Generation of transmissible hepatitis C virions from a molecular clone in chimpanzees
Generation of transmissible hepatitis C virions from a molecular clone in chimpanzees
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DOI：
10.1006/viro.1999.9603
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发表时间：
1999-03-30
期刊：
影响因子：
3.7
通讯作者：
Lau, JYN
中科院分区：
文献类型：
--
作者：
Hong, Z;Beaudet-Miller, M;Lau, JYN
对来自6种不同基因型的丙型肝炎病毒（HCV）多聚蛋白进行多重比对，在来自哈钦森（H77）分离株的克隆中共发现22个非共有突变。这些突变，共同地，可能导致在早期尝试中产生“功能正确”或“感染性”克隆的失败。系统修复这些突变后，构建了一个共识克隆，直接肝内接种体外转录的RNA后，在黑猩猩中产生感染性病毒体。这只RNA感染的黑猩猩已经患上了肝炎，并在超过11个月的时间里保持HCV阳性。为了进一步验证这种RNA衍生的感染性，将从第一只黑猩猩收集的血清静脉注射到第二只未实验过的黑猩猩身上。对第二只黑猩猩进行的感染性分析表明，HCV感染成功传播，这明确地验证了我们修复的分子克隆的感染性。氨基酸序列比较显示，我们修复的感染性克隆与Kolykhalov等人（1997 Science 277，570-574）报道的等基因克隆具有4个错配，并且与Yanagi等人（1997，Proc.Natl. Acad. Sci. USA 94，8738-8743）。在RNA水平上，发现了更多的错配（分别为43和67），其中大部分是同义替换。与16个不同基因型的分离株的进一步比较表明，我们的修复克隆共享更大的共识比报告的同基因克隆。这种产生感染性HCV RNA的方法证实了氨基酸序列一致性在HCV生物学中的重要性。(C)北京：科学出版社.
Multiple alignments of hepatitis C virus (HCV) polyproteins from six different genotypes identified a total of 22 nonconsensus mutations in a clone derived from the Hutchinson (H77) isolate. These mutations, collectively, may have contributed to the failure in generating a "functionally correct" or "infectious" clone in earlier attempts. A consensus clone was constructed after systematic repair of these mutations, which yielded infectious virions in a chimpanzee after direct intrahepatic inoculation of in vitro transcribed RNAs. This RNA-infected chimpanzee has developed hepatitis and remained HCV positive for more than 11 months. To further verify this RNA-derived infectivity, a second naive chimpanzee was injected intravenously with serum collected from the first chimpanzee. Infectivity analysis of the second chimpanzee demonstrated that the HCV infection was successfully transmitted, which validated unequivocally the infectivity of our repaired molecular clone. Amino acid sequence comparisons revealed that our repaired infectious clone had 4 mismatches with the isogenic clone reported by Kolykhalov et al. (1997 Science 277, 570-574) and 8 mismatches with that reported by Yanagi et ai (1997, Proc. Natl. Acad. Sci. USA 94, 8738-8743). At the RNA level, more mismatches (43 and 67, respectively) were identified; most of them were synonymous substitutions. Further comparisons with 16 isolates from different genotypes demonstrated that our repaired clone shares greater consensus than the reported isogenic clones. This approach of generating infectious HCV RNA validates the importance of amino acid sequence consensus in relation to the biology of HCV. (C) 1999 Academic Press.