Molecular characterization of non structural proteins of Avian Influenza Virus

by Hagag, Ibrahim Thabet

Abstract (Summary)
Highly pathogenic avian influenza virus (HPAIV) H5N1 has been endemic in Egypt since 2006 and raised concern recently for its potential to evolve and be of highly transmissible among humans. Infection of HPAIV H5N1 has been described in experimentally challenged birds. However, pathogenicity of HPAIV H5N1, isolated in Egypt, has not been reported in naturally infected chickens and ducks, which could be unique due to distinct transmission routes and dosage of infection. Here we report a recent outbreak of HPAIV H5N1 in 2013, in commercial poultry farms in Sharkia Province, Egypt. The main symptoms were ecchymoses on the shanks and feet, cyanosis of the comb and wattles and subcutaneous edema of head and neck for chickens, and nervous signs (torticollis) for ducks. Within 48-72 hrs of the onset of illness, the average mortality rates were 22.8-30% and 28.5-40% in vaccinated chickens and non-vaccinated ducks, respectively. Tissue samples of chickens and ducks were collected for cross-section immunohistochemistry and realtime RT-PCR for specific viral RNA transcripts. Higher viral RNA transcripts were detected in tissues of chicken broilers and layers, including trachea, lung, spleen, intestine, brain, and serum, than those of ducks which have only viral RNA transcripts in trachea, lung, and liver tissues. In chickens, the highest viral RNA levels appeared to be in brain, trachea, and serum with significant differences detected between chicken broilers and layers in theses tissues in particular. Significant differences of the viral RNA were observed in trachea, lung, and liver tissues of ducks than those of chickens, indicating that HPAI H5N1 replicates with distinct tissue tropism between chickens and ducks. However, these samples from different birds cannot be compared because they came from natural outbreaks with uncertain timing of the course of infection. While the viral RNA was nearly detected in all tissues and serum collected indicating viral pan-tropism, the viral antigen was detected almost ubiquitously accordingly in all tissues including testicular tissues. Interestingly, viral antigen was also observed in endothelial cells of the most organs, and seen clearly in trachea and brain in particular as well as in mononuclear cells of various tissues particularly lungs. We performed phylogenetic analyses and compared the genomic sequences of the surface hemagglutinin (H) and non structural protein 1(NS1) among the isolated viruses, the HPAI H5N1 viruses circulated in Egypt in the past and currently, and some available commercial vaccinal strains. Analysis of deduced amino acids of both HA and NS1 revealed that our isolates carry molecular determinants of HPAI viruses, including the multibasic amino acids at the cleavage site in HA and glutamate at position 92 (D92E), C – terminus E-S-E-V motif, and the deletion at position 80-84 in NS1 protein. Taken together, this is the first study about pathogenicity of the HPAIV H5N1 strain, currently circulating in Egypt, from naturally infected poultry, which provides unique understanding of the viral pathogenesis in HPAIV H5N1 infected chickens and ducks. In conclusion, this study, firstly, identified that the outbreak that appeared in commercial chickens and backyard ducks in Sharkia province, Egypt, 2013 was attributed to HPAIV H5N1infection. Secondly, genetic and amino acid analysis of H gene at cleavage site indicated that they carry molecular determinants of HPAIV strains. Moreover, H protein of our isolates belongs to genotype (5J) which is different genotype from those strains used in some available commercial vaccines currently used in Egypt. Thirdly, genetic and amino acid analysis NS gene of viral isolates indicated that they belong to genotype NS1E with no reassortment between H5N1 subtype and other subtypes currently circulating in Egypt. The amino acids residues of NS-1 and NS-2 proteins of our strain did not show progressive evolution as we did not detect amino acids substitution, deletion and or insertion at the most important motifs of the NS1 protein. Fourthly, by IHC we confirmed the pan-tropism of the Egyptian HPAIV H5N1 in naturally infected chickens where endothelial cells, mononuclear cells, and testicular tissues expressed obvious viral antigen. Detection of viral antigen in endothelial and mononuclear cells reflects that the virus may have disseminated in all birds tissues via these cells. Moreover, expression of viral antigen in brain tissues suggests that severe necrotizing encephalopathy may be at least one of the possible causes of death of birds, if not the only cause. Last but not least, this study gives insights into pathogenesis of HPAIV in naturally infected birds which may be different from that obtained from experimentally infected birds due to distinct viral dosage and route of infection, distinct age and immunity of birds, and possibility of presence of contaminant infection. Thus it can serve as an augmentation to and in comparison with experimental studies. This study is also important to the veterinarians to perform accurate diagnosis on the actual field samples. By end of this study, we recommend further studies on continued subtyping and full genome characterization of IA viruses currently circulating in the Egyptian poultry field and also, functional characterization of NS1 protein to identify its specific role in virulence of HPAI viruses. Vaccine efficacy studies, possibility of sexual transmission of IAV viruses, and pathogenicity of HPAIV in naturally infected ducks are also necessary studies.
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Bibliographical Information:

Advisor:Ali A. Salama

School:Zagazig University

School Location:Egypt

Source Type:Master's Thesis

Keywords:HPAIV H5N1, Egypt, NS1, Influenza


Date of Publication:05/11/2015

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