Laboratory of Virology, Wageningen University, Wageningen, The Netherlands.
PLoS One. 2011;6(10):e25816. doi: 10.1371/journal.pone.0025816. Epub 2011 Oct 3.
Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ~17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production.
胰腺病（PD）和昏睡病（SD）是大西洋鲑和虹鳟水产养殖中的重要病毒性疾病。PD 和 SD 的病原体是鲑鱼甲病毒（SAV），它是披膜病毒科（属甲病毒属）的一个不寻常成员。SAV 在鱼类中的复制温度较低。SAV 的爆发与每年约 1700 万至 5000 万美元的巨大经济损失有关。目前的控制策略依赖于使用灭活病毒制剂进行疫苗接种，这些制剂获取繁琐，具有内在的安全风险。在这项研究中，我们能够通过表达重组杆状病毒来获得 SAV 的非感染性病毒样颗粒（VLPs），这些杆状病毒编码 SAV 衣壳蛋白和两种主要的免疫显性病毒糖蛋白 E1 和 E2，在 Spodoptera frugiperda Sf9 昆虫细胞中。然而，只有在温度从 27°C 降低到较低温度时才能实现这一点。在 27°C 时，前体 E2（PE2）发生错误折叠，未被宿主弗林蛋白酶加工成成熟的 E2。因此，在感染细胞的表面或培养物上清液中均未检测到 E2。然而，当蛋白表达过程中的温度降低时，PE2 以温度依赖的方式加工成成熟的 E2，并且大量产生 VLPs。因此，合成过程中的温度下降是正确的 SAV 糖蛋白加工和重组 VLP 生产的前提条件。
