Abstract
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.
Highlights
Adenoviruses are medium-sized (70–100 nm in diameter), non-enveloped icosahedral viruses composed of double-stranded linear DNA genome with an average length of 26–45 kb [1]
The efficiency of adenoviral genome direct cloning can be influenced by the length of homology arm (HA)
We evaluated a variety of HA-lengths using a pilot experiment based on direct cloning of human adenovirus 9 genome (Figure 1)
Summary
Adenoviruses are medium-sized (70–100 nm in diameter), non-enveloped icosahedral viruses composed of double-stranded linear DNA genome with an average length of 26–45 kb [1]. In humans more than 100 adenovirus types have been identified and classified into seven species (A to G) based on hemagglutination properties, oncogenicity in rodents, DNA homology, and genomic organization [2,3]. Recent outbreaks of adenovirus infections are at least partly due to newly evolving adenovirus types from constant HAdVs molecular evolution [2,7,8]. To identify these novel clinical isolates is the very first step to study a new pathogen. For further studies, it is essential to get genetic access and to enable genetic modification of these novel adenovirus genomes
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