A DNA library was prepared using a TruSeq Nano DNA Low Throughput Library Prep Kit (Illumina, San Diego, CA, USA) and subjected to next-generation sequencing (NovaSeq 6000, Illumina). Viral DNA was extracted from the purified virus using a NucleoSpin Tissue Kit (Macherey-Nagel, Duren, Germany). Then, to determine the complete genome sequence of the viral isolate, we grew it in cells for 4 days and purified it from the culture medium by ultracentrifugation with a 20% sucrose cushion. Growth was inhibited with IUDR but not with ribavirin, suggesting that the isolate was a DNA virus. To identify the genome type of the isolate, we analyzed its growth in the presence of ribavirin or 5-iodo-2'-deoxyuridine (IUDR), which inhibit the growth of RNA and DNA viruses, respectively. This indicated that the virus had been isolated successfully. The cell supernatant was passed through a 0.22-μm filter and inoculated onto fresh Vero/TMPRSS2 cells, producing an extensive cytopathic effect (Supplementary Fig. A clear cytopathic effect was observed in the cells at 7 days post-inoculation. We then inoculated antibiotic-treated fecal suspensions onto African green monkey Vero cells expressing a transmembrane protease, serine 2 (Vero/TMPRSS2), which may support the replication of certain unknown viruses. To investigate bat viruses, we collected fecal samples from apparently healthy insectivorous Rhinolophus cornutus bats with permission from the local government in Shizuoka Prefecture of Japan in September 2020. BtAdVs are divided into three groups depending on host family classification: group 1, comprised of members of species A, B, G, and J, isolated from Vespertilionidae bats and genetically closely related to canine adenoviruses group 2, comprised of members of species C from Rhinolophidae bats and group 3, comprised of members of species D, E, F, H, and I from Miniopteridae and Pteropodidae bats. Bat mastadenoviruses (BtAdVs) have been classified as members of the species Bat mastadenovirus A-J by the International Committee on Taxonomy of Viruses (ICTV). Mastadenoviruses infect a variety of mammalian hosts, including bats. The family Adenoviridae is divided into six genera ( Atadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus, Siadenovirus, and Testadenovirus). Adenoviruses are non-enveloped icosahedral viruses with a double-stranded DNA genome that infect a wide range of vertebrate hosts from amphibians and fish to mammals. Bats are an important reservoir of many zoonotic viruses including adenoviruses.
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