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Front Microbiol. 2018 Dec 4;9:2950. doi: 10.3389/fmicb.2018.02950. eCollection 2018.

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.

Chen M1,2, Zheng F1,2, Yuan G3, Duan X1,2, Rong L1,2, Liu J3, Feng S1,2, Wang Z1,2, Wang M4, Feng Y1,2, Zhou Q1,2, Li J1,2, Deng K1,2, Li C5, Xia J5, Rao G6, Zhou Y3, Fu Y4, Li YP1,2,5.

Author information

1
Institute of Human Virology and Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China.
2
Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-sen University, Guangzhou, China.
3
Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China.
4
Guangzhou Blood Center, Guangzhou, China.
5
Program of Pathobiology, The Fifth Affiliated Hospital and Zhongshan School of Medicine, Sun Yat-sen University, Guangdong, China.
6
Key Laboratory of Liver Disease, Center of Infectious Diseases, PLA 458 Hospital, Guangzhou, China.

Abstract

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.

KEYWORDS:

adaptive mutation; cell culture system; consensus sequence; direct-acting antiviral agents; genotype; hepatitis C virus

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