Swine influenza viruses in Denmark: Prevalence and genetic and antigenic characterization

Funding: CEVA Animal Health and the Danish Veterinary and Food Administration. 2021. DKK 1.054,000.

Influenza A virus infection in animals poses a threat to animal health, animal welfare, production economics, food security and has regularly given rise to pandemics in humans, as in 2009 (H1N1pdm09). In order to act quickly on new threats (early warning) and thereby minimize the consequences of emerging strains, it is necessary to ensure a quick and effective exchange of virological and epidemiological information on circulating influenza virus between national and international health authorities and veterinary authorities.

The overall purpose of the project is to investigate which influenza subtypes and variants that are circulating among Danish pigs and to identify disease causes in the pig population with a view to securing the strategic goal: to reduce antibiotic consumption in Danish pig herds.

Nose swabs, lung pieces or saliva samples are submitted from private vet practices, the DTU or SEGES Laboratory for Swine Diseases in Kjellerup. RNA is extracted from the individual samples and tested for influenza A virus with real time RT-PCR assay targeting the matrix gene. Influenza virus positive samples are then screened for viruses having the H1pdm09 variant of the HA gene with two specific real-time RT-PCR assays. The result is then reported to the submitter.

The positive samples are further subtyped using multiplex PCR analysis, which tests for the eight subtypes of HA and NA relevant to the Danish swine population. If the result is negative, the segments are sequenced, in order to detect possible introduction of a new variant of HA and / or NA.

Based on the subtype, samples are selected for full genome characterization by "next generation sequencing" (NGS). Viruses are isolated from the selected samples and cultured in MDCK or SIAT cells. NGS is performed on full-length PCR products from all eight segments. The resulting PCR products are purified and sequenced by NGS on the Illumina MiSeq sequencing platform. Raw data are processed (CLC genomics workbench, QIAGEN) and consensus sequences for each segment are analyzed with phylogenetic analyzes (CLC main workbench, QIAGEN and MrBayes).

The results are reported quarterly in tabular forms and once a year to the Danish Veterinary and Food Administration in an extensive final report.

Collaborating partners: Statens Serum Institut (SSI), Fødevarestyrelsen (FVST) CEVA AH