Detection of intrahepatic hepatitis C virus replication by strand-specific semi-quntitative RT-PCR: preliminary application to the liver transplantation model

Negro, Francesco; Giostra, Emiliano; Krawczynski, Krzysztof; Quadri, Rafael; Rubbia‐Brandt, Laura; Mentha, Gilles; Colucci, Giuseppe; Perrin, Luc; Hadengue, Antoine

doi:10.1016/s0168-8278(98)80172-4

Cited by 55 publications

(71 citation statements)

References 41 publications

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“…The ratio of genomic RNA to replicative-intermediate RNA ranged from 3 to 20 in the seven cases studied, consistent with quantitative reports for flavivirus and HCV using other methods. 28,29 The total hepatic load for the HCV genome ranged from 1.8 ϫ 10 11 to 1.8 ϫ 10 12 RNA molecules among the cases studied. The estimated percentage of cells containing active HCV infection (ie, replicative-intermediate RNA) was 6, 11, 4, 12, 21, 21, and 25 for specimens A-1, A-2, B-1, B-2, C, D, and E, respectively.…”

Section: Estimation Of Hepatic Load Of Hcv Rna Moleculesmentioning

confidence: 96%

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: Estimation Of Hepatic Load Of Hcv Rna Moleculesmentioning

confidence: 96%

Dynamics of Hepatitis C Virus Replication in Human Liver

Chang

Williams

Mittler

et al. 2003

The American Journal of Pathology

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“…16,17 Using a semiquantitative strand-specific reverse transcriptase-polymerase chain reaction in 23 liver transplant recipients with HCV infection, Negro et al were able to detect intrahepatic negative-strand HCV RNA as early as 7 days after liver transplantation. 17…”

Section: The Impact Of Liver Transplantation On Levels Of Hepatitis Cmentioning

confidence: 99%

“…24 Sreekumar et al 24 found that although HCV RNA levels continue to decline from peak values, they remain significantly higher than pretransplantation values throughout the posttransplantation period. Although the correlation of pretransplantation and posttransplantation HCV RNA levels has been inconsistent, 17 a high pretransplantation viral load (Ͼlog MEq/mL, a level previously associated with increased mortality and graft loss 8 ) has been significantly associated with greater mean posttransplantation HCV RNA levels in serum at 4 months than lower pretransplantation levels (log 0.67 versus 1.67 MEq/mL, r ϭ 0.51, P ϭ .01). 24 Tissue HCV RNA levels are on average 50-to 100-fold greater than serum levels and correlate strongly with serum levels (r ϭ 0.75 to 0.89, P Յ .01).…”

Section: Medium and Late Posttransplantation Kineticsmentioning

confidence: 99%

Liver biopsy, viral kinetics, and the impact of viremia on severity of hepatitis C virus recurrence

Charlton

2003

Liver Transplantation

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A lthough the impact of hepatitis C virus (HCV) infection on allograft histology varies substantially, 1-7 allograft failure secondary to recurrence of HCV is the most common cause of death and retransplantation among recipients with HCV. 8 In HCV and in other viral infections, including HIV 9 and hepatitis B, 10 analysis of viral dynamics has not only been helpful in understanding pathogenesis of infection but also in determining responsiveness to therapies. To date there have been relatively few studies of detailed viral kinetics after liver transplantation.All of the HCV kinetics studies that have been carried out to date have been in the nontransplantation setting and have relied on indirect measures of viral turnover incorporating mathematical models that are dependent on assumptions regarding the number of infected hepatocytes, interferon mechanisms of action, and efficacy. [11][12][13][14][15] Although these studies have undoubtedly provided insight into HCV kinetics, the reported half life (t 1/2 ) of hepatitis C virions has varied greatly among studies. Neumann et al reported the virion t 1/2 to be 2.7 hours, with production and clearance rates of 10 12 virions/day. 12 In contrast, Zeuzem et al reported the t 1/2 of hepatitis C to be 64.8 hours and clearance rates to be 6.7 ϫ 10 10 virions/day. 14 Both the Neumann and Zeusem manuscripts include discussions of the limitations of the techniques and models that were used, including the inability to discriminate between inhibition of HCV uptake into hepatocytes and inhibition of HCV RNA replication in de novo infected cells. In addition, all reports of HCV dynamics have relied on the serial determination of HCV RNA levels as a surrogate of HCV synthesis. Inherent to these methods is the unproven and unlikely assumption that viral synthesis is completely inhibited after initiation of interferon therapy. These limitations notwithstanding, important insights into the natural history of posttransplantation HCV infection can be distilled from the analysis of posttransplantation viral kinetics. The Impact of Liver Transplantation on Levels of Hepatitis C Viremia Early KineticsAlthough the interval between liver transplantation and clinical allograft infection varies, negative-strand HCV RNA, the putative sine que non of HCV replication, has been detected very early in the posttransplantation course. 16,17 Using a semiquantitative strand-specific reverse transcriptase-polymerase chain reaction in 23 liver transplant recipients with HCV infection, Negro et al were able to detect intrahepatic negative-strand HCV RNA as early as 7 days after liver transplantation. 17

