؍‬ 20) and elevated (n ‫؍‬ 19) ALT levels before and during treatment with 180 g pegylated interferon ␣-2a once weekly plus daily ribavirin. Patients with chronic hepatitis C and persistently normal ALT levels showed a trend to lower pretreatment infected cell loss (␦) (P ‫؍‬ .13) but no differences in efficacy of blocking virus production (⑀) and infected cell loss during treatment (m␦) compared with patients with elevated ALT levels. Differences were significant for ⑀ (P ‫؍‬ .02) and ␦ (P ‫؍‬ .04) when applying updated "healthy" levels for ALT (0.75 times and 0.63 times upper limit of normal for male and female patients, respectively). A significant reduction of the kinetic parameters ⑀, ␦, and m␦ was observed in patients with elevated ␥-glutamyltranspeptidase (GGT) levels compared with patients with normal GGT levels (P ‫؍‬ .02, P ‫؍‬ .005, and P ‫؍‬ .02, respectively). In conclusion, viral kinetics are similar in patients with chronic hepatitis C and persistently normal ALT levels and those with elevated ALT levels. However, in patients with elevated GGT levels, a major association with reduced efficacy of blocking virus production and lower infected cell loss was observed. These data show that virological response in patients with chronic hepatitis C is less associated with baseline ALT than with GGT levels.

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“…The liver is the main target for replication of HCV in vivo (Nouri-Aria et al, 1993 ;Gastaldi et al, 1995), which occurs via minus-strand RNA as replicative intermediate (Houghton et al, 1991). In addition, the presence of both HCV RNA molecules Author for correspondence : Stefanie Seipp.…”

Section: Introductionmentioning

confidence: 99%

Establishment of persistent hepatitis C virus infection and replication in vitro.

Seipp

Mueller

Pfaff

et al. 1997

Journal of General Virology

101

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Hepatitis C virus (HCV) is a major cause of chronic viral hepatitis. Development of anti-viral strategies has been hampered by the lack of efficient cell systems to propagate HCV in vitro. To establish a long-term culture system, we tested human hepatoma (HuH7, HepG2) and porcine non-hepatoma (PK15, STE) cell lines, as well as several culture and infection conditions. As a marker for virus replication, minus-strand HCV RNA in infected cells was detected by an enhanced detection system using nested RT-PCR followed by hybridization analysis. Short-term efficiency of HCV infection (10 days) was slightly increased by addition of polyethylene glycol (PEG) and/or dimethyl sulfoxide (DMSO) to culture media during inoculation of HuH7, PK15 and STE cells, but no augmentation in long-term culture was achieved, suggesting enhanced attachment of HCV to cells rather than more efficient infection. A

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Detection of genomic and intermediate replicative strands of hepatitis C virus in liver tissue by in situ hybridization.

Cited by 99 publications

References 36 publications

Dynamics of Hepatitis C Virus Replication in Human Liver

Dynamics of Hepatitis C Virus Replication in Human Liver

Viral kinetics during antiviral therapy in patients with chronic hepatitis C and persistently normal ALT levels

Establishment of persistent hepatitis C virus infection and replication in vitro.

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