Publications by authors named "Abdelsatar Arafa"

11 Publications

  • Page 1 of 1

Genotyping and reassortment analysis of highly pathogenic avian influenza viruses H5N8 and H5N2 from Egypt reveals successive annual replacement of genotypes.

Infect Genet Evol 2020 10 23;84:104375. Epub 2020 May 23.

Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Greifswald, Riems, Germany. Electronic address:

Highly pathogenic (HP) H5N1, clade 2.2.1, and low pathogenic avian influenza (LPAI) H9N2 viruses, G1-B lineage, are endemic in poultry in Egypt and have co-circulated for almost a decade. Surprisingly, no inter-subtypic reassortment events have been reported from the field during that time. After the introduction of HPAIV H5N8, clade 2.3.4.4b, in Egyptian poultry in 2016, suddenly HP H5N2 reassortants with H9N2 viruses emerged. The current analyses focussed on studying 32 duck flocks, 4 broiler chicken flocks, and 1 turkey flock, suffering from respiratory manifestations with moderate to high mortality reared in two Egyptian governorates during 2019. Real-time RT-PCR substantiated the presence of HP H5N8 in 21 of the 37 investigated flocks with mixed infection of H9N2 in two of them. HP H5N1 was not detected. Full hemagglutinin (HA) sequencing of 10 samples with full-genome sequencing of three of them revealed presence of a single genotype. Very few substituting mutations in the HA protein were detected versus previous Egyptian HA sequences of that clade. Interestingly, amino acid substitutions in the Matrix (M2) and the Neuraminidase (NA) proteins associated with conferring both Amantadine and Oseltamivir resistance were present. Systematic reassortment analysis of all publicly available Egyptian whole genome sequences of HP H5N8 (n = 23), reassortant HP H5N2 (n = 2) and LP H9N2 (n = 53) viruses 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 that several internal gene segments in both HP H5Nx and H9N2 viruses originated from avian influenza viruses circulating in wild bird species in Egypt. Based on the limited set of whole genome sequences available, annual replacement patterns of HP H5Nx genotypes emerged and suggested selective advantages of certain genotypes since 2016.
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http://dx.doi.org/10.1016/j.meegid.2020.104375DOI Listing
October 2020

Detection of avian influenza virus: a comparative study of the in silico and in vitro performances of current RT-qPCR assays.

Sci Rep 2020 05 21;10(1):8441. Epub 2020 May 21.

Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università, 10, Legnaro, Padova, 35020, Italy.

Avian influenza viruses (AIV) are negative sense RNA viruses posing a major threat to the poultry industry worldwide, with the potential to spread to mammals, including humans; hence, an accurate and rapid AIV diagnosis is essential. To date AIV detection relies on molecular methods, mainly RT-qPCR directed against AIV M gene segment. The evolution of AIV represents a relevant issue in diagnostic RT-qPCR due to possible mispriming and/or probe-binding failures resulting in false negative results. Consequently, RT-qPCR for AIV detection should be periodically re-assessed both in silico and in vitro. To this end, a specific workflow was developed to evaluate in silico the complementarity of primers and probes of four published RT-qPCR protocols to their target regions. The four assays and one commercially available kit for AIV detection were evaluated both for their analytical sensitivity using eight different viral dilution panels and for their diagnostic performances against clinical specimens of known infectious status. Differences were observed among the tests under evaluation, both in terms of analytical sensitivity and of diagnostic performances. This finding confirms the importance of continuously monitoring the primers and probes complementarity to their binding regions.
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http://dx.doi.org/10.1038/s41598-020-64003-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242438PMC
May 2020

Efficacy of Bivalent Inactivated Vaccine Containing Insect Cell-Expressed Avian Influenza H5 and Egg-Based Newcastle Disease Virus (NDV) Against Dual Infection with Highly Pathogenic H5N1 and Velogenic NDV in Chickens.

Avian Dis 2019 09;63(3):474-480

Boehringer-Ingelheim Veterinary Research Center, Hannover, 30559, Germany.

