Detection by in situ hybridization of hepatitis C virus positive and negative RNA strands using digoxigenin-labeled cRNA probes in human liver cells

Gastaldi, Marguerite; Massacrier, Annick; Planells, Richard; Robaglia-Schlupp, Andrée; Portal-Bartolomei, Isabelle; Bourlière, Marc; Quilici, François; Fiteni, Joëlle; Mazzella, Evelyne; Cau, Pierre

doi:10.1016/0168-8278(95)80055-7

Cited by 36 publications

(14 citation statements)

References 41 publications

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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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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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“…In this study, both positive and negative HCV-RNA strands were detected in liver biopsies from patients positive for anti-HCV antibodies and negative for serum HCV-RNA using an ISH assay. Similarly, previous studies have demonstrated the presence of positive and negative HCV-RNA strands in hepatocytes from patients with chronic HCV-infection and with occult HCV-infection by ISH [19,20,25] . The hybridization signals were detected in the cytoplasm of hepatocytes.…”

Section: Discussionmentioning

confidence: 99%

“…Although the exact mechanism of HCV replication is still not fully known, there is some evidence indicating that the virus replicates through synthesis of a complementary negative-strand RNA intermediate (anti-genomic strand). Thus, the detection of negative HCV-RNA strand can be regarded as a marker of ongoing viral replication [19][20][21][22][23][24] . In this study, both positive and negative HCV-RNA strands were detected in liver biopsies from patients positive for anti-HCV antibodies and negative for serum HCV-RNA using an ISH assay.…”

Section: Discussionmentioning

confidence: 99%

Evidences of Hepatitis C Virus Replication in Hepatocytes and Peripheral Blood Monocuclear Cells from Patients Negative for Viral RNA in Serum

Falcón¹,

Acosta‐Rivero²,

Shibayama³

et al. 2005

American J. of Infectious Diseases

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Abstract:In this study, we examined the expression of hepatitis C virus (HCV) components in hepatocytes and peripheral blood mononuclear cells (PBMC) from patients positive for anti-HCV antibodies and negative for serum HCV-RNA. The samples obtained from 25 randomly selected patients (13 of 25 patients showed no histological evidences of chronic hepatitis and had normal serum ALAT and GGTP levels) were studied by in situ Hybridization and Immunofluorescence assays. The findings show that HCV-RNA of both positive and negative polarity was carried in the hepatocytes of more than 80% of cases. The proportion of cells expressing the negative strand (mean ± SD, 10.25% ± 5.56%) was lower than those expressing positive strand (mean ± SD, 19.88% ± 9.19%) (p=0.0076; Student's t test). In addition, reaction products suggestive of HCV core, E1 and E2 antigens within hepatocytes were also observed. Both hybridization and immunofluorescence signals were localized in the cytoplasm suggesting that this is the place of active HCV replication. On the other hand, the HCV-RNA of positive polarity was detected in PBMC from 16 out of 17 samples analyzed (94%) while the HCV-RNA of negative polarity was detected in 82% of samples investigated. Positive hybridization signals were localized in the cytoplasm of PBMC. Interestingly, 12 out of 13 patients with clinical and histological resolution of hepatitis, contain HCV-RNA in either liver or PBMC. These results provide further evidences that the intermediate replicative form of the HCV genome can persist in hepatocytes and PBMC after apparently complete resolution of chronic hepatitis C.

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“…Two main techniques exist that enable cellular localization of HCV; immunohistochemistry and in situ hybridization (ISH). Detection of HCV RNA by ISH has been reported [1][2][3][4][5][6][7] ; however, findings have been contradictory because of variable sensitivity. Because ISH also requires numerous tissue controls, it is a very expensive and labor-intensive technique.…”

confidence: 99%

Nonspecificity of monoclonal antibody tordji-22 for the detection of hepatitis C virus in liver transplant recipients with cholestatic hepatitis

Doughty

McCaughan

1999

Liver Transpl

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Detection of hepatitis C virus (HCV) antigens in liver tissue provides important diagnostic and pathological information. Limited studies have been performed on tissue taken after liver transplantation for HCV. In this study, serial post-liver transplantation biopsy tissue from patients with recurrent HCV was tested, with particular interest in patients showing severe cholestatic hepatitis. HCV-related antigens were detected using the commercial monoclonal antibody, Tordji-22. Initial results were promising, showing intense positive staining, especially in areas of hepatocyte ballooning. HCV-negative donor tissue was consistently negative by staining. However, as a final control for the level of tissue damage, HCV negative posttransplantation biopsy tissue showing hepatocyte ballooning was examined. These tissues also showed positive staining. All attempts to eliminate this nonspecific interaction failed. In conclusion, Tordji-22 was associated with nonspecificity in this posttransplantation population, and care is warranted when using this monoclonal antibody.

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Detection by in situ hybridization of hepatitis C virus positive and negative RNA strands using digoxigenin-labeled cRNA probes in human liver cells

Cited by 36 publications

References 41 publications

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

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

Evidences of Hepatitis C Virus Replication in Hepatocytes and Peripheral Blood Monocuclear Cells from Patients Negative for Viral RNA in Serum

Nonspecificity of monoclonal antibody tordji-22 for the detection of hepatitis C virus in liver transplant recipients with cholestatic hepatitis

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