Novel Clade 2.3.4.4b Highly Pathogenic Avian Influenza A H5N8 and H5N5 Viruses in Denmark, 2020

Abstract

Since late 2020, outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses belonging to clade 2.3.4.4b have emerged in Europe. To investigate the evolutionary history of these viruses, we performed genetic characterization on the first HPAI viruses found in Denmark during the autumn of 2020. H5N8 viruses from 14 wild birds and poultry, as well as one H5N5 virus from a wild bird, were characterized by whole genome sequencing and phylogenetic analysis. The Danish H5N8 viruses were found to be genetically similar to each other and to contemporary European clade 2.3.4.4b H5N8 viruses, while the Danish H5N5 virus was shown to be a unique genotype from the H5N5 viruses that circulated at the same time in Russia, Germany, and Belgium. Genetic analyses of one of the H5N8 viruses revealed the presence of a substitution (PB2-M64T) that is highly conserved in human seasonal influenza A viruses. Our analyses showed that the late 2020 clade 2.3.4.4b HPAI H5N8 viruses were most likely new incursions introduced by migrating birds to overwintering sites in Europe, rather than the result of continued circulation of H5N8 viruses from previous introductions to Europe in 2016/2017 and early 2020.

Keywords: Europe; H5N5 subtype; H5N8 subtype; disease outbreaks; influenza A virus; influenza in birds; phylogeny; poultry; wild.

Figure 1

Schematic representation of the HPAI H5N8 and H5N5 viruses and geographic distribution of the viruses included in this study. The schematic representation shows the likely origin of the individual gene segments of the Danish H5N8 and H5N5 viruses. Blue bars represent gene segments that were most closely related to European HPAI H5N8 viruses from late 2020. Red bars represent segments that were closely related to Russian LPAI viruses from wild birds. Yellow bars represent gene segments that were closely related to LPAI viruses from wild birds elsewhere in Eurasia. On the map, every dot represents one bird found infected with HPAI virus. The blue dots represent dead, wild birds infected with HPAI H5N8 viruses. The red dot denotes the HPAI H5N8 detection from a commercial poultry. The green dot represents the H5N5 detection in a dead, wild peregrine falcon. The map was constructed with QGIS version 13.18.2 [20] using an outline of Denmark from Kortforsyningen (www.kortforsyningen.dk, downloaded 26 April 2012).

Figure 2

Maximum clade credibility trees of PB2, PA, HA, NP, and N5 gene segments from strict molecular clock analyses. The trees include gene segment sequences from Danish H5N8 and H5N5 viruses from 2020, representative Eurasian clade 2.3.4.4 HPAI H5 viruses and LPAI viruses. The numbers above the nodes represent time to most recent common ancestor (tMRCA). Tip labels are colored according to the background of the detections. Green: H5N5 wild bird virus; red: H5N8 virus from poultry; blue: H5N8 viruses from wild birds. The scale bar depicts the timeline. PB2, polymerase basic protein 2; PA, polymerase acidic protein; HA, hemagglutinin; NP, nucleoprotein; NA, neuraminidase.