TaqMan amplification system with an internal positive control for HCV RNA quantitation

Castelain, Sandrine; Descamps, Véronique; Thibault, Vincent; François, Clément; Bonte, Dorine; Morel, Vincent; Izopet, Jacques; Capron, Dominique; Zawadzki, Patricia; Duverlie, Gilles

doi:10.1016/j.jcv.2004.03.009

Cited by 74 publications

(52 citation statements)

References 30 publications

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“…We have exploited techniques that are widely used to determine the plasmatic load of some RNA viruses, such as HIV 28 or HCV, 29 or of prion protein. 30 One interesting finding of our study is the difference in day-to-day variability of SMN1-fl and SMN2-fl levels.…”

Section: Discussionmentioning

confidence: 99%

SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

Tiziano¹,

Pinto²,

Fiori³

et al. 2009

Eur J Hum Genet

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Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous mutations of the SMN1 gene. Three forms of SMA are recognized (type I-III) on the basis of clinical severity. All patients have at least one or more (usually 2-4) copies of a highly homologous gene (SMN2), which produces insufficient levels of functional SMN protein, because of alternative splicing of exon 7. Recently, evidence has been provided that SMN2 expression can be enhanced by pharmacological treatment. However, no reliable biomarkers are available to test the molecular efficacy of the treatments. At present, the only potential biomarker is the dosage of SMN products in peripheral blood. However, the demonstration that SMN full-length (SMN-fl) transcript levels are reduced in leukocytes of patients compared with controls remains elusive (except for type I). We have developed a novel assay based on absolute real-time PCR, which allows the quantification of SMN1-fl/SMN2-fl transcripts. For the first time, we have shown that SMN-fl levels are reduced in leukocytes of type II-III patients compared with controls. We also found that transcript levels are related to clinical severity as in type III patients SMN2-fl levels are significantly higher compared with type II and directly correlated with functional ability in type II patients and with age of onset in type III patients. Moreover, in haploidentical siblings with discordant phenotype, the less severely affected individuals showed significantly higher transcript levels. Our study shows that SMN2-fl dosage in leukocytes can be considered a reliable biomarker and can provide the rationale for SMN dosage in clinical trials.

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Section: Discussionmentioning

confidence: 99%

SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

Tiziano¹,

Pinto²,

Fiori³

et al. 2009

Eur J Hum Genet

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“…HCV RNA positive-and negative-strand-specific quantification was carried out by real-time PCR using TaqMan, as previously reported (31). The primer pair and the probe were located in the 5Ј HCV noncoding region (9). Briefly, 0.5 g of total RNA was used for cDNA synthesis in a 20-l reaction mixture containing 7.5 U of Thermoscript (Invitrogen) and 10 pmol of reverse transcription (RT) primer.…”

Section: Methodsmentioning

confidence: 99%

Identification of GBF1 as a Cellular Factor Required for Hepatitis C Virus RNA Replication

Goueslain

Alsaleh

Horellou

et al. 2010

J Virol

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125

157

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In infected cells, hepatitis C virus (HCV) induces the formation of membrane alterations referred to as membranous webs, which are sites of RNA replication. In addition, HCV RNA replication also occurs in smaller membrane structures that are associated with the endoplasmic reticulum. However, cellular mechanisms involved in the formation of HCV replication complexes remain largely unknown. Here, we used brefeldin A (BFA) to investigate cellular mechanisms involved in HCV infection. BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARF), which can lead to a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways. Our data show that HCV RNA replication is highly sensitive to BFA. Individual knockdown of the cellular targets of BFA using RNA interference and the use of a specific pharmacological inhibitor identified GBF1, a guanine nucleotide exchange factor for small GTPases of the ARF family, as a host factor critically involved in HCV replication. Furthermore, overexpression of a BFA-resistant GBF1 mutant rescued HCV replication in BFA-treated cells, indicating that GBF1 is the BFA-sensitive factor required for HCV replication. Finally, immunofluorescence and electron microscopy analyses indicated that BFA does not block the formation of membranous web-like structures induced by expression of HCV proteins in a nonreplicative context, suggesting that GBF1 is probably involved not in the formation of HCV replication complexes but, rather, in their activity. Altogether, our results highlight a functional connection between the early secretory pathway and HCV RNA replication.Hepatitis C virus (HCV) is an important human pathogen. It mainly infects human hepatocytes, and this often leads to chronic hepatitis, cirrhosis, or hepatocarcinoma. HCV studies have been hampered for many years by the difficulty in propagating this virus in vitro. Things have recently changed with the development of a cell culture model referred to as HCVcc (34,60,65), which allows the study of the HCV life cycle in cell culture and facilitates studies of the interactions between HCV and the host cell.HCV is an enveloped positive-strand RNA virus belonging to the family Flaviviridae (35). The viral genome contains a single open reading frame, which is flanked by two noncoding regions that are required for translation and replication. All viral proteins that are produced after proteolytic processing of the initially synthesized polyprotein are membrane associated (15,43). This reflects the fact that virtually all steps of the viral life cycle occur in close association with cellular membranes.Interactions of HCV with cell membranes begin during entry. Several receptors, coreceptors, and other entry factors have been discovered over the years, which link HCV entry to specialized domains of the plasma membrane, such as tetraspanin-enriched microdomains and tight junctions (8,16,59). The internalization of the viral particle...

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“…The HCV genotype was determined by direct sequencing of the 5Ј noncoding region (1: n ϭ 61, 2: n ϭ 10, 3: n ϭ 17, 4: n ϭ 3, 5: n ϭ 3), and the viral loads were determined by an in-house TaqMan assay. 28 They were expressed as the logarithmic values of IU/mL of HCV RNA (6.193 Ϯ 0.085 log IU/mL). The usual concentration of SAA in the sera of normal patients is estimated to be below 5 g/mL.…”

Section: Antiviral Activity Of Saa Can Be Attenuated In the Presence mentioning

confidence: 99%

Serum amyloid A has antiviral activity against hepatitis C virus by inhibiting virus entry in a cell culture system

et al. 2006

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Serum amyloid A (SAA) is an acute phase protein produced by the liver. SAA concentration increases markedly in the serum following inflammation and infection. Large increases in SAA concentration during the acute phase response suggest that SAA has a beneficial role in host defense. This study sought to determine the effect of SAA on hepatitis C virus (HCV) infectivity using retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the recently developed cell culture system for HCV (HCVcc). SAA inhibited HCVpp and HCVcc infection in a dose-dependent manner by affecting an early step of the virus life cycle. Further characterization with HCVpp indicated that SAA blocks virus entry by interacting with the viral particle. In addition, the antiviral activity of SAA was strongly reduced when high-density lipoproteins (HDL) were coincubated with SAA. However, HDL had only a slight effect on the antiviral activity of SAA when HCVpp was first preincubated with SAA. Furthermore, analyses of SAA in sera of chronic HCV patients revealed the presence of variable levels of SAA with abnormally elevated concentrations in some cases. However, no obvious clinical correlation was found between SAA levels and HCV viral loads. In conclusion, our data demonstrate an antiviral activity for SAA and suggest a tight relationship between SAA and HDL in modulating HCV infectivity. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/ jpages/0270-9139/suppmat/index.html).

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TaqMan amplification system with an internal positive control for HCV RNA quantitation

Cited by 74 publications

References 30 publications

SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

Identification of GBF1 as a Cellular Factor Required for Hepatitis C Virus RNA Replication

Serum amyloid A has antiviral activity against hepatitis C virus by inhibiting virus entry in a cell culture system

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