Karlsen Marius, Villoing Stephane, Ottem Karl F, Rimstad Espen, Nylund Are
Department of Biology, University of Bergen, Thor Møhlens gate 55, 5020 Bergen, Norway.
BMC Res Notes. 2010 Sep 21;3:241. doi: 10.1186/1756-0500-3-241.
Salmonid alphavirus (SAV) is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed.
Infectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF) encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2197, nsP3263 and nsP3323 severely reduced infectivity, a serine to proline mutation in E2206 appeared to enhance the virus titer production.
We have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.
鲑鱼α病毒（SAV）是欧洲鲑鱼养殖中广泛存在的病原体。基于序列比较已提出了不同的病毒亚型，其中一些具有不同的地理分布。在挪威，迄今为止仅鉴定出SAV亚型3。对于对发病机制和传播重要的病毒机制了解甚少。因此，需要用于详细探索SAV基因组的工具。
构建了感染性cDNA克隆，其中3型SAV的基因组受CMV启动子控制。这些克隆被设计用于表达推测与先前报道的SAVH20/03毒株相同的蛋白质。制备了针对E2糖蛋白一部分的多克隆抗血清，以检测编码病毒结构蛋白的亚基因组开放阅读框（ORF）的表达。cDNA克隆的转染通过间接荧光抗体试验（IFAT）显示了E2蛋白的表达，并且在上清液的连续传代中，通过逆转录-聚合酶链反应（RT-PCR）、IFAT以及出现与野生型SAV感染期间所见相似的细胞病变效应，证实了感染性重组病毒的存在。通过对引入的遗传标签进行序列鉴定，确认回收的病毒源自感染性质粒。当添加另一个在第二个亚基因组α病毒启动子控制下编码增强绿色荧光蛋白（EGFP）报告基因的ORF时，重组病毒也具有感染性。最后，我们使用该系统研究了选定的点突变对奇努克鲑鱼胚胎细胞感染性的影响。虽然在非结构蛋白2（nsP2）197、非结构蛋白3（nsP3）263和nsP3323中引入的突变严重降低了感染性，但E2206处的丝氨酸到脯氨酸突变似乎提高了病毒滴度的产生。
我们基于3型基因组构建了SAV的感染性克隆。这些克隆可作为进一步功能研究的平台。
