Novel amplicon-based sequencing method for HRSV genotyping

Abstract

INTRODUCTION: Human Respiratory Syncytial Virus (HRSV) is considered the etiologic agent of diseases of the respiratory system with the greatest paediatric importance in the world. It is identified in children aged between zero and five years, who have respiratory failure, pneumonia, and bronchiolitis. It affects about 95% of children in their first two years of life and some adults although mainly the elderly and immunocompromised. Two antigenic subgroups of HRSV have been described, named A and B, both of which circulate separately or simultaneously during epidemics.

The COVID-19 pandemic has led to significant changes in the transmission dynamics of other respiratory viruses including HRSV in children. The autumn and winter seasons of 2020/21 saw almost no cases of HRSV in the United Kingdom and Ireland. An increase in HRSV cases was observed in July2021 and peaked in August, which is out of season for the UK as cases usually occur in November/December. In response to this, a National Institute for Health and Care Research (NIHR) study was funded called The BronchStart and which collects data from patients with HRSV infection such as age, sex, clinical severity on presentation, treatment received and outcomes (discharge/hospital admission/PICU admission). Also in 2021, the WHO has established an online repository of HRSV whole genome sequences (GISAID). To contribute to these studies and to aid laboratories worldwide to be able to readily sequence HRSV, we designed a novel short amplicon approach for sequencing complete HRSV genomes, which can be adapted from existing SARS-CoV-2 sequencing methodologies.

METHODS: We developed our own primer scheme for a shorter amplicon-based sequencing approach to be used in the existing SARS-CoV-2 sequencing infrastructure. A total of 6 HRSV A and 6 HRSV B genomes, with collection dates spanning 2019 to 2021, and locations spanning the UK, Australia and South Africa were used to develop the primers using Primal Scheme, resulting in two tiled ~400bp amplicon schemes for HRSV A and HRSV B. To validate the method, we selected NHS Lothian (Edinburgh, Scotland) HRSV samples from 2019 to 2022 and followed the ARTIC-loCost-v3 nanopore library preparation method, with modifications. Data analysis was performed using an in-house version of the “fieldbioinformatics” pipeline.

RESULTS AND DISCUSSION: The short amplicon-based approach provided PCR products which could be taken forward to nanopore library preparation and sequencing. We tested these in a subsection of our sample pool from 2019 to 2022 with a range of different Ct values. From 45 clinical samples for HRSV A, 39 (86.7%) showed genome completeness above 90%, with a median genome completeness across all HRSV A samples of 96.6%. For HRSV B, 39 (86.7%) out of 45 clinical samples showed genome completeness above 90%, with a median genome completeness of 97.2%. Future work includes determining the Ct cut off to find the upper limit for successful sequencing, testing of primer schemes on Illumina sequencing platforms, and efforts will be made to identify primers that can be spiked in to combat amplicon dropouts.

CONCLUSION: There is a clear need to develop more protocols for Whole Genome Sequencing for HRSV and to submit new data to GISAID aiming to contribute to the study of epidemiology of HRSV. This will assist in the knowledge of viral circulation and in the prevention of future public health problems. We developed an effective and easy-to-use tiled amplicon approach for HRSV which will be able to be used in existing SARS-CoV-2 sequencing infrastructures, allowing high throughput HRSV WGS.