Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells.

Lerat, Hervé; Berby, Françoise; Trabaud, Mary‐Anne; Vidalin, Olivier; Major, Marian E.; Trépo, C.; Inchauspé, Geneviève

doi:10.1172/jci118485

Cited by 260 publications

(201 citation statements)

References 36 publications

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“…Indeed, the possibility still exists that the detection of the negativestranded HCV RNA is an artifact caused by the high titer of positive-stranded HCV RNA in the sample. 53 In this experiment, the titer of positive sense HCV RNA at 4, 5, and 14 weeks posttransfusion were 10 7 ,10 6 , and 10 6 , respectively. However, a strong band was detected only at 5 weeks posttransfusion in spite of the lower titer than that at 4 weeks posttransfusion.…”

Section: Discussionmentioning

confidence: 68%

Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample†

et al. 1998

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We constructed a full-length complementary DNA (cDNA) clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier. The blood from the carrier was eventually transfused to a patient who later developed typical posttransfusion hepatitis C. It was also shown to be infectious to chimpanzees. We obtained 12 overlapping cDNA fragments altogether, covering the entire HCV genome. By subcloning and sequencing, clones considered to constitute the major population were selected. We could also detect 98 base pairs of extra sequences at the 3Ј end of the genome. After confirming the overlapping sequences, we combined the fragments to make a full-length cDNA. The HCV population in the donor was heterogeneous, as determined by their nucleotide sequences of the hypervariable region in envelope protein, but a few virus clones were selected in the recipient after transmission. The similar convergence of the virus population was previously observed when the same blood sample was injected into a chimpanzee. Interestingly, virus clones isolated during the acute phase in the recipient and the chimpanzee had sequences in the hypervariable region identical to that of the full-length cDNA clone. The full-length cDNA clone of HCV constructed in this study may originate from infectious virus clones.(HEPATOLOGY 1998;27:621-627.)Comparative study of the amino acid sequence of hepatitis C virus (HCV) with those of flaviviruses and pestiviruses and gene expression experiments in bacteria, yeast, and animal cells have revealed that the proteins of HCV are processed by a host-derived signalase and cleaved by virus-coded proteases. [1][2][3][4][5] In vitro culture systems that support partial replication of this virus have been developed from human T-and B-cell lines, 6,7 human fetal hepatocytes, 8 chimpanzee hepatocytes, 9 and human primary hepatocytes. 10 However, fully efficient, long-term viral replication has not yet been established. To study the biology of HCV and its pathogenesis, it is essential to establish an efficient in vitro cell culture system or an infectious complementary DNA (cDNA) clone to support the complete replication of HCV.As a first step to construct an infectious cDNA clone of HCV, it is important to select an adequate sample containing infectious HCV. To date, a considerable number of ''complete'' clones of the HCV genome have been reported. However, it is not certain that those clones have truly originated from infectious HCV, because the materials used usually were pooled plasma samples. Furthermore, the plasma of HCV carriers or patients is generally composed of quasispecies of HCV population. 11 To construct an infectious cDNA clone of RNA viruses, it is crucial to retain both 5Ј and 3Ј ends of the sequence, which are highly conserved and are considered to play essential roles for RNA synthesis, transcription, and translation. 12 Many have reported that the 3Ј untranslated region (UTR) of HCV consisted of poly(U) [13][14][15][16] or poly(A) 17 homopolymer tracts. A novel 98-nucleotide (nt) sequence down...

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Section: Discussionmentioning

confidence: 68%

Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample†

et al. 1998

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“…To assess the active replication of HCV demonstrated by the detection of negative sense molecules, and to avoid false-positive results caused by the lack of specificity of the techniques using primers from the 5Ј-noncoding region, reverse transcription and amplification of negative-strand templates were performed using capsid-derived specific primers, according to recent reported findings. 21 cDNAs were synthetized with specific primer CAP 3 (the samples were heated at 100°C for 1 hour before the PCR), and amplified with the outer primers during the first PCR: CAP 3 (5Ј TGT TGC CGC GCA GGG GCCC 3Ј) (position 448-466) and 185 (5Ј CCG CAC GTA GGG TAT CGA TGA C 3Ј) (position 732-751), then with the inner primers during the second PCR: CAP 4 (5Ј AGG TTG GGT GTG CGC GCG 3Ј) (position 468-485) and 186 (5Ј ATG TAC CCC ATG AGG TCG GC 3Ј) (position 705-728). The expected size of the amplification product is 260 bp.…”

Section: Methodsmentioning

confidence: 99%

“…This result agrees with previous observations from the literature that showed that self-priming of HCV RNA leads to false-positive results for HCV-RNA negativestrand detection mostly in patient samples with a high viral load. 21 Serial Inoculations. To perform the ''blood passage,'' blood samples were taken from 2 mice (injected with ALL cells in 1 case and with PBMC from a patient with chronic hepatitis C in the other case) at the moment of their killing, and then were reinjected subsequently into 2 other mice.…”