The genetic evolution of highly pathogenic avian influenza (HPAI) in Egypt has developed a new clade H5N1 (2.2.1.2) since 2014. Meanwhile, the new avian influenza virus (AIV) clade mutually with the velogenic Newcastle disease virus (NDV) isolate of genotype VII in Egypt (genotype VII) has resulted in severe economic losses in the broiler industry. An inactivated bivalent vaccine containing H5 (belonging to H5N1 clade 2.3.2) recombinant baculovirus expressed by insect cell (recH5) and egg-based NDV LaSota strain (recH5/NDV vaccine) was evaluated for protection against the challenge of dual HPAIV H5N1 clade 2.2.1.2 and vNDV infection in commercial broiler chickens. Vaccination was performed when chickens were 10 days old, and then birds of the respective groups were challenged with 10 50% egg infective dose per chicken of each virus in 100 µl of allantoic fluid via the intranasal route at 21 days postvaccination in a single or sequential infection of both viruses. Results showed that the recH5/NDV vaccine was able to protect chickens against single or dual challenges of both viruses ranging up to 90%-100%. Unvaccinated chickens have demonstrated 100% mortalities to a single virus challenge. Vaccinated chickens showed significant decreases in both viruses, shedding titers up to <2 log after challenge in comparison with unvaccinated ones. Cessation of viral shedding was obtained at 7 to 10 days postchallenge. The vaccinated chickens showed high hemagglutination inhibition antibody titers >6 log against both H5N1 and NDV antigens at 2 wk postvaccination. The single vaccination of bivalent inactivated recH5-NDV vaccine at 10 days old in commercial chickens has provided significant clinical protective immunity against single or dual challenge with HPAI-H5N1clade 2.2.1.2 and vNDV-genotype VII.
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http://dx.doi.org/10.1637/12017-122618-Reg.1DOI Listing
September 2019

Disentangling the role of Africa in the global spread of H5 highly pathogenic avian influenza.

Nat Commun 2019 11 22;10(1):5310. Epub 2019 Nov 22.

Department of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy.

The role of Africa in the dynamics of the global spread of a zoonotic and economically-important virus, such as the highly pathogenic avian influenza (HPAI) H5Nx of the Gs/GD lineage, remains unexplored. Here we characterise the spatiotemporal patterns of virus diffusion during three HPAI H5Nx intercontinental epidemic waves and demonstrate that Africa mainly acted as an ecological sink of the HPAI H5Nx viruses. A joint analysis of host dynamics and continuous spatial diffusion indicates that poultry trade as well as wild bird migrations have contributed to the virus spreading into Africa, with West Africa acting as a crucial hotspot for virus introduction and dissemination into the continent. We demonstrate varying paths of avian influenza incursions into Africa as well as virus spread within Africa over time, which reveal that virus expansion is a complex phenomenon, shaped by an intricate interplay between avian host ecology, virus characteristics and environmental variables.
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http://dx.doi.org/10.1038/s41467-019-13287-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874648PMC
November 2019

Molecular pathogenic and host range determinants of reassortant Egyptian low pathogenic avian influenza H9N2 viruses from backyard chicken.

Int J Vet Sci Med 2019 11;7(1):10-19. Epub 2019 Jul 11.

Virology Dept., Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.

Since the introduction of H9N2 low pathogenic avian influenza virus in Egypt, it became an endemic disease causing considerable economic losses in different poultry sectors especially in the presence of other secondary bacterial and viral infections. The H9N2 viruses in Egypt are in continuous evolution that needs deep analysis for their evolution pattern based on the genetic constitutions of the pathogenic determinant genes (HA, PB2, PB1, PA, and NS). In this work, samples were collected from the backyard chickens from 3 Egyptian governorates. Five selected viruses were sequenced and analyzed for the hemagglutinin gene which showed genetic relatedness to the Asian G1 lineage group B, similar to the circulating H9N2 viruses in Egypt since 2013. The sequence for PB2, PB1, PA, HA and NS genes of the selected five viruses indicate a natural re-assortment event with recent Eurasian subtypes and similar to Egyptian H9N2 virus isolated from pigeon in Egypt during 2014. The Egyptian viruses of our study possess amino acids signatures including S42, V127, L550, L672 and V504 in the internal genes NS1, PA, and PB2, of respectively of an impact on virus transmission and replication. This work indicates that the H9N2 is in continuous evolution with alarming to the reassortment occurrence.
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http://dx.doi.org/10.1080/23144599.2019.1637046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776986PMC
July 2019

In silico thermodynamic stability of mammalian adaptation and virulence determinants in polymerase complex proteins of H9N2 virus.

