Mori Ken-Ichi, Matsumoto Akihiro, Maki Noboru, Ichikawa Yuki, Tanaka Eiji, Yagi Shintaro
R&D Department, Advanced Life Science Institute, Inc., 2-10-23 Maruyamadai, Wako, Saitama, 351-0112, Japan.
Department of Medicine, Division of Hepatology and Gastroenterology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan.
BMC Microbiol. 2016 Sep 27;16(1):224. doi: 10.1186/s12866-016-0846-9.
Despite the high prevalence of genotype 1b hepatitis C virus (HCV) among patients, a cell culture system that permits entire viral life cycle of genotype 1b isolates is limited. To develop a cell-cultured hepatitis C virus (HCVcc) of genotype 1b, the proper combination of HCV genomic variants and host cells is essential. HCV genomes isolated from patients with distinctive symptoms may provide the variants required to establish an HCVcc of genotype 1b.
We first established subgenomic replicons in Huh7 cells using HCV cDNAs isolated from two patients: one with fulminant hepatitis after liver transplantation (TPF1) and another with acute hepatitis and moderate symptoms (sAH). Replicons established from TPF1 and sAH showed mutations in NS4B and in NS3 and NS5A, respectively. Using these replication machineries, we constructed HCV genomic RNAs for each isolate. Virus infectivity was evaluated by a focus-forming assay, which is dependent on the intracellular expression of core antigen, and production of virus particles was assessed by density-gradient centrifugation. Infectious virus was only observed in the culture medium of cells transfected with TFP1 HCV RNA. A chimeric genome with the structural segment (5'-untranslated region [UTR] through NS2) from sAH and the replication machinery (NS3 through 3'-UTR) from TPF1 exhibited greater infectivity than did TFP1, despite formation of deficient virus particles in sAH, suggesting that this genomic segment potentiates virus particle formation. To identify the responsible variants, infectious virus formation was assessed in a chimeric genome carrying parts of the sAH structural segment of the TPF1 genome. A variant in NS2 (M170T) was identified that enhanced infectious virus formation. HCVcc carrying an NS2 gene encoding the M170T substitution and adaptive mutations in NS4B (referred to as TPF1-M170T) infected naïve cured Huh7 cells in a CD81-dependent manner.
We established a novel HCVcc of genotype 1b in Huh7 cells by introducing an amino acid variant in NS2 and adaptive mutations in NS4B from HCV genomic RNA isolated from a patient with fulminant HCV after liver transplantation.
尽管基因型1b丙型肝炎病毒（HCV）在患者中普遍存在，但允许基因型1b分离株完整病毒生命周期的细胞培养系统有限。要开发基因型1b的细胞培养丙型肝炎病毒（HCVcc），HCV基因组变体与宿主细胞的适当组合至关重要。从具有独特症状的患者中分离出的HCV基因组可能提供建立基因型1b HCVcc所需的变体。
我们首先使用从两名患者分离出的HCV cDNA在Huh7细胞中建立了亚基因组复制子：一名在肝移植后发生暴发性肝炎（TPF1），另一名患有急性肝炎且症状中等（sAH）。从TPF1和sAH建立的复制子分别在NS4B以及NS3和NS5A中显示出突变。利用这些复制机制，我们为每个分离株构建了HCV基因组RNA。通过依赖于核心抗原细胞内表达的焦点形成试验评估病毒感染性，并通过密度梯度离心评估病毒颗粒的产生。仅在转染了TFP1 HCV RNA的细胞培养基中观察到感染性病毒。具有来自sAH的结构片段（5'-非翻译区[UTR]至NS2）和来自TPF1的复制机制（NS3至3'-UTR）的嵌合基因组表现出比TFP1更高的感染性，尽管在sAH中形成了缺陷病毒颗粒，这表明该基因组片段增强了病毒颗粒的形成。为了鉴定相关变体，在携带TPF1基因组部分sAH结构片段的嵌合基因组中评估感染性病毒的形成。鉴定出NS2中的一个变体（M170T）增强了感染性病毒的形成。携带编码M170T替代的NS2基因和NS4B中的适应性突变的HCVcc（称为TPF1-M170T）以CD81依赖性方式感染未感染过的治愈Huh7细胞。
我们通过在NS2中引入氨基酸变体以及在从肝移植后暴发性HCV患者分离的HCV基因组RNA的NS4B中引入适应性突变，在Huh7细胞中建立了一种新型的基因型1b HCVcc。
