Joint R&D Division of Virology, Department of Virology, Immunobiology and Parasitology, The National Veterinary Institute (SVA), Uppsala, Sweden.
Division of Veterinary Diagnostics and Research, National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark.
PLoS One. 2013 Dec 23;8(12):e82978. doi: 10.1371/journal.pone.0082978. eCollection 2013.
Astroviruses are becoming a growing concern in veterinary and public health. To date there are no registered vaccines against astrovirus-induced disease, mostly due to the difficulty to cultivate astroviruses to high titer for vaccine development using conventional techniques. As means to circumvent this drawback, we have developed stably transfected mink fetal cells and BHK21 cells constitutively expressing the full-length and truncated capsid proteins of two distinct genotypes of mink astrovirus. Protein expression in these stably transfected cells was demonstrated by strong signals as evaluated by in-situ PLA and IFA, and confirmed by Western blotting. The recombinant full-length and truncated proteins induced a high level of antibodies in mink, evaluated by ELISA, demonstrating their immunogenicity. In a challenge experiment in mink, a reduction in presentation clinical signs and virus shedding was observed in mink kits born from immunized females. The gene integration and protein expression were sustained through cell passage, showing that the used approach is robust and reliable for expression of functional capsid proteins for vaccine and diagnostic applications.
星状病毒在兽医和公共卫生领域日益受到关注。迄今为止，尚无针对星状病毒感染疾病的注册疫苗，主要是因为使用传统技术难以将星状病毒培养到高滴度以用于疫苗开发。为了克服这一缺点，我们开发了稳定转染的水貂胎儿细胞和 BHK21 细胞，这些细胞持续表达两种不同基因型的水貂星状病毒的全长和截短衣壳蛋白。通过原位 PLA 和 IFA 评估，这些稳定转染细胞中的蛋白表达显示出强烈的信号，Western blot 进一步证实了这一点。重组全长和截短蛋白在水貂中诱导了高水平的抗体，通过 ELISA 进行评估，证明了它们的免疫原性。在水貂攻毒实验中，从免疫雌性所生的水貂幼崽中观察到临床症状和病毒脱落减少。基因整合和蛋白表达在细胞传代过程中得以维持，表明该方法对于疫苗和诊断应用的功能性衣壳蛋白表达是稳健可靠的。