J Genet Eng Biotechnol 2018 Dec 23;16(2):757-767. Epub 2018 Feb 23.

VirologyDept, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt.

The polymerase complex proteins (PB2, PB1, and PA) are responsible primarily for the replication of avian influenza virus and play an important role in virus virulence, mammalian adaptation, and interspecies transmission. In this study; eight Egyptian LPAI-H9N2 viruses isolated from apparent healthy chickens and quails from 2014 to 2016. Characterization of complete nucleotide sequences, phylogenetic and mutation analysis were carried out. The measurement of thermodynamic stability of the H9N2 polymerase protein in comparison to human H3N2 and H1N1 proteins was carried out using in silico method. Phylogenetic analysis of these viruses revealed a close relationship to viruses isolated from neighboring Middle Eastern countries with an average of 96-99% homology. They are sharing the common ancestor A/quail/Hong Kong/G1/1997 (G1-Like) without any evidence for genetic reassortment. In addition, eight markers related to virulence were identified, including the combination of 627V and 391E in the PB2 gene with full-length PB1-F2 and PA-X proteins were observed in all viruses and the substitution N66S in PB1-F2 which suggest increasing virus virulence. Moreover, six markers that may affect the virus replication and transmission in mammalian hosts were identified. Five mutations related to mammalian adaptation show a structural stabilizing effect on LPAI-H9N2 polymerase complex protein according to the free-energy change (ΔΔG). Three out of those six adaptive mutations shown to increase polymerase complex protein stability were found in Egyptian LPAI-H9N2 viruses similar to Human H3N2 and H1N1 (661 in PB2, 225 and 409 in PA genes). Our results suggested that the stabilizing mutations in the polymerase complex protein have likely affected the protein structure and induced favorable conditions for avian virus replication and transmission in mammalian hosts. Indeed, the study reports the mutational analysis of the circulating LPAI-H9N2 strains in Egypt.
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http://dx.doi.org/10.1016/j.jgeb.2018.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6353722PMC
December 2018

Molecular characterization of full fusion protein (F) of Newcastle disease virus genotype VIId isolated from Egypt during 2012-2016.

Vet World 2018 Jul 15;11(7):930-938. Epub 2018 Jul 15.

Department of Virology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.

Aim: The aim of this work was to study the full F gene sequence of Newcastle disease virus (NDV) in regard to pathotyping and genotyping and to study the evolution of this NDV in Egypt.

Materials And Methods: The present study was conducted using samples from seven suspected NDV flocks of vaccinated chickens during 2012-2016 from six governorates in Egypt. The NDV was successfully isolated from pathological specimens through inoculation in specific pathogen-free embryonated chicken eggs.

Results: Pathogenicity of the NDV isolates has been estimated through intracerebral pathogenicity index and ranged from 1.66 to 1.73 which indicates the velogenic type of NDV isolates. Pathotyping and genotyping of these isolates were done through sequencing of full-length F gene. Results indicated that the seven NDV isolates showed characteristic cleavage site motif (112RRQKRF117) for the velogenic strains of NDV. Phylogenetic analysis of the F gene clustered these isolates within Group I of genotype VIId within Israeli strains NDV/IS/2015, NDV-Ch/SD883, and most of the Middle East strains. Six of seven sequenced isolates have six potential N-linked glycosylation sites. The neutralization epitope on the five antigenic sites of fusion is conserved in all Egyptian strains of this study except NDV-KFR-B7-2012 which has a substitution at D 170 N in epitope A4. In all our strains, 10 cysteine residues are recorded, except one loss of cysteine at residue 370 in both NDV-EG-35-2014 and NDV-GHB-328F-2016.

