الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
Egypt is incrusting subsequent challenges correlated to the spread of avian respiratory viruses with special reference to influenza viruses since it is introduction in 2006. Mainly due to high death toll in poultry farms resulting in severe economic losses. In addition to capability of certain AIVs to cross species barriers inducing fatal illness in humans. Successive introduction of new AIVs followed by enzootic co-circulation of several subtypes and clades convince the control measures. Besides wide spread of additional avian respiratory viruses such as vND and IBV. Numerous IBV variants were detected and circulating in the Egyptian poultry farms causing high mortalities especially in case of co infection with LPAIV H9N2. All efforts toward control AI in Egypt were failed.
Therefore, this study was carried out for providing an updated epidemiological situation concerning avian respiratory diseases including molecular characterization, genotyping and reassortment analysis of circulating AI subtypes in poultry in Egypt in period between 2017 till 2019. Besides, identifying the consequence of co-circulation between different AIV subtypes in reassortment pattern, in addition role of co-infection with other respiratory viruses such as vND and IBV in exacerbate the respiratory disease. As a necessity for this work, the diagnostic tools were improved and evaluated for AIV detection.
In Chapter 2, several combinations of respiratory viruses’ co-infections were detected in investigated poultry farms. These co-infections either between different AIV subtypes (three) and/or other respiratory viruses like IBV and vND in 32 out of 39 investigated farms. The LPAIV H9N2 detection percentage was high (27/39), especially it is co‐presence with HPAIV H5N8 in (22/39) investigated farms. Interestingly, the HPAIV H5N1 was infrequently detected. Sequence and phylogenetic analyses of the HA protein revealed that both H5 and H9 viruses were at the tips of the respective cluster branches designating continuing genetic drift. Several coding mutations in hemadsorping sites of N2 toward mammalian host’s adaptation. For H9N2 viruses sensitive detection using RT-qPCRs an updating of molecular diagnostic tools was conducted.
Samples from 11 commercial broiler chicken farms in Egypt collected
in early 2019 exhibited circulation of new reassortant AIVs (Chapter 3.1). Two different reassortants of HPAIV H5N8 were identified for first time in Egypt. Similar to novel reassortant LPAI H9N2 viruses in chicken farms that also were characterized for the first time in Egypt. In addition toa novel reassortant HPAIV H5N2 in three chicken farms in different regions in Egypt. The Latter virus carried the HA gene segment of HPAI H5 clade 2.3.4.4b viruses, besides four genome segments (PB1, PB2, PA, and NS) derived from another novel reassortant H9N2 viruses firstly recognized in 2014 in pigeons . While the (NP, NA, and M) gene segments were donated from classical H9N2 viruses circulating in Egypt poultry farms since 2010. The emergence of new reassortants and/or subtypes may induce troubles in diagnosis and control.
In chapter 3.2.1, out of total number 37 investigated poultry farms in Egypt in 2019 by rt-qPCR, all AI positive farms (n=21) were identified as HP H5N8. While HPAIV H5N1 was not detected at all. By conducting full-genome sequencing to three H5N8 viruses, number of coded mutations were detected in the M2 and NA proteins suggesting antiviral drugs Amantadine and Oseltamivir resistance. Seemingly, such new mutations occurred during virus circulation within Egypt. Systematic reassortment analysis of all available full genome sequences of Egyptian HP H5N8 (n=23), HP H5N2 (n=2) and LP H9N2 (n=53) viruses exposed existence of seven genotypes of HPAI H5Nx viruses of clade 2.3.4.4b since 2016 in Egypt. While, three genotypes were distinguishable for LP H9N2 since 2010. Heat mapping and tanglegram analyses revealed an annual pattern of genotypes replacement regarding HP H5N8 of clade 2.3.4.4b. A number of internal segments of both HP H5Nx and H9N2 viruses derived from circulating AIVs in wild bird in Egypt designating reassortment occurrence within Egypt.
Deep molecular analysis was conducted for Egyptian IBV isolates for virus genotyping (Chapter 3.2.2). In new genotyping based on S1 gene and full genome analysis isolate EGY/NR725/2016 (NR725/16) found related to genotype I, clade 23 (S1:GI-23). Depending on similar S1 gene analysis the historical IBV strains were divided to three subclades (S1: GI-23.1, S1: GI-23.2.1 and S1: GI-23.2.2) and confirmed NR725/16 as being part of a separate fourth subclade (S1: GI-23.3). Isolates related to years 2018 and 2019 revealed presence of new subclade prevails in Egypt, carrying similar mutations within S1 protein hypervariable regions (HVR) 1-3 that affect two neutralization sensitive epitopes at sites 294F, 297S and 306Y (48.2) and 329R (62.1). Furthermore, an intra-and inter genomic recombination was identified in isolate NR 725/16. The intra-subtype mixing was detected for the entire genes 3ab and E, moreover inter-subtype mixing for the entire gene 6b with a close relation to QX like viruses of genotype GI-19. Interestingly, NR725/16 isolate sharing 3ab: C gene with 2013 2016, 2018 and 2019 Egyptian IBV viruses.
In chapter 4, recent H9N2-G1 isolates carrying different cleavage sites were in vitro, in ovo and in vivo experimentally estimated. An in vitro assay, the tribasic HACS of H9N2 was processed by furin-like proteases similar to bona fide H5 HPAIV while some dibasic sites showed increased cleavability but monobasic HACS none. Yet, all viruses remained trypsin-dependent in cell culture. In ovo, showed enhanced pathogenicity of Egyptian H9N2 viruses, also tribasic H9N2 viruses were found to replicate in a grossly extended spectrum of embryonic organs. In contrast to all subtype H5/H7 HPAI viruses, tribasic H9N2 viruses neither replicated in endothelial cells in the chorio-allantoic membrane nor in other embryonic tissues. By IVPI, all H9Nx isolates proved to be of low pathogenicity.
Continuous introduction of new AIVs and co-circulation of different AI subtypes in parallel with other respiratory viruses such as IBV pushing forward further future expected reassortment and recombination evidence that might ended with emerging of novel viruses with negative impact on poultry production, also regarding to AI may have potential zoonotic significance. Therefore, continuous monitoring and updating of molecular diagnostic tools is advisable.
Upon this study conclusions and recommendations suggested regulations are provided aiming to build a stone in the way to control notifiable avian diseases in Egypt.