Monaghan Sean J, Thompson Kim D, Bron James E, Bergmann Sven M, Jung Tae S, Aoki Takashi, Muir K Fiona, Dauber Malte, Reiche Sven, Chee Diana, Chong Shin M, Chen Jing, Adams Alexandra
Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, UK.
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, EH26 0PZ, UK.
Vet Res. 2016 Jan 8;47:8. doi: 10.1186/s13567-015-0297-6.
Cyprinid herpesvirus 3 (CyHV-3), also called koi herpesvirus (KHV), is the aetiological agent of a fatal disease in carp and koi (Cyprinus carpio L.), referred to as koi herpesvirus disease. The virus contains at least 40 structural proteins, of which few have been characterised with respect to their immunogenicity. Indirect immunofluorescence assays (IFAs) using two epitope-specific monoclonal antibodies (MAbs) were used to examine the expression kinetics of two potentially immunogenic and diagnostically relevant viral antigens, an envelope glycoprotein and a capsid-associated protein. The rate of expression of these antigens was determined following a time-course of infection in two CyHV-3 susceptible cell lines. The results were quantified using an IFA, performed in microtitre plates, and image analysis was used to analyse confocal micrographs, enabling measurement of differential virus-associated fluorescence and nucleus-associated fluorescence from stacks of captured scans. An 8-tenfold increase in capsid-associated protein expression was observed during the first 5 days post-infection compared to a ≤ 2-fold increase in glycoprotein expression. A dominant protein of ~100 kDa reacted with the capsid-associated MAb (20F10) in western blot analysis. This band was also recognised by sera obtained from carp infected with CyHV-3, indicating that this capsid-associated protein is produced in abundance during infection in vitro and is immunogenic to carp. Mass spectrometry carried out on this protein identified it as a previously uncharacterised product of open reading frame 84. This abundantly expressed and immunogenic capsid-associated antigen may be a useful candidate for KHV serological diagnostics.
鲤疱疹病毒3型（CyHV-3），也称为锦鲤疱疹病毒（KHV），是鲤鱼和锦鲤（Cyprinus carpio L.）中一种致命疾病的病原体，该疾病被称为锦鲤疱疹病毒病。该病毒至少包含40种结构蛋白，其中很少有蛋白的免疫原性得到表征。使用两种表位特异性单克隆抗体（MAb）进行间接免疫荧光分析（IFA），以检测两种潜在具有免疫原性且与诊断相关的病毒抗原，即包膜糖蛋白和衣壳相关蛋白的表达动力学。在两种对CyHV-3敏感的细胞系中，在感染的时间进程之后确定这些抗原的表达速率。使用在微量滴定板中进行的IFA对结果进行定量，并使用图像分析来分析共聚焦显微镜照片，从而能够测量从捕获扫描堆栈中获得的与病毒相关的荧光和与细胞核相关的荧光差异。与糖蛋白表达增加≤2倍相比，在感染后的前5天观察到衣壳相关蛋白表达增加了8到10倍。在蛋白质印迹分析中，一种约100 kDa的主要蛋白与衣壳相关单克隆抗体（20F10）发生反应。该条带也被感染CyHV-3的鲤鱼血清识别，表明这种衣壳相关蛋白在体外感染期间大量产生，并且对鲤鱼具有免疫原性。对该蛋白进行的质谱分析确定它是开放阅读框84的一种先前未表征的产物。这种大量表达且具有免疫原性的衣壳相关抗原可能是KHV血清学诊断的有用候选物。
