Deletions of the Hypervariable Region (HVR) in Open Reading Frame 1 of Hepatitis E Virus Do Not Abolish Virus Infectivity: Evidence for Attenuation of HVR Deletion Mutants In Vivo
Deletions of the Hypervariable Region (HVR) in Open Reading Frame 1 of Hepatitis E Virus Do Not Abolish Virus Infectivity: Evidence for Attenuation of HVR Deletion Mutants In Vivo
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DOI：
10.1128/jvi.01854-08
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发表时间：
2009-01-01
影响因子：
5.4
通讯作者：
Meng, X. J.
中科院分区：
文献类型：
--
作者：
Pudupakam, R. S.;Huang, Y. W.;Meng, X. J.
戊型肝炎病毒（HEV）是一种重要的人类病原体，尽管对其生物学和复制知之甚少。比较序列分析显示，HEV开放阅读框1中存在一个高变区（HVR），其序列存在广泛的变异。为了阐明HVR在HEV复制中的作用，我们首先构建了两个HVR缺失突变体，hHVRd 1和hHVRd 2，其在基因型1的人HEV复制子的HVR中具有氨基酸（aa）711至777和747至761的框内缺失。HEV复制的证据在Huh 7细胞中检测到转染的RNA转录从突变hHVRd 2，增强的绿色荧光蛋白的表达证明。为了证实体外结果，我们构建了三种具有不同HVR缺失的禽HEV突变体：缺失aa 557至585（Delta 557-585）的突变体aHVRd 1; aHVRd 2（Delta 612-641）;和aHVRd 3（Delta 557-641）。用突变体aHVRd 1和aHVRd 2的加帽RNA转录物肝内接种的鸡发生了活跃的病毒感染，如血清转化、病毒血症和粪便病毒脱落所证明的，尽管具有完全HVR缺失的突变体aHVRd 3在鸡中明显减毒。为了进一步验证结果，我们使用基因型3猪HEV作为骨架构建了四个额外的HVR缺失突变体。突变体sHVRd 2（Delta 722-781）、sHVRd 3（Delta 735-765）和sHVRd 4（Delta 712-765）显示出耐受缺失，并且在肝内接种来自突变体的加帽RNA转录物的猪中具有感染性，而突变体sHVRd 1（Delta 712-790）具有几乎完全的HVR缺失，在感染的猪中表现出减毒表型。来自这些研究的数据表明，HVR中的缺失并不能消除HEV在体外或体内的感染性，尽管在具有较大或几乎完全HVR缺失的HEV突变体中观察到了减毒的证据。
Hepatitis E virus (HEV) is an important human pathogen, although little is known about its biology and replication. Comparative sequence analysis revealed a hypervariable region (HVR) with extensive sequence variations in open reading frame 1 of HEV. To elucidate the role of the HVR in HEV replication, we first constructed two HVR deletion mutants, hHVRd1 and hHVRd2, with in-frame deletion of amino acids (aa) 711 to 777 and 747 to 761 in the HVR of a genotype 1 human HEV replicon. Evidence of HEV replication was detected in Huh7 cells transfected with RNA transcripts from mutant hHVRd2, as evidenced by expression of enhanced green fluorescent protein. To confirm the in vitro results, we constructed three avian HEV mutants with various HVR deletions: mutants aHVRd1, with deletion of aa 557 to 585 (Delta 557-585); aHVRd2 (Delta 612-641); and aHVRd3 (Delta 557-641). Chickens intrahepatically inoculated with capped RNA transcripts from mutants aHVRd1 and aHVRd2 developed active viral infection, as evidenced by seroconversion, viremia, and fecal virus shedding, although mutant aHVRd3, with complete HVR deletion, was apparently attenuated in chickens. To further verify the results, we constructed four additional HVR deletion mutants using the genotype 3 swine HEV as the backbone. Mutants sHVRd2 (Delta 722-781), sHVRd3 (Delta 735-765), and sHVRd4 (Delta 712-765) were shown to tolerate deletions and were infectious in pigs intrahepatically inoculated with capped RNA transcripts from the mutants, whereas mutant sHVRd1 (Delta 712-790), with a nearly complete HVR deletion, exhibited an attenuation phenotype in infected pigs. The data from these studies indicate that deletions in HVR do not abolish HEV infectivity in vitro or in vivo, although evidence for attenuation was observed for HEV mutants with a larger or nearly complete HVR deletion.