Three Amino Acid Mutations (F51L, T59A, and S390L) in the Capsid Protein of the Hepatitis E Virus Collectively Contribute to Virus Attenuation
Three Amino Acid Mutations (F51L, T59A, and S390L) in the Capsid Protein of the Hepatitis E Virus Collectively Contribute to Virus Attenuation
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DOI：
10.1128/jvi.02278-10
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发表时间：
2011-06-01
影响因子：
5.4
通讯作者：
Meng, Xiang-Jin
中科院分区：
文献类型：
--
作者：
Cordoba, Laura;Huang, Yao-Wei;Meng, Xiang-Jin
戊型肝炎病毒（Hepatitis E virus，HEV）是一种重要的人类病原体，其复制和致病机制尚不清楚。我们先前鉴定了减毒的基因型3型HEV突变体（pSHEV-1），其在衣壳蛋白中含有三个独特的氨基酸突变（F51 L、T59 A和S390 L）。为了确定这些突变中的每一个的作用，我们构建了三个HEV单突变体（rF 51 L、rT 59 A和rS390 L），它们都被发现在Huh 7肝细胞中具有复制能力。为了确定突变体的致病性，我们利用了特定的无病原体（SPF）的猪模型的HEV和一个独特的接种程序，绕过需要在体外繁殖感染性HEV。通过超声引导技术，用来自每个单突变体、pSHEV-1三重突变体、野生型pSHEV-3或磷酸盐缓冲盐水（PBS）缓冲液的感染性克隆的体外转录的全长加帽RNA转录物肝内接种总共60头猪（n = 10）。结果表明，F51 L突变部分地有助于病毒减毒，而T59 A和S390 L突变导致猪中HEV的更剧烈减毒，如在三个不同尸检时间收集的肝脏、胆汁和肠内容物中的病毒载量显著降低的病毒血症发生率、粪便病毒脱落和病毒血症的延迟出现和持续时间较短以及较低的病毒载量所证明的。结果表明，衣壳蛋白中的三个突变共同有助于HEV减毒。本研究对戊型肝炎减毒活疫苗的研制具有重要意义。
Hepatitis E virus (HEV) is an important but extremely understudied human pathogen, and the mechanisms of HEV replication and pathogenesis are largely unknown. We previously identified an attenuated genotype 3 HEV mutant (pSHEV-1) containing three unique amino acid mutations (F51L, T59A, and S390L) in the capsid protein. To determine the role of each of these mutations, we constructed three HEV single mutants (rF51L, rT59A, and rS390L) which were all found to be replication competent in Huh7 liver cells. To determine the pathogenicities of the mutants, we utilized the specific-pathogen-free (SPF) pig model for HEV and a unique inoculation procedure that bypasses the need for propagating infectious HEV in vitro. A total of 60 pigs were intrahepatically inoculated, via an ultrasound-guided technique, with in vitro-transcribed full-length capped RNA transcripts from the infectious clones of each single mutant, the pSHEV-1 triple mutant, wild-type pSHEV-3, or phosphate-buffered saline (PBS) buffer (n = 10). The results showed that the F51L mutation partially contributed to virus attenuation, whereas the T59A and S390L mutations resulted in more drastic attenuation of HEV in pigs, as evidenced by a significantly lower incidence of viremia, a delayed appearance and shorter duration of fecal virus shedding and viremia, and lower viral loads in liver, bile, and intestinal content collected at three different necropsy times. The results indicate that the three mutations in the capsid protein collectively contribute to HEV attenuation. This study has important implications for developing a modified live-attenuated vaccine against HEV.