Conclusion: All viruses in this study have 52 amino acid substitutions within fusion gene in compared with Lasota strain that reveals importance for its antigenic and structural function. The present work highlights the important need to sequence F gene of NDV genotype VIId to investigate the evolution of this NDV in Egypt.
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http://dx.doi.org/10.14202/vetworld.2018.930-938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097568PMC
July 2018

Mutation signature in neuraminidase gene of avian influenza H9N2/G1 in Egypt.

Virusdisease 2017 Jun 23;28(2):164-173. Epub 2017 May 23.

Virology Department, Faculty of Veterinary Medicine, Cairo University, Giza, 12211 Egypt.

The low pathogenic avian influenza (LPAI) H9N2 subtype has become the most prevalent and widespread in many Asian and Middle Eastern countries. It causes an enzootic situation in commercial poultry and known as a potential facilitator virus that can be transmitted to human from birds. The neuraminidase (NA) gene plays an important role the release and spread of the virus from infected cells and throughout the bird. The complete nucleotide sequences of the NA gene of seven H9N2 viruses collected from apparent healthy chicken and quail flocks in Egypt during 2014-2015, were amplified and sequenced. The phylogenetic relationships were investigated and all viruses were belonging to the A/Q/HK/G1/97 strain (G1-like). There were no insertions or deletions or shortening in NA stalk regions when compared to Y280-lineage and the human H9N2 isolates. No obvious changes NA interactions with antiviral drugs. We found that the Egyptian H9N2 viruses have seven glycosylation sites like the most recorded H9N2 viruses in the country, except A/Q/Egypt/14864V/2014 virus which has only six. The NA has four amino acid substitutions distributed in different parts of the hemadsorbing site. The most characteristic substitutions in this site were S372A and W403R these substitutions were a distinctive feature resembling to human H9N2, H2N2 and H3N2 viruses but differs from the other avian influenza viruses. These Special features of surface glycoproteins of LPAI-H9N2 viruses refer to the tendency for enhanced introductions into humans and ensuring the importance of poultry in the transfer influenza viruses.
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http://dx.doi.org/10.1007/s13337-017-0367-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5510632PMC
June 2017

Molecular and antigenic traits on hemagglutinin gene of avian influenza H9N2 viruses: Evidence of a new escape mutant in Egypt adapted in quails.

Res Vet Sci 2017 Jun 13;112:132-140. Epub 2017 Feb 13.

Virology Dept, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt.

The LPAI viruses of H9N2 subtype became widely distributed in Middle Eastern countries, causing great economic losses in poultry industry especially when complicated with other pathogens. The H9N2 viruses in Egypt have a wide spread nature since its first occurrence in 2011. In this study, we collected cloacal and tracheal samples from 19 flocks for detection and propagation of H9N2 virus using real-time RT-PCR and egg inoculation. We studied the molecular evolution of the Hemagglutinin gene of H9N2 viruses by full HA gene sequencing, then the antigenic characterization was implemented using the cross HI assay and analyzed using 3D Bioinformatics cartography software. The phylogenetic analysis of the HA gene of Egyptian H9N2 viruses clearly points out the presence of only one group (Egy/G1) of originally introduced viruses in 2011 related to the G1 lineage within group B, with the presence of multiple minor clusters includes viruses from 2011 to 2015. However, a new variant (Egy/G1var) cluster was detected in quails since 2012. Genetically, Egy/G1var viruses characterized by presence of 20 amino acid substitutions within and adjacent to the antigenic sites in comparison to other Egyptian viruses. In addition, two glycosylation sites at amino acid residues 127 and 189 were determined in close to the receptor binding and antigenic sites. The antigenic analysis based on 3D antigenic mapping showed that the Egy/G1var cluster was clearly distinct from the original Egy/G1 viruses. In conclusion, Egy/G1var is shown to be a new escape mutant variant cluster with an adaptive evolution in quails.
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http://dx.doi.org/10.1016/j.rvsc.2017.02.003DOI Listing
June 2017

Predominance and geo-mapping of avian influenza H5N1 in poultry sectors in Egypt.

Geospat Health 2016 11 28;11(3):492. Epub 2016 Nov 28.

Emergency Center of Transboundary Animal Diseases, Food and Agriculture Organization of the United Nations, Giza; National Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Giza.

