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© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

Abstract

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.

Details

Title
Low-Input, High-Resolution 5′ Terminal Filovirus RNA Sequencing with ViBE-Seq
Author
Ross, Stephen J 1   VIAFID ORCID Logo  ; Hume, Adam J 2   VIAFID ORCID Logo  ; Olejnik, Judith 2   VIAFID ORCID Logo  ; Turcinovic, Jacquelyn 2   VIAFID ORCID Logo  ; Honko, Anna N 2   VIAFID ORCID Logo  ; McKay, Lindsay G A 2   VIAFID ORCID Logo  ; Connor, John H 2 ; Griffiths, Anthony 2   VIAFID ORCID Logo  ; Mühlberger, Elke 2   VIAFID ORCID Logo  ; Cifuentes, Daniel 3   VIAFID ORCID Logo 

 Department of Virology, Immunology & Microbiology, Chobanian and Avedisian School of Medicine, Boston University, Boston, MA 02215, USA; [email protected] (S.J.R.); [email protected] (A.J.H.); [email protected] (J.O.); [email protected] (J.T.); [email protected] (A.N.H.); [email protected] (L.G.A.M.); [email protected] (J.H.C.); [email protected] (A.G.); National Emerging Infectious Diseases Laboratories, Boston University, Boston, MA 02215, USA; Department of Biochemistry & Cell Biology, Chobanian and Avedisian School of Medicine, Boston University, Boston, MA 02215, USA 
 Department of Virology, Immunology & Microbiology, Chobanian and Avedisian School of Medicine, Boston University, Boston, MA 02215, USA; [email protected] (S.J.R.); [email protected] (A.J.H.); [email protected] (J.O.); [email protected] (J.T.); [email protected] (A.N.H.); [email protected] (L.G.A.M.); [email protected] (J.H.C.); [email protected] (A.G.); National Emerging Infectious Diseases Laboratories, Boston University, Boston, MA 02215, USA 
 Department of Virology, Immunology & Microbiology, Chobanian and Avedisian School of Medicine, Boston University, Boston, MA 02215, USA; [email protected] (S.J.R.); [email protected] (A.J.H.); [email protected] (J.O.); [email protected] (J.T.); [email protected] (A.N.H.); [email protected] (L.G.A.M.); [email protected] (J.H.C.); [email protected] (A.G.); Department of Biochemistry & Cell Biology, Chobanian and Avedisian School of Medicine, Boston University, Boston, MA 02215, USA 
First page
1064
Publication year
2024
Publication date
2024
Publisher
MDPI AG
e-ISSN
19994915
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
3084992772
Copyright
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.