Section: Hcv-rna Negative-strand Detection In Circulating Cells Isolatedmentioning

confidence: 99%

Hepatitis C virus persistence in human hematopoietic cells injected into SCID mice

et al. 1998

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The issue of infection of peripheral blood mononuclear cells (PBMC) by the hepatitis C virus (HCV) has potentially important implications, but is still debated. We have used the severe combined immunodeficiency (SCID) mouse model to test for the persistence of HCV in PBMC. Hematopoietic cells isolated from 14 subjects infected by HCV were inoculated intraperitoneally into SCID mice. Serum and blood cell samples from these mice were obtained with a mean follow-up of 8 weeks. As controls, human fibroblasts and sheep PBMC, preincubated with a human HCV-positive serum, were inoculated concomitantly into mice and analyzed. HCV-RNA positive strands were detected in 7 of 26 serum samples and 8 of 26 cell fractions from SCID mice inoculated with HCV-positive PBMC, after 8 weeks of follow-up. In contrast, no HCV RNA was detectable in the 10 control mice. HCV-RNA negative strands were detected in only 2 of 10 tested samples from 2 mice, and both positive mice had been inoculated with PBMC from HCVpositive subjects with malignant hematopoietic syndrome. Our study offers strong evidence for the persistence of HCV infection in mononuclear cells. Our results are also consistent with a low rate of HCV multiplication. This SCID mouse model might therefore be useful in analyzing the mechanisms of HCV persistence in mononuclear cells. (HEPATOLOGY 1998;28:211-218.)The development of a chronic carrier state is a main feature of infection by the hepatitis C virus (HCV), and in fact, up to 60% to 70% of acutely infected patients show chronic infection during follow-up. The mechanisms involved are not known. It has been shown that chronic infection develops despite a strong and polyclonal humoral response to several structural and nonstructural viral proteins. In addition, a proliferative CD4-and CD8-positive T-cell response against various HCV proteins can also be detected among circulating or liver-derived mononuclear cells. 1,2 However, it is not known to what extent these immunologic parameters correlate with the outcome of the viral infection. HCV, as an RNA virus and a member of the Flaviviridae, shows marked genetic variability, and it has been proposed that this variability might favor its escape from the immune response. All the same, despite the evidence that supports this possibility, at least for the humoral response, the actual impact of this variability in viral persistence remains to be established (for review, see Bukh et al., 3 Simmonds, 4 and Bréchot 5 ).Another intriguing feature of HCV infection regards the clinical and virological profiles of infections by different genotypes of HCV. 6,7 Infection of mononuclear cells by HCV might be a further important parameter to be considered in the pathogenesis of HCV infection. Positive-strand HCV RNA has indeed been detected by reverse-transcription polymerase chain reaction (RT-PCR) in peripheral blood mononuclear cells (PBMC) in several studies investigating patients at various stages of the viral infection. [8][9][10][11][12][13][14][15][16][17][18][19][20][21] Furthermo...

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“…Numerous publications have analyzed extrahepatic compartments of viral replication that could potentially contribute to plasma viremia, most frequently peripheral blood mononuclear cells (PBMC). [7][8][9][10][11][12][13][14] The contribution of PBMC-based HCV replication to total viremia remains unclear, however, as the level of negative-strand HCV RNA in PBMC is very low with respect to that in liver. [15][16][17][18] The continued presence of viral RNA in the PBMC of subjects who had either spontaneously cleared their plasma viremia or cleared viremia following antiviral therapy has recently been reported, raising concerns that PBMC may serve as a long-lived HCV reservoir capable of rekindling systemic infection.…”

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confidence: 99%

Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia

et al. 2008

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We determined whether hepatitis C virus (HCV) RNA could be detected associated with peripheral blood mononuclear cells (PBMC) of seropositive blood donors who had spontaneously or therapeutically cleared their plasma viremia. Blood donor plasma viremia status was first determined with a highly sensitive transcription-mediated amplification (TMA) test performed in duplicate assays. PBMC from 69 aviremic and 56 viremic blood donors were then analyzed for the presence of HCV RNA with TMA adapted to detect viral RNA in PBMC and with a reverse transcription-nested polymerase chain reaction assay. PBMCassociated HCV RNA was detected in none of the 69 aviremic donors, including all 6 subjects with a sustained viral response following antiviral therapy. PBMC-associated HCV RNA was detected in 43 of the 56 viremic donors. The 13 viremic donors with no detectable PBMCassociated HCV RNA all had very low viral loads (6 positive only in 1 of 2 duplicate plasma TMA assays, 6 with viral loads below 100 HCV RNA copies/mL, and 1 with a viremia of 2700 HCV RNA copies/mL). The absence of detectable PBMC HCV RNA detection in all 69 aviremic donors reported here contrasts with prior studies, possibly as a result of the higher sensitivity of the TMA assay used to test for plasma viremia. Conclusion: Our results indicate that PBMC are unlikely to serve as a long-lived reservoir of HCV in aviremic subjects.

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Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells.

Cited by 260 publications

References 36 publications

Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample†

Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample†

Hepatitis C virus persistence in human hematopoietic cells injected into SCID mice

Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia

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