Isolation of a Recombinant Simian Adenovirus Encoding the Human Adenovirus G52 Hexon Suggests a Simian Origin for Human Adenovirus G52

Human adenoviruses (HAdVs) are causative agents of morbidity and mortality throughout the world. These double-stranded DNA viruses are phylogenetically classified into seven different species (A-G). HAdV-G52, originally isolated in 2008 from a patient presenting with gastroenteritis, is the sole human-derived member of species G. Phylogenetic analysis previously suggested that HAdV-G52 may have a simian origin, indicating a potential zoonotic spillover into humans. However, evidence of HAdV-G52 in either human or simian populations has not been reported since. Here, we describe the isolation and characterization of rhesus (rh)AdV-69, a novel simian AdV with clear evidence of recombination with HAdV-G52, from the stool of a rhesus macaque. Specifically, the rhAdV-69 hexon capsid protein is 100% identical to that of HAdV-G52, whereas the remainder of the genome is most similar to rhAdV-55, sharing 95.36% nucleic acid identity. A second recombination event with an unknown adenovirus (AdV) is evident at the short fiber gene. From the same sample, we also isolated a second, highly related recombinant AdV (rhAdV-68) that harbors a distinct hexon gene but nearly identical backbone compared to rhAdV-69. , rhAdV-68 and rhAdV-69 demonstrate comparable growth kinetics and tropisms in human cell lines, nonhuman cell lines, and human enteroids. Furthermore, we show that coinfection of highly related AdVs is not unique to this sample since we also isolated coinfecting rhAdVs from two additional rhesus macaque stool samples. Our data collectively contribute to elucidating the origins of HAdV-G52 and provide insights into the frequency of coinfections and subsequent recombination in AdV evolution.IMPORTANCEUnderstanding the host origins of adenoviruses (AdVs) is critical for public health as transmission of viruses from animals to humans can lead to emergent viruses. Recombination between animal and human AdVs can also produce emergent viruses. HAdV-G52 is the only human-derived member of the HAdV G species. It has been suggested that HAdV-G52 has a simian origin. Here, we isolated from a rhesus macaque, a novel rhAdV, rhAdV-69, that encodes a hexon protein that is 100% identical to that of HAdV-G52. This observation suggests that HAdV-G52 may indeed have a simian origin. We also isolated a highly related rhAdV, differing only in the hexon gene, from the same rhesus macaque stool sample as rhAdV-69, illustrating the potential for co-infection of closely related AdVs and recombination at the hexon gene. Furthermore, our study highlights the critical role of whole-genome sequencing in understanding AdV evolution and monitoring the emergence of pathogenic AdVs.