Toward a better characterisation of the genetic diversity of circulating equine influenza virus strains by long-read sequencing

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Dhorne-Pollet, Sophie | Normand, Camille | Filipe-Ferreira, Mathilde | Fortier, Christine | Hue, Erika | Delmas, Bernard | Pollet, Nicolas | Barrey, Eric | Pronost, Stéphane

Edité par CCSD -

International audience. Background: Equine influenza virus (EIV) infection is one of the most important respiratory diseases in the equine industry. Knowing viral genome variants to monitor their evolution is essential to anticipate vaccine drift.Objectives: Develop a rapid and efficient method to identify circulating strains and characterise their complete genome for enhanced epidemiological surveillance.Study design: Whole genome sequencing.Methods: The genetic diversity of eight EIVs from France (2009–2023) was assessed using long-read sequencing. The two vaccine strains A/equine/South-Africa/4/2003(H3N8) and A/equine/Richmond/2/2007(H3N8) were included as controls. RNA was extracted from nasal swabs collected for clinical purposes and amplified theeight viral genomic segments by RT-PCR using primers complementary to the 50 and 30 ends of each segment. Equimolar pools of purified PCR products were prepared and sequencing libraries made for each strain. R10.3 flow cells operated on a MinION Mk1C device (ONT) were used and processed raw sequencing data using Dorado for basecalling and demultiplexing. Reads were filtered by size and mapped to one reference EIV genome to identify variants and assemble consensus sequences.Results: An average of 70 928 reads per segment and strain was obtained, corresponding to a mean coverage of 46%. The genetic diversity of the strains circulating in France was evaluated, with between 86 and 287 genetic variants per strain. Consensus sequences were constructed for each EIV strain's segment and confirmed the circulation of clade 1 in France. The experiments were reproduced using a smaller R10 Flongle flow cell to quickly and reliably characterise HA and NA for the circulating strains.Main limitations: The number of viral particles can limit sensitivity, and the design of primers limits amplification efficiency.Conclusions: Long-read sequencing technology enables rapid and efficient characterisation of the whole genetic diversity of circulating EIV strains. This method provides crucial information for epidemiological monitoring.Ethical animal research: Not required: excess material from clinical samples was used.Informed consent: Horse owners were made aware clinical material might be used for research purposes.Competing interests: None declared.Funding: IFCE: Equ'INFLUENZA project; LABEO.

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