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Modulation of highly pathogenic avian influenza virus H5N8 infection in naturally low pathogenic avian influenza virus - exposed mallard ducks

S Koethe, L Ulrich, A. Globig, T Harder, FJ Conraths, M Beer

Abstract
The 2016/2017 highly pathogenic avian influenza virus (HPAIV) H5Nx clade 2.3.4.4b outbreak was the most severe ever reported in Germany with high mortality rates not only among domestic birds, but also in wild birds.
Mallard ducks have been kept as sentinels for circulating field AIV in an aviary located at the shallows of the Baltic coast in close contact to wild waterfowl and migratory birds. The mallards were regularly tested for AIV infection. Between August 2017 and October 2018, four low pathogenic (LP) AIV infections of individual mallards were confirmed - H1N3, H3N8, H4N6 and HxN9. One month prior to the HPAIV study, all mallard ducks were tested seropositive in an IAV NP-ELISA, and two out of seven had H5-specific antibodies.
To study dynamics of HPAIV H5N8 clade 2.3.4.4b infection after a prior natural field exposure to LPAIV, seven LPAIV exposed mallards were challenged with HPAIV H5N8. The surviving ducks were re-challenged with the homologous HPAIV H5N8. For both challenge infections, virus transmission to seronegative juvenile Pekin contact ducks was investigated. Seronegative adult Pekin ducks served as a control group.
Summarizing the results, during the first challenge infection all control group ducks died peracutely or had to be euthanized due to severe neurological symptoms. However, although one mallard duck died, the mallards showed only very few clinical symptoms, but three out of four contact juvenile Pekin ducks died. H5-specific antibodies were detectable in all surviving birds. Infectious virus could be isolated from several pond and drinking water samples. In contrast, after the homologous H5N8 re-challenge, neither inoculated nor contact ducks showed any clinical symptoms, nor was an AI-specific antibody titer increase or seroconversion of contact animals determined.
In conclusion, the study simulated an HPAIV H5N8 outbreak under semi-natural conditions with LPAIV-exposed waterfowl. Mallards with preceding LPAIV immunity, did not show any severe clinical or fatal disease, but sufficient virus shedding caused fatal illness in naïve contact ducks. In contrast, H5N8-convalescent animals developed a very robust immunity when re-challenged with the homologous virus shown by the absence of virus shedding and seroconversion of the contact animals.

Talk at the European Congress of Virology in Rotterdam, The Netherlands (28 April - 1 May 2019)

Impact of the polybasic cleavage site within the HA of a recent German H7N7 virus on its pathogenicity in different poultry species

Scheibner D., Veits J., Mettenleiter T.C. and Abdelwhab E.M.

Abstract
Virulence of avian influenza viruses (AIVs) is largely dependent on the amino acid sequence of the hemagglutinin cleavage site (CS). Low pathogenic AIV (LPAIV) carrying an HA with a monobasic CS which is activated by trypsin-like proteases in the respiratory and intestinal tracts cause only local infections with mild clinical signs, if any. Some H5 and H7 subtypes exhibit high pathogenicity (HP) by acquisition of a polybasic CS after circulation of LP precursors in terrestrial poultry. The polybasic CS of HPAIV is cleaved by ubiquitous furin-like proteases causing systemic infections and high mortality. In 2015, in an outbreak in poultry in Germany LP and HP H7N7 viruses could be isolated on the same farm indicating precursor-progeny relationship. Here, we investigated the pathogenicity of these LP and HP viruses in chickens, Muscovy ducks and turkeys via oculonasal and intravenous inoculation and analyzed virulence determinants by reverse genetics. Muscovy ducks showed no or mild clinical signs, while turkeys and chickens died after infection with HPAIV. Insertion of a polybasic CS into the HA of the LP virus (LP-poly) increased pathogenicity for chickens and turkey drastically, but was not sufficient for 100% mortality. LP-poly was less virulent in turkeys than in chickens exhibiting IVPI values of 2.8 and 1.9, re-spectively. Thus, virulence determinants differ between these two species. Our results contribute to understand the pathobiology and evolution of recent LP and HP H7N7 viruses in different poultry species.

Poster at the 29th Annual Meeting of the German Society for Virology in Düsseldorf, Germany (20-23 March, 2019)

Pathogenicity and virulence determinants of recent German H7N7 viruses in different poultry species

Scheibner, D., Salaheldin, A.H., Winter, F., Gischke, M., Veits, J., Mettenleiter, T.C., and Abdelwhab, E.M.

Abstract

Virulence of avian influenza viruses (AIVs) is largely dependent on the amino acid sequence of the hemagglutinin cleavage site (CS). Low pathogenic AIV (LPAIV) carrying an HA with a monobasic CS which is activated by trypsin-like proteases in the respiratory and intestinal tracts cause only local infections with mild clinical signs, if any. Some H5 and H7 subtypes exhibit high pathogenicity (HP) phenotype after acquisition of a polybasic CS after circulation of LP precursors in terrestrial poultry. The polybasic CS of HPAIV is cleaved by ubiquitous furin-like proteases causing systemic infections and high mortality. In 2015, in an outbreak in poultry in Germany LP and HP H7N7 viruses could be isolated on the same farm indicating precursor-progeny relationship. In this study, we investigated the pathogenicity of these LP and HP viruses in chickens, Muscovy ducks and turkeys via oculonasal and intravenous inoculation. Furthermore, virulence determinants of HPAIV were identified using reverse genetics. Muscovy ducks showed no or mild clinical signs, while turkeys and chickens died after the challenge with HP AIV. Insertion of a polybasic CS in the HA of the LP virus (LP_poly) increased pathogenicity for chicken and turkey drastically, but was not sufficient to kill all animals. LP_Poly was less virulent in turkeys than chickens exhibiting IVPI values of 2.8 and 1.9, respectively. Interestingly, LP_poly containing the NS gene from the isogenic HPAIV was as virulent and transmissible as the original HPAIV in chickens. Our results are important to better understand the pathobiology and evolution of recent LP and HP H7N7 viruses in different poultry species.

Talk at the 10th International Symposium on Avian Influenza, Brighton, UK (15 - 18 April 2018)