Abstract

Design and evaluation of RSVAB-WGS targeted PCR

For the design, we aligned 922 selected sequences obtained from the GenBank and GISAID databases. We chose a total of 12 primers for the RSVAB-WGS method and two additional primers for RSVAB-GF method (Table 1). A mix of these primers collectively cover both RSV subtypes, generating PCR fragments of an average size of 1.5–2.5 kb in the WGS and 3.0–3.5 kb in the GF protocol. Both amplification protocols are openly available on the platform protocols.io [5].

The PCR parameters and the final sensitivity of the chosen protocol were evaluated using dilutions of well-characterised reference viruses, RSV A Long and RSV B CH-18537, as control samples (Table 2). Our method exhibited a sensitivity of 20 copies/mL for RSV A and 200 copies/mL for RSV B (Table 2).

We constructed libraries following the instructions provided by the Illumina DNA library preparation kit (Illumina, United States) for 300-cycle cartridges in Illumina MiSeq and NextSeq sequencers. Sequences were analysed for viral genome reconstruction using the viralrecon pipeline v2.6.0. (https://github.com/nf-core/viralrecon) [6], implemented in Nextflow (https://www.nextflow.io). Phylogenetic analysis was conducted using FasTreeMP software [7] with a generalised time reversible (GTR) model and a bootstrap test of 1,000 iterations.

For validation, we analysed a total of 142 nasopharyngeal exudates (NPE) and nasopharyngeal aspirates (NPA) collected from RSV-positive patients during the epidemic seasons 2018/19, 2019/20 and 2021/22. To assess the RSV viral load and facilitate subsequent sequencing, we performed a real-time PCR to detect RSV and used the resulting quantification cycle (Cq) value as a preliminary indicator [1,8]. Despite the usefulness of setting a cut-off in the Cq values used to ensure the complete genome acquisition, in this work, a deliberate decision was made not to set it. The aim was to determine the limit of detection in the method by sequencing both our lowest Cq value sample from the season (14.33) and the highest (29.4).

Complete genome sequencing of respiratory syncytial virus A and B

For surveillance purposes, the RSVAB-WGS method exhibited practical versatility and proved to be applicable to specimens of both RSV subtypes across a spectrum of viral loads. Validation involved analysing 34 positive clinical specimens, 16 RSV A and 18 RSV B, previously typed [8]. These samples showed Cq values ranging from 15 to 20 (10 RSV A, eight RSV B), 21 to 26 (six samples of either subtype) and exceeding 27 (four RSV B). For samples with Cq values≤25, RSVAB-WGS achieved more than 90% genome coverage. For Cq values 26–27, coverage ranged from 60% to 90%, and for Cq values exceeding 27, coverage dropped to 50%. We append in Supplementary Table S1 the detailed sequencing parameters for RSV clinical specimens used in RSVAB WGS method including a description of subtypes, Cq values, sequencing/assembly metrics and GISAID accession numbers.

Recognising the importance of accurate RSV genomic characterisation, we developed the RSVAB-GF method to complement the genomic coverage for both major antigenic proteins in cases where WGS encountered coverage difficulties, such as in samples with low viral load or when the resources for sequencing were constrained. This method offers a simpler and more cost-effective approach to obtaining sequences of both antigens. Validation involved analysing 108 clinical specimens (74 RSV A, 34 RSV B) with Cq values ranging from 14.3 to 29.4 and in all cases, both genes were completely covered. We provide in Supplementary Table S2 a description of the samples used in the targeted RSV AB-GF method including sequence identification, subtype, Cq value and GISAID accession numbers.

Genetic analysis of the genomes obtained with RSVAB-WGS and RSVAB-GF

To compare taxonomic classifications obtained from full genome sequences and partial G and F sequences, we used 14 sequences (seven RSV A, seven RSV B) collected during the 2021/22 season (Table 3). We used the whole genome to assign RSV clades because either the complete sequence or the F gene sequence was available to us in addition to the G gene sequence. These clade definitions were based on the proposed nomenclatures by Goya et al. [9] and Ramaekers et al. [10] using Nextstrain platform. Analysis of the 14 RSV A sequences with both methods demonstrated complete agreement in clade assignation. For the seven RSV A sequences, identified subclades included A.D.1, A.D.3 and A.D.5.2. Taxonomic assignments from seasons 2018/19 and 2019/20 showed the presence of subclades A.D.1, A.D.3 (both also circulating in the 2021/22 season) and subclade A.D.2.3 (Table 3). In the RSV B subtype, clade assignment revealed total agreement in taxonomic classification. The seven sequences from the 2021/22 season were identified as subclade B.D.5.2.1.1 with both methods.

