Avian influenza infections in poultry farms in Egypt, a continuous challenge : Current problems related to pathogenesis, epidemiology and diagnosis

Since 2006, Egypt is facing huge challenges related to the spread of avian influenza viruses. This is partly due to the very high death toll of HPAI in poultry populations causing huge economic losses. The ability of some AI viruses to cross species barriers and invoke severe and often fatal illness in human beings causes further alert. Sequential introduction of new viruses and enzootic co-circulation of different AI subtypes and clades as well as presence of further avian respiratory pathogens, such as IBV and NDV, are at the basis of these problems. All previous efforts to eradicate and control AI in Egypt were unsuccessful. The main objectives of this study aim at providing an update of the epidemiological situation regarding AIV including molecular characterization, and genotyping and reassortment analysis of AIV subtypes circulating in poultry in Egypt between 2017 and 2019. In addition, an assessment was attempted of the role of co-infections with different AIV subtypes and/or other respiratory viruses such as IBV and NDV. As a prerequisite for such work, improved diagnostic tools were developed and evaluated for AIV detection. In Chapter 2.1, various combinations of co-infections in poultry flocks with three different AIV subtypes and other respiratory pathogens such as IBV and NDV were detected in 32 out of 39 farms examined. The percentage of AIV H9N2‐positive samples was high (27/39), and co‐presence of HPAIV H5N8 was detected most often (22/39). The previously dominating HPAIV subtype H5N1 was only infrequently detected. Sequence and phylogenetic analyses of the hemagglutinin gene showed that both H5 and H9 viruses were located at the tips of the respective cluster branches indicating ongoing genetic drift. Several coding mutations in the neuraminidase N2 hemadsorption site suggested some adaptation to mammalian hosts. Sensitive detection of H9N2 viruses by RT-qPCRs required an update of diagnostic tools. Examination of further Egyptian poultry samples collected in early 2019 from 11 commercial broiler chicken farms revealed the presence and circulation of new reassortant AI viruses (Chapter 2.2). This affected HPAIV H5N8 of which two different reassortants were characterized that had not been reported in Egypt before. Also, novel reassortant H9N2 viruses were detected in chicken holdings for the first time in Egypt. Most importantly, however, a novel reassortant HPAIV H5N2 was found on three chicken holdings in different areas of Egypt. The HA gene segment was donated from HPAI H5 clade 2.3.4.4b viruses whereas four genome segments (PB1, PB2, PA, and NS) originated from another novel reassortant H9N2 viruses first detected in pigeons. Other segments (NP, NA, M) were acquired from H9N2 viruses circulating in chickens in Egypt since 2010. The formation of new reassortants and subtype may cause further problems in diagnosis and control of these viruses. Real-time RT-PCR investigations substantiated the presence of HP H5N8 in 21 of a further 37 investigated flocks sampled in 2019 (Chapter 2.3). HPAIV H5N1 was detected no longer in any of those holdings. Full-genome sequencing of three H5N8 viruses revealed presence of amino acid substitutions in the M2 and NA proteins conferring resistence to antiviral drugs Amantadine and Oseltamivir. Apparently, these mutations were acquired during circulation in Egypt. Systematic reassortment analysis of all publicly available whole genome sequences of HP H5N8 (n=23), reassortant HP H5N2 (n=2) and LP H9N2 (n=53) viruses from Egypt revealed presence of at least seven different genotypes of HPAI H5Nx viruses of clade 2.3.4.4b in Egypt since 2016. For H9N2 viruses, at least three genotypes were distinguishable. Heat mapping and tanglegram analyses suggested an annual replacement pattern of HP H5 genotypes. Several internal gene segments in both HP H5Nx and H9N2 viruses originated from AIV circulating in wild bird species in Egypt indicating local reassortment processes. Constant introduction of new AIV strains, reassortment leading to the generation of new subtypes and on-going genetic drift in AIV in Egypt called for a continuing update of molecular diagnostic tools. Based on the conclusions of this study recommendations for improved regulations and control of notifiable avian diseases in Egypt are provided.