Molecular subtyping of European swine influenza viruses and scaling to high-throughput analysis

Emilie Bonin, Stéphane Quéguiner, Cédric Woudstra, Stéphane Gorin, Nicolas Barbier, Timm C. Harder, Patrick Fach, Séverine Hervé, Gaëlle Simon

Swine influenza is a respiratory infection of pigs that may have a significant economic impact in affected herds and pose a threat to the human population since swine influenza A viruses (swIAVs) are zoonotic pathogens. Due to the increasing genetic diversity of swIAVs and because novel reassortants or variants may become enzootic or have zoonotic implications, surveillance is strongly encouraged. Therefore, diagnostic tests and advanced technologies able to identify the circulating strains rapidly are critically important. Several reverse transcription real-time PCR assays (RT-qPCRs) were developed to subtype European swIAVs in clinical samples previously identified as containing IAV genome. The RT-qPCRs aimed to discriminate HA genes of four H1 genetic lineages (H1av, H1hu, H1huΔ146-147, H1pdm) and one H3 lineage, and NA genes of two N1 lineages (N1, N1pdm) and one N2 lineage. After individual validation, each RT-qPCR was adapted to high-throughput analyses in parallel to the amplification of the IAV M gene (target for IAV detection) and the β-actin gene (as an internal control), in order to test the ten target genes simultaneously on a large number of clinical samples, using low volumes of reagents and RNA extracts. The RT-qPCRs dedicated to IAV molecular subtyping enabled the identification of swIAVs from the four viral subtypes that are known to be enzootic in European pigs, i.e. H1avN1, H1huN2, H3N2 and H1N1pdm. They also made it possible to discriminate a new antigenic variant (H1huN2Δ146-147) among H1huN2 viruses, as well as reassortant viruses, such as H1huN1 or H1avN2 for example, and virus mixtures. These PCR techniques exhibited a gain in sensitivity as compared to end-point RT-PCRs, enabling the characterization of biological samples with low genetic loads, with considerable time saving. Adaptation to high-throughput analyses appeared effective, both in terms of specificity and sensitivity. This new development opens novel perspectives in diagnostic capacities that could be very useful for swIAV surveillance and large-scale epidemiological studies.