Challenge Pools of Hepatitis C Virus Genotypes 1–6 Prototype Strains: Replication Fitness and Pathogenicity in Chimpanzees and Human Liver–Chimeric Mouse Models

Bukh, Jens; Meuleman, Philip; Tellier, Raymond; Engle, Ronald E.; Feinstone, Stephen M.; Eder, Gerald; Satterfield, William C.; Govindarajan, Sugantha; Krawczynski, Krzysztof; Miller, R.H.; Leroux‐Roels, Geert; Purcell, Robert H.

doi:10.1086/651579

Cited by 69 publications

(67 citation statements)

References 50 publications

(63 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Because only the 5′-terminal sequence of the 1a (strain H77) 5′ UTR was previously determined (11), here we determined the 5′-terminal sequences of the prototype genotype strains used in development of respective 5′ UTR-NS2 recombinants, by sequence analysis of viruses recovered from plasma pools of experimentally infected chimpanzees (12) (Fig. 2 and Fig.…”

Section: Resultsmentioning

confidence: 99%

MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR

Gottwein

Scheel

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

182

173

View full text Add to dashboard Cite

MicroRNA-122 (miR-122) is believed to stimulate hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5′ untranslated region (5′ UTR). Recently, it was demonstrated that locked nucleic acid SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a and 1b infection in vivo. However, virus-producing culture systems with 5′ UTR of different HCV genotypes have not been available for testing 5′ UTR-based treatment approaches. Using JFH1-based Core-NS2 genotype recombinants, we developed 5′ UTR-NS2 recombinants of HCV genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a with efficient growth in Huh7.5 cells. Deletion mutagenesis studies demonstrated that the 5′ UTR SLI was essential for genotypes 1-6 infection. However, lack of SLI could be compensated for by insertion of other structured HCV or host RNA sequences, including U3 small nucleolar RNA. We demonstrated that SPC3649-induced miR-122 antagonism had a potent antiviral effect against HCV genotypes 1-6 5′ UTR-NS2 viruses. Strikingly, HCV recombinant virus with substitution of SLI and miR-122 binding site 1 (S1) by the U3 RNA sequence was not affected by miR-122 antagonism; this was attributed to the lack of an intact S1 by reverse genetics studies. Therefore, we engineered the corresponding U3 RNA sequences into S1 and demonstrated that HCV recombinants with wild-type SLI and single or combined mutations at four of eight nucleotides of S1 were viable in Huh7.5 cells. These mutations reduced the efficacy of SPC3649 treatment, indicating that escape variants to miR-122 antagonism-based HCV therapy could potentially occur.cell culture infection | locked nucleic acid therapy | miRNA | RNA recombination H epatitis C virus (HCV) infects ∼180 million people worldwide, often causing fatal chronic liver diseases. HCV is a ∼9.6-kb positive-sense RNA virus belonging to the genus Hepacivirus in the Flaviviridae family (1), which has been classified into seven major genotypes and numerous subtypes (2, 3). The viral genome consists of a single ORF flanked by 5′ and 3′ UTRs. The 5′ UTR consists of ∼341 nucleotides forming four major domains. Domain I [nucleotides 1-43, numbering according to reference isolate H77 (GenBank accession no. AF009606)] contains one stem-loop structure (SLI), essential for RNA replication (1), and two microRNA-122 (miR-122) binding sites (Fig. S1), through which liver-abundant miR-122 stimulates HCV replication and translation (4-6). Although the 5′ UTR is a conserved region, a number of genotype-specific nucleotides naturally exist, which have been used for HCV genotyping (3). To date, genotype-specific functional analysis of the HCV 5′ UTR has been hampered by lack of suitable culture systems.Current therapy with pegylated IFN-α and ribavirin has severe side effects and the outcome is dependent on the infecting HCV genotype. Overall, ∼50% of patients completing treatment are cured. Thus, therapeutic drugs that are more effective against diverse HCV genotypes are urgently needed. R...

show abstract

Section: Resultsmentioning

confidence: 99%

MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR

Gottwein

Scheel

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

182

173

View full text Add to dashboard Cite

show abstract

“…These chimeric genomes were shown to be highly useful to study entry, neutralization and virus assembly of all seven known HCV genotypes. They have been further validated to be infectious in vivo as human liver-chimeric mice developed high-titer infections after inoculation with HCV of genotypes 1-6 (Bukh, Meuleman et al 2010). Highest virus titers could be achieved with a J6-JFH1 chi mera designated Jc1 that allows the production of virus particles of about 10 6 infectious units per ml .…”

