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

Kato, Naoya; Sejima, Hiroe; Ueda, Youki; Mori, Kazuhiko; Satoh, Shinya; Dansako, Hiromichi; Ikeda, Masanori

doi:10.1371/journal.pone.0091156

Cited by 9 publications

(6 citation statements)

References 45 publications

(83 reference statements)

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“…Flaviviruses Can lead to viremia through vascular system and accumulate firstly into the spleen [23–25]. The spleen exhibited higher viral loads compared to other organs [22, 26].…”

Section: Introductionmentioning

confidence: 99%

The dynamic distribution of duck Tembusu virus in the spleen of infected shelducks

et al. 2019

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Background Duck Tembusu virus (DTMUV) is a novel member of Flavivirus . The isolated and purified DTMUV strain XZ-2012 was used as a strain model, to intramuscularly inject the six-month egg-laying shelducks with the infective dose of 10 4 TCID 50 . The dynamic distribution of the virus in spleen at different time post-infection (pi) was studied using RT-PCR, RT-qPCR, ELISA, immunofluorescence and transmission electron microscopy (TEM). Result The results showed that the virus occurred in the spleen after 2 hpi and lasted up to 18 dpi. The registered viral load increased from 2 hpi to 3 dpi, and then it diminished from 6 dpi to 18 dpi with a slight rise at 12 dpi. From 2 hpi to 6 dpi the DTMUV particles were mostly distributed in the periellipsoidal lymphatic sheath (PELS) of spleen white pulp, few being found in the sheathed capillary. From 9 dpi to 18 dpi, the DTMUV particles were migrating into periarterial lymphatic sheaths (PALS) around the central artery through the red pulp. Under TEM, the virus particles could be observed mostly in lymphocytes and macrophages. Conclusion It was suggested that DTMUV invaded lymphocytes and macrophages of the spleen at 2 hpi and replicated significantly from 1 dpi to 3 dpi, being eliminated from 9 dpi to 18 dpi. This is the first study on the dynamic distribution of DTMUV from invasion to elimination in duck spleen conducted by molecular and morphological methods. It could provide theoretical basis for the occurrence, development and detoxification of the virus in the organs of the immune system.

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“…Flaviviruses Can lead to viremia through vascular system and accumulate firstly into the spleen [23–25]. The spleen exhibited higher viral loads compared to other organs [22, 26].…”

Section: Introductionmentioning

confidence: 99%

The dynamic distribution of duck Tembusu virus in the spleen of infected shelducks

et al. 2019

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“…The aa sequences deduced from the nucleotide sequences of the 10 clones analyzed were compared with the deduced consensus aa sequences of the HCV–RNAs derived from ORL8 cells (Figure 6A). 10 For the selection of the aa substitutions that may contribute to N‐89(N‐251) resistance, aa substitutions derived from known adaptive mutations 19,29‐33 were first excluded from the detected aa substitutions, because adaptive mutations are known to appear naturally during the course of cell culture 34‐36 . Second, all of the aa substitutions that were detected in the HCV–RNAs obtained from the N‐89(N‐251)‐sensitive Li23‐derived cells during long‐term cell culture 7,34‐36 were also excluded from the detected aa substitutions.…”

Section: Resultsmentioning

confidence: 99%

“…Among the remaining aa substitutions, many aa substitutions in the C‐terminal region of NS5A are notable. However, this region is known to be dispensable for HCV–RNA replication 37 and the mutations in this region are intensively accumulated during the long‐term RNA replication 34‐36 . Therefore, we assumed that aa substitutions in the NS5B region, but not in the NS5A region, are involved in N‐89 resistance.…”

Section: Resultsmentioning

confidence: 99%

Antiviral mechanism of preclinical antimalarial compounds possessing multiple antiviral activities