Highly pathogenic avian influenza (HPAI) virus of the H5N1 subtype has been enzootic in the Egyptian poultry with significant human infections since 2008. This work evaluates the epidemiological and virological information from February 2006 to May 2015 in spatial and temporal terms. Only data with confirmed HPAI H5N1 sub-type were collected, and matched with the epidemiological data from various spatially and temporally-dispersed surveillances implemented between 2006 and 2015. Spatio-temporal analysis was conducted on a total of 3338 confirmed H5N1 HPAI poultry disease outbreaks and outputs described based on transmission patterns, poultry species, production types affected, trade, geographic and temporal distributions in Egypt. The H5N1 virus persists in the Egyptian poultry displaying a seasonal pattern with peak prevalence between January and March. There was no specific geographic pattern, but chickens and ducks were more affected. However, relatively higher disease incidences were recorded in the Nile Delta. Phylogenetic studies of the haemagglutinin gene sequences of H5N1 viruses indicated that multiple clusters circulated between 2006 and 2015, with significant deviations in circulation. Epidemiological dynamics of HPAI has changed with the origins of majority of outbreaks shifted to household poultry. The persistence of HPAI H5N1 in poultry with recurrent and sporadic infections in humans can influence virus evolution spatio-temporally. Household poultry plays significant roles in the H5N1 virus transmission to poultry and humans, but the role of commercial poultry needs further clarifications. While poultry trading supports the persistence and transmission of H5N1, the role of individual species may warrant further investigation. Surveillance activities, applying a multi-sectoral approach, are recommended.
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http://dx.doi.org/10.4081/gh.2016.492DOI Listing
November 2016

Phylodynamics of avian influenza clade 2.2.1 H5N1 viruses in Egypt.

Virol J 2016 Mar 22;13:49. Epub 2016 Mar 22.

Food and Agriculture Organization of the United Nations (FAO) - Emergency Center of Transboundary Animal Diseases (ECTAD), 11 Al Eslah El Zerai St, P.O. Box, 2223, Giza, Egypt.

Background: Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are widely distributed within poultry populations in Egypt and have caused multiple human infections. Linking the epidemiological and sequence data is important to understand the transmission, persistence and evolution of the virus. This work describes the phylogenetic dynamics of H5N1 based on molecular characterization of the hemagglutinin (HA) gene of isolates collected from February 2006 to May 2014.

Methods: Full-length HA sequences of 368 H5N1 viruses were generated and were genetically analysed to study their genetic evolution. They were collected from different poultry species, production sectors, and geographic locations in Egypt. The Bayesian Markov Chain Monte Carlo (BMCMC) method was applied to estimate the evolutionary rates among different virus clusters; additionally, an analysis of selection pressures in the HA gene was performed using the Single Likelihood Ancestor Counting (SLAC) method.

Results: The phylogenetic analysis of the H5 gene from 2006-14 indicated the presence of one virus introduction of the classic clade (2.2.1) from which two main subgroups were originated, the variant subgroup which was further subdivided into 2 sub-divisions (2.2.1.1 and 2.2.1.1a) and the endemic subgroup (2.2.1.2). The clade 2.2.1.2 showed a high evolution rate over a period of 6 years (6.9 × 10(-3) sub/site/year) in comparison to the 2.2.1.1a variant cluster (7.2 × 10(-3) over a period of 4 years). Those two clusters are under positive selection as they possess 5 distinct positively selected sites in the HA gene. The mutations at 120, 154, and 162 HA antigenic sites and the other two mutations (129∆, I151T) that occurred from 2009-14 were found to be stable in the 2.2.1.2 clade. Additionally, 13 groups of H5N1 HPAI viruses were identified based on their amino acid sequences at the cleavage site and "EKRRKKR" became the dominant pattern beginning in 2013.

Conclusions: Continuous evolution of H5N1 HPAI viruses in Egypt has been observed in all poultry farming and production systems in almost all regions of the country. The wide circulation of the 2.2.1.2 clade carrying triple mutations (120, 129∆, I151T) associated with increased binding affinity to human receptors is an alarming finding of public health importance.
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http://dx.doi.org/10.1186/s12985-016-0477-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802640PMC
March 2016