Section: Jfh1 and Chimeric Genomesmentioning

confidence: 99%

Cell Culture Systems for Hepatitis C Virus

Steinmann

Pietschmann

2013

Current Topics in Microbiology and Immunology

View full text Add to dashboard Cite

Due to the obligatory intracellular life style of viruses, cell culture systems for efficient viral propagation are crucial to obtain a detailed understanding of the virus host cell interaction. For hepatitis C virus (HCV) the development of permissive and authentic culture models has been and continues to be a challenging task. The first ef forts to culture HCV had limited success and range back to before the virus was molecularly cloned in 1989. Since then several major breakthroughs have gradually overcome limitations in culturing the virus and sequentially permitted analysis of viral RNA replication, cell entry and ultimately the complete replication cycle in cultured cells in 2005. Until today, basic and applied HCV research greatly benefit from these tremendous efforts which spurred multiple com plementary cell based model systems for distinct steps of the HCV replication cycle. When used in combination they now permit deep insights into the fascinating biology of HCV and its interplay with the host cell. In fact drug development has been much facilitated and our understanding of the molecular determinants of HCV replication has grown in parallel to these advances. Building on this ground work and further refining our cellular models to better mimic the ar chitecture, polarization and differentiation of natural hepatocytes should reveal novel unique aspects of HCV replication. Ultimately, models to culture primary HCV isolates across all genotypes may teach us important new lessons about viral functional adaptations that have evolved in exchange with its human host and that may ex plain the variable natural course of hepatitis C.

show abstract

“…Twenty-four hours after the first IgG infusion, CHA5A009 was challenged intravenously with 100 chimpanzee infectious doses of each of the HCV strains (with the genotype in parentheses) H77(1a), ED43(4a), SA13(5a), and HK6a(6a) (29)(30)(31), chosen because H77 is the homologous acute-phase patient H strain (31) and chronic-phase patient H sera cross-neutralized all strains in HCVpp and HCVcc assays (18,19). Based on 5=-untranslated region (UTR)-based TaqMan assay (32), CHA5A009 had serum HCV titers of 2.7 and 4.8 log 10 IU/ml at weeks 1 and 2, respectively, and peak titers of 5 to 6 log 10 IU/ml at weeks 3 to 11; the animal remained persistently infected (Fig.…”

mentioning

confidence: 99%

Immunoglobulin with High-Titer In Vitro Cross-Neutralizing Hepatitis C Virus Antibodies Passively Protects Chimpanzees from Homologous, but Not Heterologous, Challenge

Bukh

Engle

Faulk

et al. 2015

J Virol

Self Cite

View full text Add to dashboard Cite

cThe importance of neutralizing antibodies (NAbs) in protection against hepatitis C virus (HCV) remains controversial. We infused a chimpanzee with H06 immunoglobulin from a genotype 1a HCV-infected patient and challenged with genotype strains efficiently neutralized by H06 in vitro. Genotype 1a NAbs afforded no protection against genotype 4a or 5a. Protection against homologous 1a lasted 18 weeks, but infection emerged when NAb titers waned. However, 6a infection was prevented. The differential in vivo neutralization patterns have implications for HCV vaccine development. Chimpanzees have been essential for hepatitis C virus (HCV) research (1-3). Understanding the role of neutralizing antibodies (NAbs) in preventing HCV infection is important for vaccine efforts against this important pathogen, which annually infects ϳ4 million people and causes chronic liver disease (4-12). We showed that chronic-phase patient serum/plasma prevented HCV infection of chimpanzees if the virus and anti-HCV antibody were incubated prior to inoculation (13,14). Others showed that immunoglobulin (IgG) purified from anti-HIV antibodypositive blood donors prevented HCV infection when mixed with virus and then administered to a chimpanzee (15). However, the protective effect of preexisting polyclonal HCV NAbs remains to be determined.Studies with culture systems using pseudotyped particles (HCVpp) or infectious viruses (chimeric cell culture-derived HCV [HCVcc]) confirmed the existence of NAbs in acute-and chronic-phase patients, with high-level cross-neutralization of heterotypic HCV strains (16)(17)(18)(19)(20). However, differences exist in neutralization capacities of chronic-phase samples against HCV variants of the same genotype (21), and there is a constant evolution of HCV variants escaping NAbs (14,22,23). Nonetheless, these in vitro data suggest that chronic-phase HCV samples might be useful for broad protection against HCV. Effective NAbs could help define critical epitopes for vaccine development (24-27).We prepared H06 IgG from plasma obtained almost 30 years after disease onset in patient H with persistent HCV infection (28). By infusing H06 IgG with high in vitro neutralizing titers into a chimpanzee (CHA5A009) and then challenging with different HCV strains sensitive to neutralization in vitro (18, 19), our aim was to test the principle of passive immunoprophylaxis against homologous and heterologous strains in vivo.Animal experimentation and sample collection from chimpanzee CHA5A009 were performed from 2007 to 2008. The housing and care of the chimpanzee met or exceeded all requirements of the National Research Council's 1996 Guide for the Care and Use of Laboratory Animals, 7th ed. (National Academies Press, Washington, DC), which were in effect until 2010. The project license (ASP LID 64) and specific protocol (06-C-482) were separately approved by the Institutional Animal Care and Use Committees of the National Institute of Allergy and Infectious Diseases, NIH, and Bioqual, Inc., the facility housing the chimpanzee...

show abstract

Challenge Pools of Hepatitis C Virus Genotypes 1–6 Prototype Strains: Replication Fitness and Pathogenicity in Chimpanzees and Human Liver–Chimeric Mouse Models

Cited by 69 publications

References 50 publications

MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR

MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR

Cell Culture Systems for Hepatitis C Virus

Immunoglobulin with High-Titer In Vitro Cross-Neutralizing Hepatitis C Virus Antibodies Passively Protects Chimpanzees from Homologous, but Not Heterologous, Challenge

Contact Info

Product

Resources

About