et al. 2021

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We previously found that N‐89 and its derivative, N‐251, which are being developed as antimalarial compounds, showed multiple antiviral activities including hepatitis C virus (HCV). In this study, we focused on the most characterized anti‐HCV activity of N‐89(N‐251) to clarify their antiviral mechanisms. We first prepared cells exhibiting resistance to N‐89(N‐251) than the parental cells by serial treatment of HCV–RNA‐replicating parental cells with N‐89(N‐251). Then, we newly generated HCV–RNA‐replicating cells with the replacement of HCV–RNAs derived from N‐89(N‐251)‐resistant cells and parental cells. Using these cells, we examined the degree of inhibition of HCV–RNA replication by N‐89(N‐251) and found that the host and viral factors contributed almost equally to the resistance to N‐89(N‐251). To further examine the contribution of the host factors, we selected several candidate genes by cDNA microarray analysis and found that the upregulated expression of at least RAC2 and CKMT1B genes independently and differently contributed to the acquisition of an N‐89(N‐251)‐resistant phenotype. For the viral factors, we selected several mutation candidates by the genetic comparative analysis of HCV–RNAs and showed that at least one M414I mutation in the HCV NS5B contributed to the resistance to N‐89. Moreover, we demonstrated that the combination of host factors (RAC2 and/or CKMT1B) and a viral factor (M414I mutation) additively increased the resistance to N‐89. In summary, we identified the host and viral factors contributing to the acquisition of N‐89(N‐251)‐resistance in HCV–RNA replication. These findings will be useful for clarification of the antiviral mechanism of N‐89(N‐251).

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“…1 ). Therefore, we decided to next use OL8 cells, which acquired the genetic diversity of the HCV by long-term culture [ 24 ]. Thus, using the OL8(3.5Y) cells [ 20 ], which had been continuously passaged every 7 days for 3.5 years in the G418-containing medium, we tried to obtain RBV-resistant cells by repetitive RBV treatment.…”

Section: Resultsmentioning

confidence: 99%

“…By this analysis, we found six common aa substitutions: F24L, C172R, and S175P in Core; I1641M in NS3; and V2244A and T2351A in NS5A. Among these aa substitutions, I1641M in NS3 and T2351A in NS5A were also detected as the conserved aa substitutions after a 4-year culture of genome-length HCV RNA-replicating OL and OL11 cells, respectively [ 24 ], suggesting these aa substitutions occurred regardless of RBV pressure. Therefore, four remaining aa substitutions, F24L, C172R, and S175P in Core and V2244A in NS5A, are considered to have occurred because of the prolonged RBV treatment (approximately 14 weeks).…”

Section: Discussionmentioning

confidence: 99%

Establishment of Hepatitis C Virus RNA-Replicating Cell Lines Possessing Ribavirin-Resistant Phenotype

et al. 2015

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BackgroundRibavirin (RBV) is a potential partner of interferon-based therapy and recently approved therapy using direct acting antivirals for patients with chronic hepatitis C. However, the precise mechanisms underlying RBV action against hepatitis C virus (HCV) replication are not yet understood. To clarify this point, we attempted to develop RBV-resistant cells from RBV-sensitive HCV RNA-replicating cells.Methodology/Principal FindingsBy repetitive RBV (100 μM) treatment (10 weeks) of 3.5-year-cultured OL8 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates, dozens of colonies that survived RBV treatment were obtained. These colonies were mixed together and further treated with high doses of RBV (up to 200 μM). By such RBV treatment, we successfully established 12 RBV-survived genome-length HCV RNA-replicating cell lines. Among them, three representative cell lines were characterized. HCV RNA replication in these cells resisted RBV significantly more than that in the parental OL8 cells. Genetic analysis of HCV found several common and conserved amino acid substitutions in HCV proteins among the three RBV-resistant cell species. Furthermore, using cDNA microarray and quantitative RT-PCR analyses, we identified 5 host genes whose expression levels were commonly altered by more than four-fold among these RBV-resistant cells compared with the parental cells. Moreover, to determine whether viral or host factor contributes to RBV resistance, we developed newly HCV RNA-replicating cells by introducing total RNAs isolated from RBV-sensitive parental cells or RBV-resistant cells into the HCV RNA-cured-parental or -RBV-resistant cells using an electroporation method, and evaluated the degrees of RBV resistance of these developed cells. Consequently, we found that RBV-resistant phenotype was conferred mainly by host factor and partially by viral factor.Conclusions/SignificanceThese newly established HCV RNA-replicating cell lines should become useful tools for further understanding the anti-HCV mechanisms of RBV.

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

Cited by 9 publications

References 45 publications

The dynamic distribution of duck Tembusu virus in the spleen of infected shelducks

The dynamic distribution of duck Tembusu virus in the spleen of infected shelducks

Antiviral mechanism of preclinical antimalarial compounds possessing multiple antiviral activities

Establishment of Hepatitis C Virus RNA-Replicating Cell Lines Possessing Ribavirin-Resistant Phenotype

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