2nd Periodic Report (12/2018 - 05/2020)

Summary of the context and overall objectives of the project

Avian influenza is a major impediment to healthy and profitable poultry production in Europe and other parts of the world. The dynamics of avian influenza virus (AIV) infection in poultry populations have changed dramatically in recent years, resulting in major mortality of birds and significant economic losses. A striking example is the 2014-2015 emergence of highly pathogenic avian influenza virus of the subtype H5N8, which spread not only from South-East Asia to Europe, but also to North America, causing outbreaks in poultry holdings of seven countries with a loss of over 50 million heads of poultry. The subsequent introduction of H5N8 viruses in 2016/2017 was the source of numerous outbreaks in poultry farms in many European countries and underlines the importance of this virus type for farming but also wild life. The subsequent detections of the related H5N6 virus as well as novel introductions by H5N8 in early 2020 demonstrate the continuous risks. In addition to the risk for poultry holdings worldwide, the potential for animal influenza viruses to become pandemic in humans is of great concern to public health. This occurred most recently in 2009, when a reassortant H1N1 influenza virus with gene segments from human, pig, and avian viruses caused the first influenza pandemic of the 21st century.
The overall objective of DELTA-FLU is to determine the key viral, host-related, and environmental factors that determine the dynamics of avian influenza in poultry and other host species, with the goal of improving prevention and control strategies against this important disease.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Our research continued to be focused on the key questions of DELTA-FLU yielding new achievements important for influenza science. Using GSM-GPS loggers the movement of wild ducks was followed. By receiving tracks from Eurasia and North America gaps could be filled regarding the overlap of migratory birds in Siberian breeding grounds. Studies are in progress to incorporate movement ecology with pathogen dispersal. Moreover, studies with wild birds have been performed to investigate levels of virulence, patterns of excretion and effects of prior exposure to HPAIV. Results from these experiments indicated mutations which are responsible for changes in transmissibility and virulence in viruses switching from poultry to wild waterbirds and back from wild waterbirds to poultry. 
It was shown that domestic duck populations are an important intermediate host population for HPAI viruses switching from wild waterbirds to terrestrial poultry. Ecological and environmental factors associated with AIV incursion from wild birds to poultry were investigated using data from H5N8 outbreaks in Italy. Visual and audio census methods in addition to camera traps were used to generate an ornithocoenosis database which provides input for models to enable prediction of species distribution close to poultry farms in specific environments. Positive sediment and environmental samples have been collected from sheds of an H6N5 AIV infected poultry farm in the UK. Results reveal that environmental samples could serve as valuable tool for AIV surveillance and for understanding of virus ingress mechanisms in the field. Further investigations of virus contamination in feed and water supports evidence that HPAIV environmental contamination in a high biosecurity barn unit can cause infection in susceptible hosts and lead to disease spread through commercial and domestic flocks. Virus host interaction and adaptation of AIVs was studied in chickens, ducks and pigs in vitro, in ovo, in vivo and ex vivo. 
Mallard ducks were found to play a role as virus reservoir for HPAIV H5N8 effective in transmission and it was shown that LP infection protects chickens or ducks from homotypic HP challenges with H7N7 or H5N8 viruses, respectively. Virulence determinants other than the HA segment of German isolates of H7N7 and H5N8 subtype have been investigated in different host species. In conclusion, virulence determinants differ dependent on virus strain and host species. Furthermore, different models for in vitro virus characterisation have been established. Virulence studies have been carried out to investigate the pathogenicity of poultry-origin viruses in pigs. The results revealed that H9N2 and H3N1 viruses were not efficient in replication in pig nasal muscosa informing risk assessment of potential adaptation of bird viruses to mammals.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The collection and analysis of migration data will contribute to an improved understanding of connectivity between Asia and Europe via migratory birds as a potential portal of AIV incursion, which has a high impact on all future risk analyses and will allow new insights into AIV ecology. The results will form a basis for more targeted temporal and geographical surveillance of AIV in migratory waterfowl and identification and strengthening of weak spots in biosecurity around poultry holdings. The data will also allow a more precise prediction of introduction events e.g. due to bird migration.
The database on notifiable AIV outbreak data since 2005, the collection of data about the ecology of the wild bird-poultry interface, and the experimental modelling of the biosecurity barriers around poultry holdings elucidating possibly unrecognized routes by which AIV can cross the biosecurity barriers around poultry holdings, as well as variations among AIV strains in stability on fomites and ability to switch from anseriform to galliform hosts. 
Understanding the underlying innate immune characteristics of poultry and the results obtained in AIV infection experiments will elucidate the relative importance of different components of the innate immune system against AIV infections, and the impact of the pre-infection adaptive immune status of poultry on the early detection and evolution of AIV. This will provide the necessary knowledge for future risk assessments and control strategies. This is of high importance since both the number of AIV introductions into Europe and the number of connected outbreaks in wild birds and poultry are as high as never before. The socio-economic impact of our data is therefore much higher as also the impact of AIV has so much increased within the last few years. Besides the commercial poultry farming it has also a considerable influence on the private breed of poultry and also the wild bird area, especially as far as protected species are concerned. It is therefore directly influencing and helping the commercial and the private sector and people who are engaged in those areas. The knowledge can then also be used by international organizations and the measures that have now been developed can also be taken into consideration in national and EU legislation. Finally, private industry working in the poultry field might implement measures developed here within the consortium to improve the biosecurity measures and lower the introduction rates.
Our studies will also help to assess the zoonotic risk and to improve the understanding of human spill over infections or the development of influenza virus strains with pandemic potential.
The establishment of ex vivo explants from chicken and ducks and of the porcine respiratory tract explant system is an effective way of representing the in vivo conditions and of reducing the number of animals used in experiments. This is of high impact for animal welfare ethics and is very much in compliance with the 3R principles.