Specific detection of positive and negative stranded hepatitis C viral RNA using chemical RNA modification

Gunji, Toshiaki; Kato, Naoya; Hijikata, Makoto; Hayashi, Kohei; Saitoh, Shu‐ichi; Shimotohno, Kunitada

doi:10.1007/bf01310568

Cited by 85 publications

(58 citation statements)

References 17 publications

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“…In addition, an RT reaction was carried out in the absence of the antisense primer with Superscript II after chemical modification of RNA to analyze the specificity of negative-stranded HCV RNA. 32 RT reaction was conducted by use of the primer La3 (for positive-stranded RNA) and primer Ls1 (for negative-stranded RNA) (Fig. 2A).…”

Section: Determination Of the 3ј Extra Sequence In The Plasma Of Donomentioning

confidence: 99%

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: Determination Of the 3ј Extra Sequence In The Plasma Of Donomentioning

confidence: 99%

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

et al. 1998

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“…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] Furthermore, it was possible in some cases to detect the negative strand of HCV RNA, and thus to suggest that HCV RNA might replicate in these cells. [11][12][13][14][15][16][17][18][19][20][21] These findings were further substantiated by the detection of HCV genomes in PBMC and liver mononuclear cells by using in situ hybridization.…”

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confidence: 99%

“…Several investigators have now emphasized the risk of false-positive results when detecting negative HCV-RNA strands by PCR. 20,21,[24][25][26] Moreover, detection of HCV-RNA positive strands cannot be taken as firm evidence of PBMC infection because of the obvious risk of viral particle adsorption into mononuclear blood cells. We have therefore hypothesized that using the severe combined immunodeficiency (SCID) mouse model 27 might be of value to clarify some of these issues for the following reason: It has been demonstrated that this model allows long-term survival of human hematopoietic cells.…”

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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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“…The primer-dependent mechanism for RT reaction has been generally accepted over the years. However, during RT-PCR analysis of human and animal RNA viruses, bacterial operons, as well as eukaryotic cellular RNAs, it has been observed that cDNA could be synthesized by reverse transcriptase without the addition of exogenous oligonucleotide (Gunji et al, 1994;Lanford et al, 1995;Lerat et al, 1996;Schoenike et al, 1999;Guacucano et al, 2000;Peyrefitte et al, 2003;Haddad et al, 2007;Tuiskunen et al, 2010, Moison et al, 2011. This completely contradicts the well-accepted mechanism of cDNA synthesis, indicating that RT occurs in a primer-independent manner.…”

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confidence: 52%

“…However, many lines of evidence supported the possibility that this kind of cDNA synthesis is most likely primed by cellular small nucleic acids (DNA, microRNA, tRNA, etc. ) associated with the commercial reverse transcriptase as well as template RNA, or by the thermosTable hairpin structure at the 3'-end of the template RNA, the so called self-priming (Agranovsky, 1992;Gunji et al, 1994;Lerat et al, 1996;Timofeeva and Skrypina, 2001;Piche and Schernthaner, 2003;Haddad et al, 2007;Tuiskunen et al, 2010;Moison et al, 2011). Regardless, since the primer-independent RT usually contributes to non-specific cDNA synthesis that may interfere with the PCR specificity, thus resulting in misinterpretation of the final experimental data, much effort has been devoted to overcoming this unexpected event (Lerat et al, 1996;Peyrefitte et al, 2003;Haddad et al, 2007;Moison et al, 2011).…”

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confidence: 99%

Self-priming on the plant viral RNAs during reverse transcription-PCR

Zhang¹,

Wu²,

Zhang³

et al. 2015

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Summary. -The occurrence of the primer-independent cDNA synthesis during RT-PCR analysis of human and animal RNA viruses has been well documented. Conversely, there is scant knowledge about this event in plant RNA viruses. Here we show that the primer-independent cDNA synthesis occurs in all eight different plant RNA viruses tested in this study, suggesting a common phenomenon for RT-PCR analysis of plant RNA viruses. Additional experiments indicate that the event is likely contributed to by RNA self-priming, and can be effectively reduced or eliminated through increasing temperature of the RT reaction.Keywords: RT-PCR; primer-independent cDNA synthesis; self-priming; plant RNA virus; RT temperature * Corresponding author. E-mail: liweimin01@caas.cn; phone: +86-10-82106117. C. Zhang. and H.N. Wu contributed equally to this work. Abbreviations: CGMMV = cucumber green mottle mosaic virus; CMV = cucumber mosaic virus; gRNA = genomic RNA; ORSV = odontoglossum ring-spot virus; PVX = potato virus X; TCV = turnip crinkle virus; TMV U1 = tobacco mosaic virus U1 strain; TNV = tobacco necrosis virus; TRV = tobacco rattle virus RT-PCR is one of the most common methods of molecular biology used to detect and quantify RNA expression levels in both cells and small quantities of tissues. Within this process, RT is the first and fundamental step, in which the RNA template is converted into its cDNA by reverse transcriptase in the presence of exogenous oligonucleotide referred to as primer, providing template DNA for subsequent PCR amplification. The primer-dependent mechanism for RT reaction has been generally accepted over the years. However, during RT-PCR analysis of human and animal RNA viruses, bacterial operons, as well as eukaryotic cellular RNAs, it has been observed that cDNA could be synthesized by reverse transcriptase without the addition of exogenous oligonucleotide (Gunji et al

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Specific detection of positive and negative stranded hepatitis C viral RNA using chemical RNA modification

Cited by 85 publications

References 17 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

Self-priming on the plant viral RNAs during reverse transcription-PCR

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