Low-Input, High-Resolution 5' Terminal Filovirus RNA Sequencing with ViBE-Seq.

Publisher: MDPI Country of Publication: Switzerland NLM ID: 101509722 Publication Model: Electronic Cited Medium: Internet ISSN: 1999-4915 (Electronic) Linking ISSN: 19994915 NLM ISO Abbreviation: Viruses Subsets: MEDLINE

Original Publication: Basel, Switzerland : MDPI

RNA, Viral*/genetics ; High-Throughput Nucleotide Sequencing*/methods ; Genome, Viral* ; Ebolavirus*/genetics ; Sequence Analysis, RNA*/methods ; Filoviridae*/genetics; Marburgvirus/genetics ; Humans ; Animals

Although next-generation sequencing (NGS) has been instrumental in determining the genomic sequences of emerging RNA viruses, de novo sequence determination often lacks sufficient coverage of the 5' and 3' ends of the viral genomes. Since the genome ends of RNA viruses contain the transcription and genome replication promoters that are essential for viral propagation, a lack of terminal sequence information hinders the efforts to study the replication and transcription mechanisms of emerging and re-emerging viruses. To circumvent this, we have developed a novel method termed ViBE-Seq (Viral Bona Fide End Sequencing) for the high-resolution sequencing of filoviral genome ends using a simple yet robust protocol with high fidelity. This technique allows for sequence determination of the 5' end of viral RNA genomes and mRNAs with as little as 50 ng of total RNA. Using the Ebola virus and Marburg virus as prototypes for highly pathogenic, re-emerging viruses, we show that ViBE-Seq is a reliable technique for rapid and accurate 5' end sequencing of filovirus RNA sourced from virions, infected cells, and tissue obtained from infected animals. We also show that ViBE-Seq can be used to determine whether distinct reverse transcriptases have terminal deoxynucleotidyl transferase activity. Overall, ViBE-Seq will facilitate the access to complete sequences of emerging viruses.

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R21AI147285 National Institute of Allergy and Infectious Diseases; R21AI169646 National Institute of Allergy and Infectious Diseases; R01AI133486 National Institute of Allergy and Infectious Diseases; Agmt 9/16/22 Howard Hughes Medical Institute, Emerging Pathogens Initiative

Keywords: 5′ end RNA sequencing; Ebola virus; Marburg virus; ViBE-Seq; emerging viruses; filovirus; terminal deoxynucleotidyl transferase; viral bona fide end sequencing

0 (RNA, Viral)

Date Created: 20240727 Date Completed: 20240728 Latest Revision: 20240729

20240729

PMC11281615

10.3390/v16071064

39066227