Evolutionary and temporal dynamics of emerging influenza D virus in Europe (2009-22)

Influenza D virus (IDV) is an emerging influenza virus that was isolated for the first time in 2011 in the USA from swine with respiratory illness. Since then, IDV has been detected worldwide in different animal species, and it was also reported in humans. Molecular epidemiological studies revealed the circulation of two major clades, named D/OK and D/660. Additional divergent clades have been described but have been limited to specific geographic areas (i.e. Japan and California). In Europe, IDV was detected for the first time in France in 2012 and subsequently also in Italy, Luxembourg, Ireland, the UK, Switzerland, and Denmark. To understand the time of introduction and the evolutionary dynamics of IDV on the continent, molecular screening of bovine and swine clinical samples was carried out in different European countries, and phylogenetic analyses were performed on all available and newly generated sequences. Until recently, D/OK was the only clade detected in this area. Starting from 2019, an increase in D/660 clade detections was observed, accompanied by an increase in the overall viral genetic diversity and genetic reassortments. The time to the most recent common ancestor (tMRCA) of all existing IDV sequences was estimated as 1995-16 years before its discovery, indicating that the virus could have started its global spread in this time frame. Despite the D/OK and D/660 clades having a similar mean tMRCA (2007), the mean tMRCA for European D/OK sequences was estimated as January 2013 compared to July 2014 for European D/660 sequences. This indicated that the two clades were likely introduced on the European continent at different time points, as confirmed by virological screening findings. The mean nucleotide substitution rate of the hemagglutinin-esterase-fusion (HEF) glycoprotein segment was estimated as 1.403 × 10^-3 substitutions/site/year, which is significantly higher than the one of the HEF of human influenza C virus (P < 0.0001). IDV genetic drift, the introduction of new clades on the continent, and multiple reassortment patterns shape the increasing viral diversity observed in the last years. Its elevated substitution rate, diffusion in various animal species, and the growing evidence pointing towards zoonotic potential justify continuous surveillance of this emerging influenza virus.