Quantitation of hepatitis C virus RNA by competitive polymerase chain reaction

Kaneko, Shuichi; Murakami, Seishi; Unoura, Masashi; Kobayashi, Kenichi

doi:10.1002/jmv.1890370408

Cited by 100 publications

(41 citation statements)

References 19 publications

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“…HCV RNA titer was measured by competitive PCR. 24 When the titers were less than 10 3 genome Eq/mL, the presence of HCV RNA was examined by RT-nested PCR using the 5Ј UTR. 25 RNA Preparation, cDNA Synthesis, and PCR.…”

Section: Methodsmentioning

confidence: 99%

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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: Methodsmentioning

confidence: 99%

“…To clone the cDNA in the 5Ј UTR, we adopted the 5Ј rapid 24 HCV RNA, whose titer was less than 10 3 , was detected by using RT-nested PCR of the 5Ј noncoding region. 25 Arrows denote the times when plasma specimens were obtained for analysis of HCV RNA.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 1998

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“…The multiple regression analysis showed measured the amount of HCV in serum. 9,10,12,16,[18][19][20][21] Ideno effect of length of HCV exposure, risk factors, degree ally, HCV-RNA viral load should be estimated directly of bile duct damage, steatosis, or total Scheuer or Kno-from liver tissue itself, because serum levels of virus dell score on RNA levels. No significant confounding ef-may be contributed to and influenced by extrahepatic fect of HCV genotype on the degree of liver injury was sources.…”

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confidence: 99%

Intrahepatic hepatitis C RNA levels do not correlate with degree of liver injury in patients with chronic hepatitis C

et al. 1996

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jury. Intrahepatic viral load appears to be significantly The balance between a direct cytopathic effect by hepincreased in patients with genotype 1b. IFN-a treatment atitis C virus (HCV) and immune-mediated injury redoes significantly lower intrahepatic HCV load. (HEPAmains unclear. This report aims to test the following TOLOGY 1996;23:676-687.) hypotheses: (1) that intrahepatic HCV load would correlate with the degree of liver injury; (2) that interferon alfa (IFN-a) would decrease intrahepatic HCV-RNA levPersistent hepatitis C infection results in varying deels. Liver tissues (n Å 56) were obtained from 47 patients grees of chronic liver injury. [1][2][3][4][5][6] Healthy carriers of the with chronic HCV (9 before and after IFN-a therapy). hepatitis C virus (HCV) have been described but seem Total RNA was isolated and quantitated for specific HCV uncommon. 7 The pathogenesis of HCV-induced hepatic RNA by dot-blot polymerase chain reaction (DB-PCR) injury remains unclear and could be attributable to either using a standard curve created from synthetic HCV RNA direct cytopathic damage by HCV 8-10 or immune-mediof known titer to calculate actual RNA levels. A multivar-ated hepatic injury induced by HCV. 1,[11][12][13][14][15] It is possible iate analysis was undertaken to determine the relationthat both may act simultaneously. 16,17ship of intrahepatic HCV-RNA levels with risk factors,One way to identify whether liver damage is attributlength of HCV exposure, and histological injury scores.The confounding effect of HCV genotype was examined able to direct cytopathic damage is to examine whether by direct sequencing of the NS5b region. Liver HCV RNA the degree of viral load correlates with the degree of ranged from 10 2 to 3.1 1 10 7 molecules per microgram liver injury. Most studies addressing this question have total liver RNA. The multiple regression analysis showed measured the amount of HCV in serum. 9,10,12,16,[18][19][20][21] Ideno effect of length of HCV exposure, risk factors, degree ally, HCV-RNA viral load should be estimated directly of bile duct damage, steatosis, or total Scheuer or Kno-from liver tissue itself, because serum levels of virus dell score on RNA levels. No significant confounding ef-may be contributed to and influenced by extrahepatic fect of HCV genotype on the degree of liver injury was sources. Also, the presence of serum immune complexes observed. However, genotype 1b had a significantly may introduce further variables. Doubt has also been higher mean intrahepatic HCV-RNA load compared raised as to the validity of HCV-RNA quantitation in with the other genotypes detected. In the 9 patients who received IFN-a, treatment, 7 had no detectable HCV serum compared with plasma. after treatment. This was associated with a significantAs well as the sample type, the method of HCV quandecrease in intrahepatic HCV-RNA levels (7.57 { 2.53 titation itself is important. A number of techniques 1 10 5 to 1.82 { 1.80 1 10 3 molecules per microgram total have been reported for quantitation of HCV by...

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“…These data suggest that the ALTlevels alone do not yield an accurate assessment of the long-term efficacy of IFN. In recent years, an objective evaluation of the anti-viral effect of IFN has become possible by using quantitative analysis of the HCV RNA (5,6). In addition, it has been reported that the anti-HCV antibody titer is also clinically related to the therapeutic effect of IFN (7,8), and we have reported that the presence of HCV RNAis closely associated with that of anti-N14 antibody (9).…”

Section: Introductionmentioning

confidence: 98%

Hepatitis C Virus RNA and Anti-N14 Antibody Levels during Interferon Alpha Therapy for Chronic Hepatitis C.

Oketani

Tanaka

et al. 1994

Intern. Med.

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To investigate the markers useful for evaluating the long-term efficacy of interferon (IFN) therapy, the quantity of hepatitis C virus (HCV) RNAand two anti-HCV antibody titers (anti-N14 and anti-C-100-3 antibody) in 21 chronic hepatitis C patients were determined. In all complete responders, a sustained clearance of the virus and reductions in the anti-HCVantibody titers were observed during and after therapy. In most of the temporary responders, reductions in the HCV RNAlevels and in both anti-HCV antibody titers were observed temporarily during the therapy, and relapse followed. In nonresponders, although the HCVRNAlevels and anti-N14 antibody titer tended to remain unchanged or increased during and after therapy, the anti-C-100-3 antibody titers showed no tendency. These results demonstrate that the monitoring of the HCVRNAlevel and anti-N14 antibody titer is clinically useful for following the patient's response to IFN therapy for chronic hepatitis C. (Internal Medicine 33: 588-592, 1994)

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Quantitation of hepatitis C virus RNA by competitive polymerase chain reaction

Cited by 100 publications

References 19 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†

Intrahepatic hepatitis C RNA levels do not correlate with degree of liver injury in patients with chronic hepatitis C

Hepatitis C Virus RNA and Anti-N14 Antibody Levels during Interferon Alpha Therapy for Chronic Hepatitis C.

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