For the phylogenetic analysis (Figure), we selected sequences from the main antigens G and F due to the high number of sequences with good quality and coverage in the GISAID database in comparison with those of the complete genome. We constructed a separate tree for each RSV subtype. In both subtypes, sequence clustering was primarily determined by mutations spanning consecutive seasons rather than by year or geographical location. The phylogenetic analysis of 2,314 RSV A sequences, including 74 sequences from this study, and 2,875 RSV B sequences, with the 34 G and F sequences obtained from this work, corroborated previous taxonomic results (Figure) [11].

Open in a separate window

Figure

Phylogenetic tree of respiratory syncytial virus G and F sequences published in GISAID (n=2,314 RSV A; n=2,875 RSV B)

F: fusion protein; G: glycoprotein; RSV: respiratory syncytial virus.

The numbers represent percentage bootstrap support. The sequences obtained in this work were represented by triangles. Clusters, where these sequences were located, are identified by three colours (red, blue and green) according to the defining mutations. The colours yellow and purple mark the nodes with the defining mutations of the group.

Discussion

Several methods have already been designed for sequencing RSV isolates or samples, employing diverse approaches [12-16]. However, the primary challenges in successful RSV sequencing often involve obtaining complete genomes from clinical samples with low viral loads [17] and high diversity of RNA genomes [18]. Previous methods often required prior subtyping and quantification due to differences in primer design based on subtypes [13,19], making the process expensive and labour-intensive. PCR amplicon sequencing, using amplicons generated through specific primers [20,21] or sequence-independent single primer amplification (SISPA) [13], exhibits notable advantages, particularly in clinical specimens with low viral loads [13]. The effectiveness of double-stranded cDNA generation before targeted amplification has proven effective for other respiratory viruses like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [22,23], and it was also validated in this work for RSV. The use of cDNA in our RSVAB- WGS method provided an augmented amount of viral nucleic acids using only a few microlitres of the clinical sample extract, optimising sample usage. Amplicon size is critical, as shorter amplicons can introduce biases in genome reconstruction and prevent the successful assembly of a full genome sequence for unknown viruses. The method described in this work produces amplicons compatible with both long and short-read sequencers, offering a versatile solution for laboratories, irrespective of the type of sequencer available.

To demonstrate the utility of our RSVAB-WGS and RSVAB-GF methods in molecular epidemiology studies of RSV, we conducted phylogenetic and genomic analyses using samples collected during the 2018/19, 2019/20 and 2021/22 epidemic RSV seasons. The phylogenetic analysis revealed that changes in the G protein gene had a larger impact on grouping than the timing of virus collection or geographical location. While WGS for monitoring viral transmission chains can offer great accuracy, studies indicate that the relationships identified with the G region are similar to the patterns observed with full-genome sequences [16]. Furthermore, a previous world-wide analysis of the RSV clades [11] demonstrated that entirely sequenced complete G gene sequences yielded a phylogenetic tree topology comparable to that obtained using whole genomes. This underscores the utility of the RSVAB-GF method in providing evolutionary information through a simplified system that could be used when the viral load is low and also when the resources for sequencing are not enough to support a WGS system.

In addition, we currently find it highly pertinent to characterise the F protein gene variability with an accurate and robust complementary method such as the RSVAB-GF PCR. This is especially crucial since the F protein is the main target for most vaccines, which are either already approved [1] or soon expected to be. Moreover, there are several monoclonal antibodies for RSV prevention [3], such as nirsevimab or palivizumab, all of which also target the F protein.

Despite the advantages presented by these methods, we also have to address limitations in the WGS method, such as a decrease in coverage starting from Cq = 25 and the need to improve the complete genome sequencing in RSV B. Currently, the method is being improved to solve these issues. Although this work was confined to the sequences received in our laboratory, we believe this does not restrict the capability of these methods to sequence any type of RSV because the primer design was performed using sequences from various locations and different seasons.

The World Health Organization has started a global effort to homogenise RSV surveillance, based on the Global Influenza Surveillance and Response System [11]. The surveillance system needs to be supported by reliable and practical methods.

Conclusions

While implementing WGS of RSV remains technically demanding and expensive, many laboratories express the need for guidance. Our universal amplicon-based system RSVAB-WGS is an accurate and cost-effective means to obtain genomic data. Coupled with the RSVAB-GF method, it enhances capacity and performance for high-quality sequence acquisition from both genes in a single run, even when processing hundreds of samples. These methods to characterise the complete RSV genome and the variability of G and F protein genes can serve as a useful tool for RSV surveillance, including monitoring of preventive measures to limit the spread of the virus.

Ethical statement

Data availability statement

Funding statement

Acknowledgements

Supplementary Data

Notes

Notes

Conflict of interest: None declared.

References