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.

High pathogenicity avian influenza viruses clade 2.3.4.4 H5 have spread among wild birds worldwide during recent years causing annual die-offs among wild birds and outbreaks in poultry in multiple European countries. The outbreaks significantly impact the poultry and game bird sectors. Infected game birds may act as a bridging species potentially enabling spread of virus into commercial and backyard premises. In this study, the pathogenesis and transmission of a HPAIV clade 2.3.4.4b H5N6 virus was investigated in pheasants and chickens. Efficient virus transmission was detected between pheasants over multiple rounds of naïve pheasant introductions and onwards to chickens. Mortality of up to 100% was observed for both infected pheasants and chickens. Intra-species transmission from chicken to chicken was less efficient. The study confirmed that clade 2.3.4.4b H5N6 HPAIV is highly virulent in pheasants and emphasises the role of pheasants as a bridging host for the infection of commercial poultry.

Beginning in late 2017, highly pathogenic avian influenza (HPAI) H5N6 viruses caused outbreaks in wild birds and poultry in several European countries. H5N6 viruses were detected in 43 wild birds found dead throughout Denmark. Most of the Danish virus-positive dead birds were found in the period from February to April 2018. However, unlike the rest of Europe, sporadic HPAI H5N6-positive dead wild birds were detected in Denmark in July, August, September, and December 2018, with the last positive bird being found in January 2019. HPAI viruses were not detected in active surveillance of apparently healthy wild birds. In this study, we use full genome sequencing and phylogenetic analysis to investigate the wild bird HPAI H5N6 viruses found in Denmark. The Danish viruses were found to be closely related to those of contemporary HPAI H5N6 viruses detected in Europe. Their sequences formed two clusters indicating that at least two or more introductions of H5N6 into Denmark occurred. Notably, all viruses detected in the latter half of 2018 and in 2019 grouped into the same cluster. The H5N6 viruses appeared to have been maintained undetected in the autumn 2018.

Influenza D virus (IDV) was first described in 2011 and has been found to mainly circulate among cattle and swine populations worldwide. Nasal swab samples were collected from 100 Danish calf herds (83 dairy and 17 veal herds) from 2018–2020. Influenza D virus was detected in 12 of the herds. Samples with the lowest cycle quantification value were selected for full genome sequencing. A hemagglutinin-esterase fusion (HEF) gene sequence from a Danish IDV collected in 2015 was also included in this study. Phylogenetic analysis showed that viruses from seven of the IDV-positive herds belonged to the D/OK lineage and clustered together in the HEF tree with the IDV collected in 2015. Viruses from the four other herds belonged to the D/660 lineage, where three of the viruses clustered closely together, while the fourth virus was more phylogenetically distant in all gene segments. The high level of genetic similarity between viruses from two different herds involved in calf trading suggests that transmission occurred through the movement of calves. This study is, to our knowledge, the first to describe the characterization of IDV in calves in Denmark.

In 2021 and 2022, clade 2.3.4.4b H5Nx high pathogenicity avian influenza viruses were detected in one harbor seal and in one adult and three fox cubs in Denmark. The viruses were closely related to contemporary viruses found in Europe, and some had obtained amino acid substitutions related to mammalian adaptation. Notably, the virus distribution appeared to have been different in the infected fox cubs, as one exclusively tested positive for the presence of HPAIV in the brain and the other two only in the lung. Collectively, these findings stress the need for increased disease surveillance of wild and farmed mammals.