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HCV and HIV infection and co-infection: injecting drug use ...

SciELO - Scientific Electronic Library Online ... Infecções e co-infecções pelos vírus HCV e HIV: uso de drogas injetáveis e comportamento ... drug-use behaviors in injecting drug users (IDUs) in relation to single hepatitis C virus (HCV) and ... The Hepatitis C Support Group is here for anyone looking for support in dealing with Hepatitis C. You ... When we first started dating everything was wonderful. Simultaneous Targeting of HCV Replication and Viral Binding with a Single .... in Huh-7 replicon cell line after 3-day transduction with single shRNA vectors. Here, we establish single-cell analysis of viral RNA sequence diversity by utilizing a model ... Huh7 cells from the cell line 2/1 (25) constitutively expressing the ...


Nonstructural protein precursor NS4A/B from hepatitis C ...

Author contributions: Y.-P.L., S.R., J.M.G., T.K.H.S., R.H.P., and J.B. designed research; Y.-P.L., S.R., J.M.G., T.K.H.S., L.M., and J.B. performed research; R.H.P. contributed new reagents/analytic tools; Y.-P.L., S.R., J.M.G., T.K.H.S., and J.B. analyzed data; and Y.-P.L., S.R., and J.B. wrote the paper.

Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases worldwide, but treatment options are limited. Basic HCV research required for vaccine and drug development has been hampered by inability to culture patient isolates, and to date only the JFH1 (genotype 2a) recombinant replicates spontaneously in hepatoma cells and releases infectious virus. A JFH1 chimera with the 5′ end through NS2 from another genotype 2a strain, J6, had enhanced infectivity. However, the full-length J6 clone (J6CF), which we previously found to be fully functional in vivo, was replication incompetent in vitro. Through a systematic approach of culturing J6 with minimal JFH1 sequences, we identified three mutations in NS3, NS4A, and NS5B that permitted full-length J6 propagation and adaptation with infectivity titers comparable to JFH1-based systems. The most efficient recombinant, J6cc, had six adaptive mutations and did not accumulate additional changes following viral passage. We demonstrated that HCV NS3/NS4A protease-, NS5A- and NS5B polymerase-directed drugs respectively inhibited full-length J6 infection dose dependently. Importantly, the three J6-derived mutations enabled culture adaptation of the genetically divergent isolate J8 (genotype 2b), which differed from the J6 nucleotide sequence by 24%. The most efficient recombinant, J8cc, had nine adaptive mutations and was genetically stable after viral passage. The availability of these robust JFH1-independent genotype 2a and 2b culture systems represents an important advance, and the approach used might permit culture development of other isolates, with implications for improved individualized treatments of HCV patients and for development of broadly efficient vaccines.

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The outcome of infection is associated with genetic variability of HCV and host factors (). No vaccine is available, and current IFN-based treatment is suboptimal, with many side effects, with low efficacy against the most prevalent HCV variants (–), and with differential influence from host factors (). Directly acting antivirals (DAA) might improve treatment outcome but also have differential efficacy in treatment of patients with different HCV genotypes (). The HCV positive sense single-strand RNA genome (∼9.6 kb) contains a single ORF flanked by 5′ and 3′ untranslated regions (UTRs). The ORF encodes virus structural proteins (Core, E1, and E2), p7, and six nonstructural (NS) proteins (). HCV isolates are classified into seven major genotypes and numerous subtypes differing by 31–33% and 20–25%, respectively ().

The high heterogeneity of HCV and the lack of representative culture systems have hampered HCV vaccine development, preclinical drug testing, assessment of neutralizing antibodies, and basic HCV research. Although a number of HCV full-length genomes were shown to be infectious in chimpanzees (–), to date only the JFH1 strain (genotype 2a) could replicate autonomously in Huh7 human hepatoma cells (, ); efficient growth depended on adaptive mutations (–). The low probability of isolating a replication-competent HCV genome demands alternative approaches to develop culture systems for HCV isolates. Using the unique replication capacity of JFH1, inter- and intragenotypic recombinants including Core-NS2 (–), 5′ UTR-NS2 (), NS3 protease/NS4A (, ), and NS5A () of various genotypes have been developed. Besides permitting functional studies of specific regions in a genotype-specific manner, these culture systems have been used for testing HCV inhibitors (–), assessment of neutralizing antibodies (, , , ), host microRNA-122 silencing (), animal model development (), and HCV entry receptor discovery (). The JFH1 recombinants with Core-NS2 () or 5′ UTR-NS2 () from another in vivo infectious genotype 2a clone, J6CF (), did not require adaptation for efficient growth (, ). Both J6 () and JFH1 () were isolated from Japanese hepatitis C patients. Studies on recombinants containing various JFH1 () and J6CF () elements demonstrated that the JFH1 NS3 helicase, NS5B polymerase, and 3′ UTR (), as well as specific amino acids, nucleotides, and structural features in NS5B and the 3′ UTR (), are important for the replication capacity of JFH1 in Huh7 cells. Substitutions with JFH1-specific NS5B residues and a nucleotide in the 3′ UTR enhanced the J6CF NS5B RNA polymerase activity and the replication of J6CF replicons with JFH1 elements, respectively ().