A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses

Xiaomin Dong; Yi-Mo Deng; Ammar Aziz; Paul Whitney; Julia Clark; Patrick Harris; Catherine Bautista; Anna-Maria Costa; Gregory Waller; Andrew J Daley; Megan Wieringa; Tony Korman; Ian G Barr

doi:10.1016/j.jcv.2023.105423

A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses

J Clin Virol. 2023 Apr:161:105423. doi: 10.1016/j.jcv.2023.105423. Epub 2023 Mar 12.

Authors

Affiliations

¹ WHO Collaborating Centre for Reference and Research on Influenza, Royal Melbourne Hospital, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, 3000, Australia; Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, 3000, Australia.
² Queensland Children's Hospital, Brisbane, QLD, 4101, Australia; Children's Health Queensland Hospital & Health Service, Brisbane, QLD, 4101, Australia.
³ UQ Centre for Clinical Research, Faculty of Medicine, University of Queensland, Herston, QLD, 4029, Australia; Central Microbiology, Pathology Queensland, Royal Brisbane & Women's Hospital, Herston, QLD, 4006, Australia.
⁴ Central Microbiology, Pathology Queensland, Royal Brisbane & Women's Hospital, Herston, QLD, 4006, Australia.
⁵ Department of Microbiology and Infectious Diseases, The Royal Children's Hospital Melbourne, Parkville, VIC, 3052, Australia.
⁶ Department of Microbiology, Infection Prevention and Control, The Royal Children's and Royal Women's Hospitals, Parkville, VIC, 3052, Australia.
⁷ Monash Health, Clayton, VIC, 3168, Australia.
⁸ WHO Collaborating Centre for Reference and Research on Influenza, Royal Melbourne Hospital, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, 3000, Australia; Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, 3000, Australia. Electronic address: ian.barr@influenzacentre.org.

PMID: 36934591
DOI: 10.1016/j.jcv.2023.105423

Abstract

Background: Human Respiratory Syncytial Virus (RSV) infections pose a significant risk to human health worldwide, especially for young children. Whole genome sequencing (WGS) provides a useful tool for global surveillance to better understand the evolution and epidemiology of RSV and provide essential information that may impact on antibody treatments, antiviral drug sensitivity and vaccine effectiveness.

Objectives: Here we report the development of a rapid and simplified amplicon-based one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) for WGS of both human RSV-A and RSV-B viruses.

Study design: Two mRT-PCR reactions for each sample were designed to generate amplicons for RSV WGS. This new method was tested and evaluated by sequencing 206 RSV positive clinical samples collected in Australia in 2020 and 2021 with RSV Ct values between 10 and 32.

Results: In silico analysis and laboratory testing revealed that the primers used in the new method covered most of the currently circulating RSV-A and RSV-B. Amplicons generated were suitable for both Illumina and Oxford Nanopore Technologies (ONT) NGS platforms. A success rate of 83.5% with a full coverage for the genome of 98 RSV-A and 74 RSV-B was achieved from all clinical samples tested.

Conclusions: This assay is simple to set up, robust, easily scalable in sample preparation and relatively inexpensive, and as such, provides a valuable addition to existing NGS RSV WGS methods.

Keywords: One-step multiplex RT-PCR; Respiratory syncytial virus; Whole genome sequencing.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antiviral Agents
Child
Child, Preschool
Humans
Multiplex Polymerase Chain Reaction
Respiratory Syncytial Virus Infections* / diagnosis
Respiratory Syncytial Virus Infections* / epidemiology
Respiratory Syncytial Virus, Human* / genetics
Sensitivity and Specificity

Substances

Antiviral Agents