Hepatitis C virus (HCV) strains belong to seven genotypes with numerous subtypes that respond differently to antiviral therapies. Genotype 1, and primarily subtype 1b, is the most prevalent genotype worldwide. The development of recombinant HCV infectious cell culture systems for different variants, permitted by the high replication capacity of strain JFH1 (genotype 2a), has advanced efficacy and resistance testing of antivirals. However, efficient infectious JFH1-based cell cultures of subtype 1b are limited and comprise only the 5' untranslated region (5'UTR)-NS2, NS4A, or NS5A regions. Importantly, it has not been possible to develop efficient 1b infectious systems expressing the NS3/4A protease, an important target of direct-acting antivirals. We developed efficient infectious JFH1-based cultures with genotype 1b core-NS5A sequences of strains DH1, Con1, and J4 by using previously identified HCV cell culture adaptive substitutions A1226G, R1496L, and Q1773H. These viruses spread efficiently in Huh7.5 cells by acquiring additional adaptive substitutions, and final recombinants yielded peak supernatant infectivity titers of 4 to 5 log10 focus-forming units (FFU)/ml. We subsequently succeeded in adapting a JFH1-based 5'UTR-NS5A DH1 recombinant to efficient growth in cell culture. We evaluated the efficacy of clinically relevant NS3/4A protease and NS5A inhibitors against the novel genotype 1b viruses, as well as against previously developed 1a viruses. The inhibitors were efficient against all tested genotype 1 viruses, with NS5A inhibitors showing half-maximal effective concentrations several orders of magnitude lower than NS3/4A protease inhibitors. In summary, the developed HCV genotype 1b culture systems represent valuable tools for assessing the efficacy of various classes of antivirals and for other virological studies requiring genotype 1b infectious viruses.

Efficient Hepatitis C Virus Genotype 1b Core-NS5A Recombinants Permit Efficacy Testing of Protease and NS5A Inhibitors.

Development of cell culture infectious clones for hepatitis C virus genotype 1b and transcription analysis of 1b-infected hepatoma cells.

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines. 

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A Hepatitis C virus genotype 1b post-transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo

Adaptive mutations promote hepatitis C virus assembly by accelerating core translocation to the endoplasmic reticulum

Dengue virus (DENV) is primarily transmitted by the bite of an infected mosquito of Aedes aegypti and Aedes albopictus, and symptoms caused may range from mild dengue fever to severe dengue hemorrhagic fever and dengue shock syndrome. Reverse genetic system represents a valuable tool for the study of DENV virology, infection, pathogenesis, etc. Here, we generated and characterized an eukaryotic-activated full-length infectious cDNA clone for a DENV serotype 1 (DENV-1) isolate, D19044, collected in 2019. Initially, nearly the full genome was determined by sequencing overlapping RT-PCR products, and was classified to be genotype I DENV-1. D19044 wild-type cDNA clone (D19044_WT) was assembled by four subgenomic fragments, in a specific order, into a low-copy vector downstream the CMV promoter. D19044_WT released the infectious virus at a low level (1.26 × 103 focus forming units per milliliter [FFU/mL]) following plasmid transfection of BHK-21 cells. Further adaptation by consecutive virus passages up to passage 37, and seven amino acid substitutions (7M) were identified from passage-recovered viruses. The addition of 7M (D19044_7M) greatly improved viral titer (7.5 × 104 FFU/mL) in transfected BHK-21 culture, and virus infections in 293T, Huh7.5.1, and C6/36 cells were also efficient. D19044_7M plasmid was genetically stable in transformant bacteria after five transformation-purification cycles, which did not change the capacity of producing infectious virus. Moreover, the D19044_7M virus was inhibited by mycophenolic acid in a dose-dependent manner. In conclusion, we have developed a DNA-launched full-length infectious clone for a genotype I isolate of DENV-1, with genetic stability in transformant bacteria, thus providing a useful tool for the study of DENV-1.

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Development and Characterization of a Genetically Stable Infectious Clone for a Genotype I Isolate of Dengue Virus Serotype 1

Hepatitis C virus (HCV) is classified into seven major genotypes, and genotype 6 is commonly prevalent in Asia, thus reverse genetic system representing genotype 6 isolates in prevalence is required. Here, we developed an infectious clone for a Chinese HCV 6a isolate (CH6a) using a novel strategy. We determined CH6a consensus sequence from patient serum and assembled a CH6a full-length (CH6aFL) cDNA using overlapped PCR product-derived clones that shared the highest homology with the consensus. CH6aFL was non-infectious in hepatoma Huh7.5 cells. Next, we constructed recombinants containing Core-NS5A or 5'UTR-NS5A from CH6a and the remaining sequences from JFH1 (genotype 2a), and both were engineered with 7 mutations identified previously. However, they replicated inefficiently without virus spread in Huh7.5 cells. Addition of adaptive mutations from CH6a Core-NS2 recombinant, with JFH1 5'UTR and NS3-3'UTR, enhanced the viability of Core-NS5A recombinant and acquired replication-enhancing mutations. Combination of 22 mutations in CH6a recombinant with JFH1 5'UTR and 3'UTR (CH6aORF) enabled virus replication and recovered additional four mutations. Adding these four mutations, we generated two efficient recombinants containing 26 mutations (26m), CH6aORF_26m and CH6aFL_26m (designated "CH6acc"), releasing HCV of 104.3-104.5 focus-forming units (FFU)/ml in Huh7.5.1-VISI-mCherry and Huh7.5 cells. Seven newly identified mutations were important for HCV replication, assembly, and release. The CH6aORF_26m virus was inhibited in a dose- and genotype-dependent manner by direct-acting-antivirals targeting NS3/4A, NS5A, and NS5B. The CH6acc enriches the toolbox of HCV culture systems, and the strategy and mutations applied here will facilitate the culture development of other HCV isolates and related viruses.

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Development of an Infectious Cell Culture System for Hepatitis C Virus Genotype 6a Clinical Isolate Using a Novel Strategy and Its Sensitivity to Direct-Acting Antivirals.

Guirong Rao

Papers

Development of an Infectious Cell Culture System for Hepatitis C Virus Genotype 6a Clinical Isolate Using a Novel Strategy and Its Sensitivity to Direct-Acting Antivirals.

Development of cell culture infectious clones for hepatitis C virus genotype 1b and transcription analysis of 1b-infected hepatoma cells.