In this study, we employed short- and long-read sequencing technologies to delineate the transcriptional architecture of the human monkeypox virus and to identify key regulatory elements that govern its gene expression. Specifically, we conducted a transcriptomic analysis to annotate the transcription start sites (TSSs) and transcription end sites (TESs) of the virus by utilizing Cap Analysis of gene expression sequencing on the Illumina platform and direct RNA sequencing on the Oxford Nanopore technology device. Our investigations uncovered significant complexity in the use of alternative TSSs and TESs in viral genes. In this research, we also detected the promoter elements and poly(A) signals associated with the viral genes. Additionally, we identified novel genes in both the left and right variable regions of the viral genome.IMPORTANCEGenerally, gaining insight into how the transcription of a virus is regulated offers insights into the key mechanisms that control its life cycle. The recent outbreak of the human monkeypox virus has underscored the necessity of understanding the basic biology of its causative agent. Our results are pivotal for constructing a comprehensive transcriptomic atlas of the human monkeypox virus, providing valuable resources for future studies.

Bernhard Nocht Institute for Tropical Medicine WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Reference and Research Hamburg Germany

Department of Experimental Biology Faculty of Science Masaryk University Brno Czechia

Department of Medical Biology Albert Szent Györgyi Medical School University of Szeged Szeged Hungary

Institute of Biology Faculty of Sciences University of Pécs Pécs Hungary

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Ceske Budejovice Czechia

National Laboratory of Virology Szentágothai Research Centre University of Pécs Pécs Hungary

Veterinary Research Institute Brno Czechia

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