Beard M R, Abell G, Honda M, Carroll A, Gartland M, Clarke B, Suzuki K, Lanford R, Sangar D V, Lemon S M
Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, TX, USA.
Hepatology. 1999 Jul;30(1):316-24. doi: 10.1002/hep.510300137.
We describe an infectious molecular clone of a Japanese genotype 1b strain of hepatitis C virus (HCV-N). The molecularly cloned sequence of HCV-N was compared with alignments of other HCV sequences, leading to the identification of 15 unique, nonconservative amino acid substitutions within the HCV-N open reading frame (ORF). These were repaired to the consensus genotype 1b residue, and the infectivity of RNA transcribed from the repaired clone was assessed by intrahepatic inoculation of a chimpanzee. Viral RNA was first detected in the serum of this chimpanzee 3 weeks following inoculation, and was intermittently present over the next 14 weeks. A strong and persistent anti-HCV serological response developed 13 weeks following inoculation, with seroconversion in the recombinant immunoblot assay (RIBA). A weaker, transient serological response, characterized by seroconversion in a third-generation enzyme-linked immunosorbent assay (ELISA) but not RIBA, occurred between weeks 1 and 5. This may have represented an anamnestic response to HCV antigens translated directly from the intrahepatically inoculated RNA, because the animal previously had undergone 2 unsuccessful attempts at rescue of HCV by intrahepatic RNA inoculation. There was neither biochemical nor histological evidence of liver disease. Although this is within the range of expected outcomes in an HCV-naive chimpanzee, prior immunologic priming may have modified the infection in this animal. The HCV-N clone is the first infectious molecular clone of HCV that is comprised entirely of genotype 1b sequence, and it contains an ORF sequence that is significantly divergent from that of a previously described genotype 1a/1b chimera.
我们描述了一株日本1b基因型丙型肝炎病毒（HCV-N）的感染性分子克隆。将HCV-N的分子克隆序列与其他HCV序列比对，在HCV-N开放阅读框（ORF）内鉴定出15个独特的、非保守的氨基酸替换。将这些替换修复为1b基因型的共有残基，并通过对黑猩猩进行肝内接种来评估修复后的克隆转录的RNA的感染性。接种后3周在这只黑猩猩的血清中首次检测到病毒RNA，并在接下来的14周内间歇性出现。接种后13周出现强烈且持续的抗HCV血清学反应，重组免疫印迹分析（RIBA）出现血清转化。在第1至5周之间出现较弱的、短暂的血清学反应，其特征是在第三代酶联免疫吸附测定（ELISA）中出现血清转化，但在RIBA中未出现。这可能代表了对直接从肝内接种的RNA翻译的HCV抗原的回忆反应，因为这只动物之前曾有2次通过肝内RNA接种拯救HCV的尝试均未成功。既没有肝病的生化证据也没有组织学证据。尽管这在未感染HCV的黑猩猩预期结果范围内，但先前的免疫致敏可能改变了这只动物的感染情况。HCV-N克隆是首个完全由1b基因型序列组成的HCV感染性分子克隆，其ORF序列与先前描述的1a/1b基因型嵌合体的序列有显著差异。
