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Here, we report on the evolutionary, biological, and structural characterization of a novel bat adenovirus (BtAdV) recovered from a Rafinesque's big-eared bat (<jats:named-content content-type=\"genus-species\">Corynorhinus<\/jats:named-content><jats:named-content content-type=\"genus-species\">rafinesquii<\/jats:named-content>) in Kentucky, USA, which is the first adenovirus isolated from North American bats. This virus (BtAdV 250-A) exhibits a close phylogenetic relationship with<jats:named-content content-type=\"genus-species\">Canine mastadenovirus A<\/jats:named-content>(CAdV A), as previously observed with other BtAdVs. To further investigate the relationships between BtAdVs and CAdVs, we conducted mass spectrometric analysis and single-particle cryo-electron microscopy reconstructions of the BtAdV 250-A capsid and also analyzed the<jats:italic>in vitro<\/jats:italic>host ranges of both viruses. Our results demonstrate that BtAdV 250-A represents a new mastadenovirus species that, in contrast to CAdV, has a unique capsid morphology that contains more prominent extensions of protein IX and can replicate efficiently in a phylogenetically diverse range of species. These findings, in addition to the recognition that both the genetic diversity of BtAdVs and the number of different bat species from disparate geographic regions infected with BtAdVs appears to be extensive, tentatively suggest that bats may have served as a potential reservoir for the cross-species transfer of adenoviruses to other hosts, as theorized for CAdV.<\/jats:p><jats:p><jats:bold>IMPORTANCE<\/jats:bold>Although many adenoviruses are host specific and likely codiverged with their hosts over millions of years, other adenoviruses appear to have emerged through successful cross-species transmission events on more recent time scales. The wide geographic distribution and genetic diversity of adenoviruses in bats and their close phylogenetic relationship to<jats:named-content content-type=\"genus-species\">Canine mastadenovirus<\/jats:named-content><jats:italic>A<\/jats:italic>(CAdV A) has raised important questions about how CAdV A, and possibly other mammalian adenoviruses, may have emerged. Although most adenoviruses tend to cause limited disease in their natural hosts, CAdV A is unusual in that it may cause high morbidity and sometimes fatal infections in immunocompetent hosts and is thus an important pathogen of carnivores. Here, we performed a comparative evolutionary and structural study of representative bat and canine adenoviruses to better understand the relationship between these two viral groups.<\/jats:p>","DOI":"10.1128\/jvi.01504-16","type":"journal-article","created":{"date-parts":[[2016,11,3]],"date-time":"2016-11-03T01:23:24Z","timestamp":1478136204000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":31,"title":["Evolution and Cryo-electron Microscopy Capsid Structure of a North American Bat Adenovirus and Its Relationship to Other Mastadenoviruses"],"prefix":"10.1128","volume":"91","author":[{"given":"Nicole","family":"Hackenbrack","sequence":"first","affiliation":[{"name":"Department of Medicine, The Pennsylvania State College of Medicine, Hershey, Pennsylvania, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthew B.","family":"Rogers","sequence":"additional","affiliation":[{"name":"Department of Computational and Systems Biology, Center for Vaccine Research, University of Pittsburgh, Pittsburgh, Pennsylvania, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robert E.","family":"Ashley","sequence":"additional","affiliation":[{"name":"Department of Medicine, The Pennsylvania State College of Medicine, Hershey, Pennsylvania, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M. 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