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“…Intrahepatic genomic-and minus-strand HCV-RNA titers were normalized to an arbitrary ␤-actin mRNA titer of 1,024, as measured on the same specimen. 11,15 The intrahepatic ␤-actin titer was expressed as the last fourfold dilution giving a visible band by ethidium bromide staining of RT-PCR products obtained from 100 ng of total liver RNA and run on a 1.6% agarose gel.…”

mentioning

confidence: 99%

Detection of genomic- and minus-strand of hepatitis C virus RNA in the liver of chronic hepatitis C patients by strand-specific semiquantitative reversetranscriptase polymerase chain reaction

Negro

Krawczynski

Quadri³

et al. 1999

Hepatology

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Studies aimed at correlating the intrahepatic hepatitis C virus (HCV)-RNA level and anatomo-clinical features have been difficult because of sensitivity and specificity shortcomings of available techniques. We titered the genomic-and minus-strand HCV RNAs by a strand-specific, semiquantitative, genotype-independent reverse-transcriptase polymerase chain reaction (RT-PCR) in the liver tissue of 61 patients with chronic hepatitis C. Findings were correlated with the levels of HCV RNA in the serum, the HCV genotype, the expression of intrahepatic HCV antigens, the histological activity (using separate scores for the lobular and the portal/periportal necroinflammatory activity and for the fibrosis), and the response to interferon alfa (IFN-␣) treatment. Genomic-and minus-strand HCV RNA were detected in 59 and 57 liver specimens, respectively. The pathogenesis of the hepatitis C virus (HCV)-associated liver disease is believed to be mainly mediated by the immune system. 1 However, some studies have shown a direct correlation between the viremia level and the hepatitis activity, 2-6 thus suggesting a direct cytopathogenic effect of HCV on hepatocytes. Ideally, the correlation between HCV replicative levels and pathological findings (as well as with other clinical features) should rely on the quantitation of HCV RNA (or antigens) in the liver. Unfortunately, correlative studies between the semiquantitation of HCV replicative levels in the liver and the disease activity have been difficult because of the technical shortcomings of the published techniques. In particular, the detections of HCV RNA by commercially available (semi)quantitative assays have some limitations as to their sensitivity or variation coefficient, 7,8 and may amplify different viral genotypes with a different efficiency. 9 Moreover, all diagnostic assays are aimed at detecting the genomicstrand HCV RNA; therefore, they may also detect the virions' RNA trapped in the liver biopsy specimens at the time of sampling. On the other hand, the specificity of the detection of the minus-strand HCV RNA (the putative HCV replication intermediate RNA) has been repeatedly questioned. 10 Suitably modified reverse-transcriptase polymerase chain reaction (RT-PCR)-based assays of sufficient specificity are now available 10 and can be applied to the analysis of clinical specimens.We have previously detected the intrahepatic minus-strand HCV RNA by a semiquantitative, strand-specific RT-PCR in liver transplant recipients. 11 In that study, we showed a lack of correlation between the HCV-RNA level in the serum, the genomic-and the minus-strand HCV-RNA titers in the liver on the one hand, and the histological grading and staging of the liver disease on the other. 11 In the present work, we applied the same assay to a series of liver biopsy specimens taken from chronic hepatitis C patients. Our aim was to assess whether clinically significant correlations could be established between the intrahepatic titers of either strand of HCV RNA and some histopathological and clinica...

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Detection of intrahepatic hepatitis C virus replication by strand-specific semi-quntitative RT-PCR: preliminary application to the liver transplantation model

Cited by 55 publications

References 41 publications

Dynamics of Hepatitis C Virus Replication in Human Liver

Dynamics of Hepatitis C Virus Replication in Human Liver

Liver biopsy, viral kinetics, and the impact of viremia on severity of hepatitis C virus recurrence

Detection of genomic- and minus-strand of hepatitis C virus RNA in the liver of chronic hepatitis C patients by strand-specific semiquantitative reversetranscriptase polymerase chain reaction

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