Establishment of a reverse genetics system for Schmallenberg virus, a newly emerged orthobunyavirus in Europe.
Establishment of a reverse genetics system for Schmallenberg virus, a newly emerged orthobunyavirus in Europe.
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DOI：
10.1099/vir.0.049981-0
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发表时间：
2013-04
期刊：
影响因子：
--
通讯作者：
Szemiel AM
中科院分区：
文献类型：
--
作者：
Elliott RM;Blakqori G;van Knippenberg IC;Koudriakova E;Li P;McLees A;Shi X;Szemiel AM
施马伦贝格病毒(Schmallenberg Virus，SBV)是一种新出现的正病毒，在欧洲牛、绵羊和山羊中引起了广泛的疾病。与其他正丁型病毒一样，SBV的特征是由三个负向RNA基因组组成，编码四个结构蛋白和两个非结构蛋白。本研究表明，SBV具有广泛的体外宿主范围，BHK-21细胞是SBV增殖和空斑滴定检测的适宜宿主。将SBV基因组片段克隆为cDNA3个重组体，构建了回收传染性病毒的三质粒拯救系统。重组病毒在细胞培养中的表现与真病毒相似。编码在最小基因组片段上的非结构NSS蛋白的ORF被相应的cDNA中的翻译终止密码子干扰，当将该质粒用于反向遗传学时，恢复了一个缺乏NSS表达的重组病毒。与野生型病毒相比，这种病毒关闭宿主细胞蛋白质合成的能力降低了。此外，NSS缺失的病毒可诱导细胞中的干扰素，这表明，与其他正位病毒一样，NSS作为干扰素拮抗剂发挥作用，最可能的方式是全局抑制宿主细胞的新陈代谢。建立一个强大的SBV反向遗传学系统将有助于研究其致病机制，并创造出可能成为候选疫苗的减毒株。
Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that has caused widespread disease in cattle, sheep and goats in Europe. Like other orthobunyaviruses, SBV is characterized by a tripartite negative-sense RNA genome that encodes four structural and two non-structural proteins. This study showed that SBV has a wide in vitro host range, and that BHK-21 cells are a convenient host for both SBV propagation and assay by plaque titration. The SBV genome segments were cloned as cDNA and a three-plasmid rescue system was established to recover infectious virus. Recombinant virus behaved similarly in cell culture to authentic virus. The ORF for the non-structural NSs protein, encoded on the smallest genome segment, was disrupted by introduction of translation stop codons in the appropriate cDNA, and when this plasmid was used in reverse genetics, a recombinant virus that lacked NSs expression was recovered. This virus had reduced capacity to shut-off host-cell protein synthesis compared with the wild-type virus. In addition, the NSs-deleted virus induced interferon (IFN) in cells, indicating that, like other orthobunyaviruses, NSs functions as an IFN antagonist, most probably by globally inhibiting host-cell metabolism. The development of a robust reverse genetics system for SBV will facilitate investigation of its pathogenic mechanisms as well as the creation of attenuated strains that could be candidate vaccines.
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