Bats are an important reservoir of many zoonotic viruses [1,2,3,4] including adenoviruses [5, 6]. 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. The family Adenoviridae is divided into six genera (Atadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus, Siadenovirus, and Testadenovirus). Mastadenoviruses infect a variety of mammalian hosts, including 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) [7]. 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 [8].

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. 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 [9]. A clear cytopathic effect was observed in the cells at 7 days post-inoculation. 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. S1). This indicated that the virus had been isolated successfully. 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. Growth was inhibited with IUDR but not with ribavirin, suggesting that the isolate was a DNA virus. 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. Viral DNA was extracted from the purified virus using a NucleoSpin Tissue Kit (Macherey-Nagel, Duren, Germany). 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). The sequence dataset was assembled de novo using CLC Genomics Workbench software (v.8, CLC bio, Aarhus, Denmark), and sequence comparisons indicated that the isolate was a BtAdV strain, which was named Rc-kw20. The complete genome of Rc-kw20 is 36,506 nucleotides long with 54% G+C content. The complete nucleotide sequence has been deposited in the DDBJ/GenBank database under the accession number MZ683934.

The full genomic nucleotide sequence was analyzed phylogenetically (Fig. 1a). The phylogenetic tree indicated that strain Rc-kw20 was positioned in the same cluster with previous BtAdV-C strains, all of which had been detected in Rhinolophus sinicus bats in China, suggesting that Rc-Kw20 belongs to the species Bat mastadenovirus C in the genus Mastadenovirus. The full genomic nucleotide sequence of Rc-kw20 was 83.8%, 83.9%, and 83.7% identical to BtAdV-C strains, WIV9, WIV10, and WIV11, respectively (GenBank accession numbers KT698853-5).

Fig. 1
figure 1

Genetic analysis of bat mastadenoviruses. Phylogenetic trees based on (a) full-length nucleotide sequences, (b) DNA polymerase amino acid sequences, and (c) hexon amino acid sequences were constructed by the neighbor-joining method with 1,000 bootstrap replicates using MEGA X software. Bootstrap values are indicated as percentages at the nodes. Bars indicate evolutionary distance in units of substitutions per site. All bat mastadenoviruses are underlined, and the novel isolate identified in this study is shown in bold red. (d) Gene map of BatAdV-Rc-kw20 in which genes are shown as grey arrows and ITR sequences are shown as black rectangles

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Open reading frames (ORFs) were predicted using SnapGene Viewer software (SnapGene v.5; GSL Biotech, San Diego, CA, USA) (Table 1), and 33 putative ORFs were identified. Comparisons of the DNA polymerase amino acid sequences of Rc-kw20 and other BtAdV-C strains, which provide one demarcation criterion for adenoviral species [10], showed that they were nearly 90% identical (Table 1). Phylogenetic analysis confirmed that the Rc-kw20 strain was positioned in the BtAdV-C cluster (Fig. 1b), and this was supported by analysis using hexon protein amino acid sequences (Fig. 1c), confirming that Rc-Kw20 is a BtAdV-C strain and that Rhinolophus cornutus should be included as a susceptible host for group 2 BtAdV.

Table 1 Amino acid sequence identity of Rc-kw20 proteins to those of other BtAdV-C strains
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We also generated a genomic map, which included the 54-bp-long inverted terminal repeat (ITR) sequences (Fig. 1d). Twenty of the 33 proteins encoded by Rc-kw20 shared high sequence similarity (more than 80% identity) with those of previously characterized BtAdV-C strains, whereas the fiber protein and several E3- and E4-related proteins shared moderate similarity (ranging from 48.0% to 67.2% sequence identity), and only the large E3 (E3l) protein, which contains host immune-suppression-related motifs [11], showed considerably lower similarity (31.8% to 34.6% sequence identity) (Table 1). Possible functional differences involving proteins with lower sequence similarity might determine the host specificity of BtAdV-C strains.

Recent studies have indicated that Rhinolophus spp. bats may represent an ancestral host for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19. Indeed, related bat sarbecoviruses have been detected in Rhinolophus affinis, R. sinicus, R. pusillus, R. malayanus, R. acuminatus, and R. shameli in Asian countries [12,13,14,15,16,17]. In addition, we detected a bat sarbecovirus in R. cornutus in Japan [18]. Further studies are needed to assess the zoonotic potential of bat viruses inhabiting these Rhinolophus species, including BtAdV-C.