分离安大略省水生鸟类博尔纳病毒 1 并鉴定其在永生化禽类细胞系中的复制情况。
Isolation of Ontario aquatic bird bornavirus 1 and characterization of its replication in immortalized avian cell lines.
机构信息
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada.
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada.
出版信息
Virol J. 2020 Jan 31;17(1):16. doi: 10.1186/s12985-020-1286-6.
BACKGROUND
Aquatic bird bornavirus 1 (ABBV-1) has been associated with neurological diseases in wild waterfowls. In Canada, presence of ABBV-1 was demonstrated by RT-qPCR and immunohistochemistry in tissues of waterfowls with history of neurological disease and inflammation of the central and peripheral nervous tissue, although causation has not been proven by pathogenesis experiments, yet. To date, in vitro characterization of ABBV-1 is limited to isolation in primary duck embryo fibroblasts. The objectives of this study were to describe isolation of ABBV-1 in primary duck embryonic fibroblasts (DEF), and characterize replication in DEF and three immortalized avian fibroblast cell lines (duck CCL-141, quail QT-35, chicken DF-1) in order to evaluate cellular permissivity and identify suitable cell lines for routine virus propagation.
METHODS
The virus was sequenced, and phylogenetic analysis performed on a segment of the N gene coding region. Virus spread in cell cultures, viral RNA and protein production, and titres were evaluated at different passages using immunofluorescence, RT-qPCR, western blotting, and tissue culture dose 50% (TCID) assay, respectively.
RESULTS
The isolated ABBV-1 showed 97 and 99% identity to European ABBV-1 isolate AF-168 and North American ABBV-1 isolates 062-CQ and CG-N1489, and could infect and replicate in DEF, CCL-141, QT-35 and DF-1 cultures. Viral RNA was detected in all four cultures with highest levels observed in DEF and CCL-141, moderate in QT-35, and lowest in DF-1. N protein was detected in western blots from infected DEF, CCL-141 and QT-35 at moderate to high levels, but minimally in infected DF-1. Infectious titre was highest in DEF (between approximately 10 to 10 FFU / 10 cells). Regarding immortalized cell lines, CCL-141 showed the highest titre between approximately 10 to 10 FFU / 10 cells. DF-1 produced minimal infectious titre.
CONCLUSIONS
This study confirms the presence of ABBV-1 among waterfowl in Canada and reported additional in vitro characterization of this virus in different avian cell lines. ABBV-1 replicated to highest titre in DEF, followed by CCL-141 and QT-35, and poorly in DF-1. Our results showed that CCL-141 can be used instead of DEF for routine ABBV-1 production, if a lower titre is an acceptable trade-off for the simplicity of using immortalized cell line over primary culture.
背景
水生鸟类 bornavirus 1（ABBV-1）已与野生水禽的神经疾病相关联。在加拿大，通过 RT-qPCR 和免疫组织化学在具有神经疾病和中枢及周围神经组织炎症病史的水禽组织中证实了 ABBV-1 的存在，尽管尚未通过发病机制实验证明因果关系。迄今为止，ABBV-1 的体外特征仅限于在原代鸭胚成纤维细胞中分离。本研究的目的是描述在原代鸭胚成纤维细胞（DEF）中分离 ABBV-1，并在 DEF 和三种永生禽类成纤维细胞系（鸭 CCL-141、鹌鹑 QT-35、鸡 DF-1）中表征复制，以评估细胞允许性并确定适合常规病毒繁殖的细胞系。
方法
对 N 基因编码区的一段序列进行测序，并进行系统发育分析。使用免疫荧光、RT-qPCR、western blot 和组织培养 50%半数感染剂量（TCID）测定法分别在不同传代时评估病毒在细胞培养物中的传播、病毒 RNA 和蛋白的产生以及滴度。
结果
分离的 ABBV-1 与欧洲 ABBV-1 分离株 AF-168 和北美的 ABBV-1 分离株 062-CQ 和 CG-N1489 的同源性分别为 97%和 99%，可感染并在 DEF、CCL-141、QT-35 和 DF-1 培养物中复制。在四种培养物中均检测到病毒 RNA，DEF 和 CCL-141 中检测到的水平最高，QT-35 中检测到的水平中等，DF-1 中检测到的水平最低。在感染的 DEF、CCL-141 和 QT-35 中western blot 检测到 N 蛋白的水平为中等到高水平，但在感染的 DF-1 中检测到的水平最低。在 DEF 中产生的感染性滴度最高（约为 10 至 10 FFU/10 个细胞）。关于永生细胞系，CCL-141 产生的滴度最高，约为 10 至 10 FFU/10 个细胞。DF-1 产生的感染性滴度最低。
结论
本研究证实了加拿大水禽中存在 ABBV-1，并报告了该病毒在不同禽类细胞系中的进一步体外特征。ABBV-1 在 DEF 中复制的滴度最高，其次是 CCL-141 和 QT-35，而在 DF-1 中复制的滴度较低。我们的结果表明，如果较低的滴度是使用永生细胞系而不是原代培养物的简单性的可接受权衡，那么 CCL-141 可用于替代 DEF 进行常规 ABBV-1 生产。
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