Molecular cloning of hepatitis C virus genome from a single Japanese carrier: sequence variation within the same individual and among infected individuals

Tanaka, Torahiko; Kato, Naoya; Nakagawa, Masanori; Ootsuyama, Yuko; Cho, Myung‐Je; Nakazawa, Takahiro; Hijikata, Makoto; Ishimura, Yuzuru; Shimotohno, Kunitada

doi:10.1016/0168-1702(92)90066-i

Cited by 134 publications

(1 citation statement)

References 14 publications

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“…An approximately 5–8% difference at the nucleotide level has been observed within a single genotype [3]. Furthermore, an approximately 1% difference at the nucleotide level is also observed among HCV genomes in an individual [13]. Although genetic analyses of HCV using in vivo specimens have estimated that the genetic mutation rate of HCV is 1.4–1.9×10 −3 base substitutions/site/year [14]–[16], the potential variability of HCV is not clear due to the selective pressure of immune system functions in vivo [17], [18].…”

Section: Introductionmentioning

confidence: 92%

Genetic Characterization of Hepatitis C Virus in Long-Term RNA Replication Using Li23 Cell Culture Systems

et al. 2014

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BackgroundThe most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells.Methodology/Principal FindingsLi23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 5′-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a time-dependent manner, and the mutation rates in the 5′-terminus-NS2 and NS3-NS5B regions were 4.0–9.0×10−3 and 2.7–4.0×10−3 base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3–105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents.Conclusions/SignificanceLong-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis.

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Section: Introductionmentioning

confidence: 92%

Genetic Characterization of Hepatitis C Virus in Long-Term RNA Replication Using Li23 Cell Culture Systems

et al. 2014

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Genotypes of hepatitis C virus in Nigeria

Oni

Harrison

1996

J. Med. Virol.

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A pilot survey of hepatitis C virus (HCV) infection in Nigeria was carried out on healthy adult blood donors and children of preschool age. Sixteen of 200 (8%) donors were positive for antibodies using a second generation enzyme-linked immunosorbent assay (ELISA) but all of the children were negative. Supplementary testing of the ELISA-positives using a recombinant immunoblot assay (RIBA-2) confirmed the presence of antibody in four and two others were indeterminate. Four of the anti-HCV-positive sera and one found positive by ELISA but which was negative by RIBA-2 were found to be positive for HCV RNA using reverse transcriptase-polymerase chain reaction (RT-PCR) and primers specific for the 5' untranslated region (5'UTR) of the HCV genome. The NS5 and core regions also were amplified and the PCR products from all three regions were sequenced. Sequences from the 5'UTR could be divided into two groups: one group comprised three isolates with greater than 95% sequence identity with published sequences of genotype 1 and the other comprised two isolates with greater than 93% sequence identity with genotype 4. Analysis of three sequences amplified from the NS5 region confirmed this assignment to genotypes 1 and 4. Pairwise comparisons of the NS5 region sequences with representatives of 1a, 1b, 1c (for the first group) and 4a-4h (for the second group) show the first group to include subtypes classifiable as 1a and a novel sequence and the second group to include a novel sequence within genotype 4. Sequence analysis of the core region was consistent with this interpretation. These data confirm the presence of at least two major HCV genotypes in Nigeria (genotypes 1 and 4) and we report two novel sequences which have been designated provisionally as genotypes 1d and 4i.

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Analysis of mother-to-infant transmission of hepatitis C virus: Quasispecies nature and buoyant densities of maternal virus populations

et al. 1997

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Mother-to-infant transmission of hepatitis C virus (HCV) was analyzed by sequencing of viral RNA and semiquantitative polymerase chain reaction following ultracentrifugation of maternal sera. In two mother-infant pairs, the hypervariable region 1 (HVR1) and carboxyl terminus of envelope 1 (E1) were sequenced. Both viral sequences in the infants were less diverse than those of their mothers. Although the E1 sequences were almost identical in each mother-infant pair, the HVR1 sequences of the infants were related, but not identical, to those of the mothers. Serial examinations of one infant revealed that the HVR1 nucleotide sequence did not change from 10 days to 3 months of age. In six mothers with uninfected infants, all of the dense fractions of sera contained significant amounts of HCV RNA, whereas in six mothers with infected infants, only two of those fractions contained significant amounts of HCV RNA. These results indicate that the strains of HCV detected in the infants were not dominant in the mothers, but were still transmissible to the infants. As dense fractions are known to contain antibody-bound HCV particles, maternal antibodies against HCV may inhibit viral transmission.

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Molecular cloning of hepatitis C virus genome from a single Japanese carrier: sequence variation within the same individual and among infected individuals

Cited by 134 publications

References 14 publications

Genetic Characterization of Hepatitis C Virus in Long-Term RNA Replication Using Li23 Cell Culture Systems

Genetic Characterization of Hepatitis C Virus in Long-Term RNA Replication Using Li23 Cell Culture Systems

Genotypes of hepatitis C virus in Nigeria

Analysis of mother-to-infant transmission of hepatitis C virus: Quasispecies nature and buoyant densities of maternal virus populations

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