Abstract：
Koi herpesvirus (KHV) is the causative agent of a koi herpesvirus disease (KHVD) inducing high mortality rates in common carp and koi (Cyprinus carpio). No widespread effective vaccination strategy has been implemented yet, which is partly due to side effects of the immunized fish. In this study, we present an evaluation of the purification of infectious KHV from host cell protein and DNA, using the steric exclusion chromatography. The method is related to conventional polyethylene glycol (PEG) precipitation implemented in a chromatographic set-up and has been applied for infectious virus particle purification with high recoveries and impurity removal. Here, we achieved a yield of up to 55% of infectious KHV by using 12% PEG (molecular weight of 6 kDa) at pH 7.0. The recoveries were higher when using chromatographic cellulose membranes with 3-5 μm pores in diameter instead of 1 μm. The losses were assumed to originate from dense KHV precipitates retained on the membranes. Additionally, the use of >0.6 M NaCl was shown to inactivate infectious KHV. In summary, we propose a first step towards a purification procedure for infectious KHV with a possible implementation in fish vaccine manufacturing.
摘要：
锦鱼疱疹病毒（KHV）是锦鱼疱疹病毒病（KHVD）的病原体，在鲤鱼和锦鱼（Cyprinuscarpio）中引起高死亡率。尚未实施广泛有效的疫苗接种策略，部分原因是免疫鱼的副作用。在这项研究中,我们提出了从宿主细胞蛋白和DNA纯化感染性KHV的评估，使用空间排阻色谱法。该方法与色谱装置中实施的常规聚乙二醇（PEG）沉淀有关，已用于高回收率和杂质去除的感染性病毒颗粒纯化。这里,通过在pH7.0下使用12％PEG（分子量6kDa），我们获得了高达55％的感染性KHV的产量。当使用直径为3-5μm而不是1μm的色谱纤维素膜时，回收率更高。假定损失源自保留在膜上的致密KHV沉淀物。此外,使用>0.6MNaCl可以灭活感染性KHV.总之,我们提出了迈向感染性KHV纯化程序的第一步，并可能在鱼类疫苗生产中实施。
