Mletzko A, Amtmann A, Bergmann S, Lee P, Christian J, Buchholz R, Becker A
Department of Chemical and Biological Engineering, Institute of Bioprocess Engineering, Faculty of Engineering, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Paul-Gordan-Str. 3, 91052, Erlangen, Germany.
Certified Veterinary Specialist for Aquaculture and for Fisheries Science, Friedrich-Loeffler Institute, Federal Research Institute for Animal Health, Institute of Infectology, Suedufer 10, 17493, Greifswald - Insel Riems, Germany.
In Vitro Cell Dev Biol Anim. 2017 Aug;53(7):579-585. doi: 10.1007/s11626-017-0170-1. Epub 2017 Jun 27.
Research of cyprinid herpesvirus 3 (CyHV-3) is focused on the infection mechanism and disease development in animals using genetic and immunological approaches to improve treatments and diagnostics. In contrast, only few tried to investigate the CyHV-3 replication behaviour in available cell cultures. Whereas, obtaining high virus yields by in vitro replication enables achieving of the mentioned above goals easier and more reliable. The following work presents an attempt to illuminate the KHV replication in common carp brain (CCB) cell cultures from the engineering point of view. The isolate KHV-TP30 was used testing the influence on process parameters, such as multiplicity of infection (MOI), time of infection (TOI) and time of harvest (TOH). Virus concentrations and infectivity at different time points of infection were examined using hydrolyzed probe qPCR (Gilad et al. 2004) and 50% tissue culture infectivity dose (TCID). The data obtained show that while the amount of the virus DNA remains constant after reaching its maximum, the infectivity of the virus decreases. Thus, especially, TOH can be crucial for generating a high-quality virus stock. Applying optimized parameters improved the infectivity of the harvested virus and reached a robust titre as high as 1.9 × 10 TCID/mL. To our knowledge, so far, there is no information in the peer-reviewed literature showing comparably high virus titres. Such virus yields not only facilitate conduction of further studies, including stability tests of the virus stock under various supplementation or disinfection trails, but also provide enough virus material to perform more detailed examinations of the infection mechanism.
鲤疱疹病毒3型（CyHV-3）的研究主要集中在动物的感染机制和疾病发展方面，采用遗传和免疫学方法来改进治疗和诊断。相比之下，只有少数研究尝试在现有的细胞培养物中研究CyHV-3的复制行为。然而，通过体外复制获得高病毒产量能够更轻松、更可靠地实现上述目标。以下工作从工程学角度尝试阐明鲤疱疹病毒（KHV）在鲤脑（CCB）细胞培养物中的复制情况。使用分离株KHV-TP30测试其对感染复数（MOI）、感染时间（TOI）和收获时间（TOH）等工艺参数的影响。在感染的不同时间点，使用水解探针定量聚合酶链反应（qPCR）（吉拉德等人，2004年）和50%组织培养感染剂量（TCID）检测病毒浓度和感染性。获得的数据表明，虽然病毒DNA量在达到最大值后保持恒定，但病毒的感染性会降低。因此，特别是收获时间对于生成高质量病毒储备可能至关重要。应用优化参数提高了收获病毒的感染性，达到了高达1.9×10 TCID/mL的稳健滴度。据我们所知，到目前为止，同行评审文献中没有信息表明有如此高的病毒滴度。这样的病毒产量不仅便于进行进一步研究，包括在各种添加或消毒试验下对病毒储备的稳定性测试，还提供了足够的病毒材料来更详细地研究感染机制。
