High-Throughput Cloning and Characterization of Emerging Adenovirus Types 70, 73, 74, and 75

• Published in: International journal of molecular sciences Vol. 21; no. 17; p. 6370
• Main Authors: Zhang, Wenli, Mese, Kemal, Schellhorn, Sebastian, Bahlmann, Nora, Mach, Nicolas, Bunz, Oskar, Dhingra, Akshay, Hage, Elias, Lafon, Marie-Edith, Wodrich, Harald, Heim, Albert, Ehrhardt, Anja
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 02.09.2020; MDPI
• Subjects: Accuracy; Adenoviruses; Adenoviruses, Human - classification; Adenoviruses, Human - genetics; Clinical isolates; Cloning; Cloning, Molecular - methods; Deoxyribonucleic acid; DNA; Efficiency; emerging adenoviruses; Gene deletion; Genes, Reporter; genetic access; Genetic engineering; Genetic modification; Genetic Vectors - genetics; Genome, Viral; Genomes; high-throughput cloning; High-Throughput Screening Assays; Homologous Recombination; Homology; Humans; Insertion; Life Sciences; linear-linear homologous recombination; Microbiology and Parasitology; Mutation; Pathogenicity; Pathogens; Plasmids; receptor usage; Reporter gene; Tropism; Vaccines; Vectors (Biology); Viruses; linear-linear homologous recombination; genetic access; high-throughput cloning; emerging adenoviruses; receptor usage; Human; High-Throughput Screening Assays; Adenoviruses; Cloning; Genes; Humans; Reporter; Viral; Homologous Recombination; Molecular; Genetic Vectors; Genome
• ISSN: 1422-0067; 1661-6596; 1422-0067; 1661-6596
• 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.