Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, Witten/Herdecke University, 58453 Witten, Germany.
Institut für Virologie, Adenovirus Konsiliarlabor, Medizinische Hochschule, 30625 Hannover, Germany.
Int J Mol Sci. 2020 Sep 2;21(17):6370. doi: 10.3390/ijms21176370.
Recently an increasing number of new adenovirus types associated with type-dependent pathogenicity have been identified. However, identification of these clinical isolates represents the very first step to characterize novel pathogens. For deeper analyses, these adenoviruses need to be further characterized in basic virology experiments or they could be applied in translational research. To achieve this goal, it is essential to get genetic access and to enable genetic modification of these novel adenovirus genomes (deletion, insertion, and mutation). Here we demonstrate a high-throughput approach to get genetic access to new adenoviruses via homologous recombination. We first defined the cloning conditions regarding homology arm-length and input adenoviral genome amounts. Then we cloned four naturally occurring adenoviruses (Ad70, Ad73, Ad74, and Ad75) into easy-to-manipulate plasmids and genetically modified them by reporter gene insertion. Three recombinant adenoviruses (Ad70, Ad73, and Ad74) containing a reporter cassette were successfully reconstituted. These novel reporter-labeled adenoviruses were further characterized using the inserted luciferase reporter with respect to receptor usage, presence of anti-adenovirus antibodies, and tropism in vitro. The identified receptor usage, the relatively low prevalence of anti-adenovirus antibodies, and the various cancer cell line transduction pattern are important features of these new pathogens providing essential information for their therapeutic application.
最近，越来越多与病毒类型相关的致病性新腺病毒被鉴定出来。然而，这些临床分离株的鉴定仅仅是对新型病原体进行特征描述的第一步。为了进行更深入的分析，这些腺病毒需要在基础病毒学实验中进一步进行特征描述，或者可以应用于转化研究。为了实现这一目标，获得遗传途径并实现这些新型腺病毒基因组的遗传修饰（缺失、插入和突变）是至关重要的。在这里，我们展示了一种通过同源重组获得新型腺病毒遗传途径的高通量方法。我们首先确定了同源臂长度和腺病毒基因组输入量的克隆条件。然后，我们将四种天然存在的腺病毒（Ad70、Ad73、Ad74 和 Ad75）克隆到易于操作的质粒中，并通过插入报告基因进行遗传修饰。成功构建了三个含有报告基因盒的重组腺病毒（Ad70、Ad73 和 Ad74）。使用插入的荧光素酶报告基因，进一步对这些新型报告标记腺病毒进行了受体利用、抗腺病毒抗体存在和体外嗜性的特征描述。鉴定的受体利用、相对较低的抗腺病毒抗体流行率以及不同的癌细胞系转导模式是这些新病原体的重要特征，为它们的治疗应用提供了重要信息。
