Novel Infectious cDNA Clones of Hepatitis C Virus Genotype 3a (Strain S52) and 4a (Strain ED43): Genetic Analyses and In Vivo Pathogenesis Studies
Novel Infectious cDNA Clones of Hepatitis C Virus Genotype 3a (Strain S52) and 4a (Strain ED43): Genetic Analyses and In Vivo Pathogenesis Studies
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DOI：
10.1128/jvi.02667-09
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发表时间：
2010-05-01
影响因子：
5.4
通讯作者：
Bukh, Jens
中科院分区：
文献类型：
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作者：
Gottwein, Judith M.;Scheel, Troels K. H.;Bukh, Jens
此前，发现丙型肝炎病毒（HCV）基因型1a（H77、HCV-1和HC-TN株）、1b（HC-J4、Con1和HCV-N）和2a（HC-J6和JFH1）的cDNA克隆的RNA转录本在黑猩猩中具有传染性。然而，只有 JFH1 在人肝癌 Huh7 细胞中具有感染性。我们对 HCV 基因型 3a（菌株 S52）和 4a（菌株 ED43）原型菌株进行了遗传分析，并生成了全长共有 cDNA 克隆（pS52 和 pED43）。用这些克隆的RNA转录物转染Huh7.5细胞没有产生表达HCV Core的细胞。然而，黑猩猩的肝内转染导致强烈感染，HCV RNA 滴度峰值接近 5.5 log(10) 国际单位 (IU)/ml。在病毒滴度达到峰值时从血清中回收的基因组共有序列与亲本质粒的序列相同。两只黑猩猩都出现了急性肝炎，肝酶升高，肝脏出现明显的坏死性炎症变化，同时检测到了分泌γ干扰素的肝内T细胞。然而，两种动物肝内T细胞反应的开始时间和广度差异很大，ED43感染的动物（在病毒控制的第一个证据之前3周）出现早期（第4周）多特异性反应，而S52感染的动物（没有病毒控制的证据）出现晚期（第11周）反应，反应的广度有限。在任一动物的急性感染期间均未检测到自体血清中和抗体。两只动物都受到持续感染。总之，我们生成了 HCV 基因型 3a 和 4a 的全功能感染性 cDNA 克隆。所有基因功能的证明可能会进一步开发这些重要基因型的重组细胞培养系统。
Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic analysis of HCV genotype 3a (strain S52) and 4a (strain ED43) prototype strains and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield cells expressing HCV Core. However, intrahepatic transfection of chimpanzees resulted in robust infection with peak HCV RNA titers of similar to 5.5 log(10) international units (IU)/ml. Genomic consensus sequences recovered from serum at the times of peak viral titers were identical to the sequences of the parental plasmids. Both chimpanzees developed acute hepatitis with elevated liver enzymes and significant necroinflammatory liver changes coinciding with detection of gamma interferon-secreting, intrahepatic T cells. However, the onset and broadness of intrahepatic T-cell responses varied greatly in the two animals, with an early (week 4) multispecific response in the ED43-infected animal (3 weeks before the first evidence of viral control) and a late (week 11) response with limited breadth in the S52-infected animal (without evidence of viral control). Autologous serum neutralizing antibodies were not detected during the acute infection in either animal. Both animals became persistently infected. In conclusion, we generated fully functional infectious cDNA clones of HCV genotypes 3a and 4a. Proof of functionality of all genes might further the development of recombinant cell culture systems for these important genotypes.