Evaluation of Hepatitis C Virus Core Antigen Assays in Detecting Recombinant Viral Antigens of Various Genotypes

Saeed, Mohsan; Suzuki, Ritsuro; Kondo, Madoka; Aizaki, Hideki; Kato, Takanobu; Mizuochi, Toshiaki; Wakita, Takaji; Watanabe, Haruo; Suzuki, Tetsuro

doi:10.1128/jcm.01437-09

Cited by 11 publications

(14 citation statements)

References 17 publications

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“…S4 in the supplemental material). These results are consistent with our previous study, which suggested that core Ag levels of HCV strain JFH-1 were underestimated by the Lumipulse-Ag kit in comparison to the ELISA-Ag assay (28). Strain JFH-1 harbors an Ala-to-Thr substitution at aa 48; conversion of Thr to Ala at this position in JFH-1 was sufficient to overcome this underestimation.…”

Section: Discussionsupporting

confidence: 82%

“…Although core Ag levels are thought to be related closely to HCV RNA titers, the correlation and linearity of core Ag levels have not yet been fully evaluated. In addition, these quantitative parameters are known to be affected by nucleotide and amino acid sequences at the target regions of the assays (5,6,28,34), and this sequence variation depends on genotypes or predominant strains in specific geographical regions.…”

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confidence: 99%

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Japanese Reference Panel of Blood Specimens for Evaluation of Hepatitis C Virus RNA and Core Antigen Quantitative Assays

et al. 2012

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An accurate and reliable quantitative assay for hepatitis C virus (HCV) is essential for measuring viral propagation and the efficacy of antiviral therapy. There is a growing need for domestic reference panels for evaluation of clinical assay kits because the performance of these kits may vary with region-specific genotypes or polymorphisms. In this study, we established a reference panel by selecting 80 donated blood specimens in Japan that tested positive for HCV. Using this panel, we quantified HCV viral loads using two HCV RNA kits and five core antigen (Ag) kits currently available in Japan. The data from the two HCV RNA assay kits showed excellent correlation. All RNA titers were distributed evenly across a range from 3 to 7 log IU/ml. Although the data from the five core Ag kits also correlated with RNA titers, the sensitivities of individual kits were not sufficient to quantify viral load in all samples. As calculated by the correlation with RNA titers, the theoretical lower limits of detection by these core Ag assays were higher than those for the detection of RNA. Moreover, in several samples in our panel, core Ag levels were underestimated compared to RNA titers. Sequence analysis in the HCV core region suggested that polymorphisms at amino acids 47 to 49 of the core Ag were responsible for this underestimation. The panel established in this study will be useful for estimating the quality of currently available and upcoming HCV assay kits; such quality control is essential for clinical usage of these kits.

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Section: Discussionsupporting

confidence: 82%

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confidence: 99%

Japanese Reference Panel of Blood Specimens for Evaluation of Hepatitis C Virus RNA and Core Antigen Quantitative Assays

et al. 2012

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“…9B). This discrepancy may be due to the lower detection efficiency of the JFH-1 core protein in the immunoassay, as reported previously (31). Infectivity in the culture medium was also determined.…”

Section: Resultsmentioning

confidence: 92%

“…Synthesized HCV RNA (10 g) was transfected into Huh-7.5.1 or Huh7-25 cells. HCV core protein levels in the culture medium were measured by immunoassay (31). HCV RNA levels in the culture medium were quantified as described above.…”

Section: Figmentioning

confidence: 99%

Novel Cell Culture-Adapted Genotype 2a Hepatitis C Virus Infectious Clone

Date

Kato

Katô

et al. 2012

J Virol

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Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones. Hepatitis C virus (HCV) is a principal agent in posttransfusion and sporadic acute hepatitis (6,19). HCV belongs to the Flaviviridae family and Hepacivirus genus. Infection with HCV leads to chronic liver diseases, including cirrhosis and hepatocellular carcinoma (16). HCV is a major public health problem, infecting an estimated 170 million people worldwide (6,16,19). Current standard therapy for HCV-related chronic hepatitis is based on the combination of interferon (IFN) and ribavirin although virus eradication rates are limited to around 50% (7,24,30). Telaprevir and boceprevir were approved by the U.S. Food and Drug Administration in 2011 in combination with pegylated alpha interferon and ribavirin for the treatment of genotype 1 chronic hepatitis C (34, 35). Both agents inhibit the NS3-NS4A serine protease essential for replication of HCV (25,36). It is important to develop more anti-HCV drugs with different modes of action to achieve greater efficacy and to avoid the emergence of drug-resistant viruses. To that end, a detailed understanding of the viral replication mechanism is needed to discover novel antiviral targets. An efficient virus culture system is indispensable for detailed analysis of HCV life cycles. In an important development, a subgenomic HCV RNA replicon system has been developed (22) to assess HCV replication in cultured cells. Furthermore, an efficient HCV culture system was established by using a JFH-1 strain virus isolated from a fulminant hepatitis patient (20,38,41). By transfection of in vitro transcribed full-length JFH-1 HCV RNA into HuH-7 cells, efficient JFH-1 RNA replication and infectious viral particle production were detected. However, this effic...

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“…The resultant fragments were cloned into pCAGGS. Plasmids for expression of FLAG-tagged core derives from genotype 1a, 2a and 3a were described previously (Saeed et al, 2009). Pol I-driven HCV genome plasmid (genotype 2a) pHHJFH1am was described previously (Suzuki et al, 2012).…”

Section: Methodsmentioning

confidence: 99%

Single-domain intrabodies against hepatitis C virus core inhibit viral propagation and core-induced NFκB activation

Suzuki

Saito

Matsuda

et al. 2016

Journal of General Virology

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Hepatitis C virus (HCV) core plays a key role in viral particle formation and is involved in viral pathogenesis. Here, constructs for single-domain intrabodies consisting of variable regions derived from mouse mAbs against HCV core were established. Expressed single-domain intrabodies were shown to bind to HCV core, and inhibit the growth of cell culture-produced HCV derived from JFH-1 (genotype 2a) and a TH (genotype 1b)/JFH-1 chimera. Adenovirus vectors expressing intrabodies were also capable of reducing HCV propagation. Intrabody expression did not affect viral entry or genome replication of single-round infectious trans-complemented HCV particles. However, intrabody expression reduced intracellular and extracellular infectious titres in CD81-defective Huh7-25 cells transfected with the HCV genome, suggesting that these intrabodies impair HCV assembly. Furthermore, intrabody expression suppressed HCV core-induced NFk B promoter activity. These intrabodies may therefore serve as tools for elucidating the role of core in HCV pathogenesis.

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Evaluation of Hepatitis C Virus Core Antigen Assays in Detecting Recombinant Viral Antigens of Various Genotypes

Cited by 11 publications

References 17 publications

Japanese Reference Panel of Blood Specimens for Evaluation of Hepatitis C Virus RNA and Core Antigen Quantitative Assays

Japanese Reference Panel of Blood Specimens for Evaluation of Hepatitis C Virus RNA and Core Antigen Quantitative Assays

Novel Cell Culture-Adapted Genotype 2a Hepatitis C Virus Infectious Clone

Single-domain intrabodies against hepatitis C virus core inhibit viral propagation and core-induced NFκB activation

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