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

Abstract

BackgroundSwine 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.ResultsSeveral 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (10.1186/s12985-018-0920-z) contains supplementary material, which is available to authorized users.

Spatiotemporal Distribution and Evolution of the A/H1N1 2009 Pandemic Influenza Virus in Pigs in France from 2009 to 2017: Identification of a Potential Swine-Specific Lineage

“…The LSI VetMAX swine influenza A/H1N1/2009-H1 detection kit (Life Technologies, Carlsbad, CA, USA) and the Adiavet A/H1N1 (2009) real-time kit (Bio-X Diagnostics, Rochefort, Belgium) were used interchangeably to amplify the H1pdm gene, whereas the LSI VetMAX swine influenza A/H1N1/2009-N1 detection kit (Life Technologies, Carlsbad, CA, USA) was used to amplify the N1pdm gene (51). In-house RT-PCR assays specific for the HA or NA genes from other swine IAVs circulating in France and/or in Europe were run in parallel to identify either swine IAVs other than H1N1pdm, reassortant viruses, or virus mixtures (52,53).…”

Influenza A Virus in Swine: Epidemiology, Challenges and Vaccination Strategies

“…This virus was the result of reassortment between a North American "triplereassortant" swine influenza virus and a European H1avN1 (14). After its introduction in Europe, this H1N1pdm09 became established and widely reassorted with pre-existing H1N1, H3N2, and H1N2 subtypes, further complicating swine influenza epidemiology (6,10,(15)(16)(17)(18)(19). Moreover, the H1N1pdm09 internal gene cassette extensively reassorted with domestic viruses in the UK and became the dominant backbone there (13).…”

Evaluation of the Pathogenicity and the Escape from Vaccine Protection of a New Antigenic Variant Derived from the European Human-Like Reassortant Swine H1N2 Influenza Virus

“…Given the impact of swIAV in terms of animal health and public health due to zoonotic potential, monitoring the circulation and evolution of virus strains has been encouraged in many countries after the last influenza pandemic in 2009. In France, swIAV surveillance highlighted the emergence in 2012 of a new antigenic variant of H1N2 subtype, which results from a genetic drift in the H1 hu N2 virus circulating since the ‘90s (“Scotland/94” lineage; HA clade 1B.1.2.3) [ 2 , 3 ]. The haemagglutinin (HA)-encoding gene of the variant exhibits mutations in several antigenic sites, as well as two successive amino acid deletions at residue positions 146–147 (129–130 when H1 numbering without signal peptide), which are located in the cell receptor binding site (RBS) [ 2 , 4 ].…”

“…In France, swIAV surveillance highlighted the emergence in 2012 of a new antigenic variant of H1N2 subtype, which results from a genetic drift in the H1 hu N2 virus circulating since the ‘90s (“Scotland/94” lineage; HA clade 1B.1.2.3) [ 2 , 3 ]. The haemagglutinin (HA)-encoding gene of the variant exhibits mutations in several antigenic sites, as well as two successive amino acid deletions at residue positions 146–147 (129–130 when H1 numbering without signal peptide), which are located in the cell receptor binding site (RBS) [ 2 , 4 ]. The proportion of new variant H1 hu N2 Δ14–147 strains that were detected over the country among the H1 hu N2 viruses increased until reaching 50% in 2013–2014 [ 3 ].…”