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Abdelwhab, E. M., M. K. Hassan, A. S. Abdel-Moneim, M. M. Naguib, A. Mostafa, I. T. M. Hussein, A. Arafa, A. M. Erfan, W. H. Kilany, M. G. Agour, et al., "Introduction and enzootic of A/H5N1 in Egypt: Virus evolution, pathogenicity and vaccine efficacy ten years on.", Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, vol. 40, pp. 80-90, 2016 06. Abstract

It is almost a decade since the highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 was introduced to Egypt in 2005, most likely, via wild birds; marking the longest endemic status of influenza viruses in poultry outside Asia. The endemic A/H5N1 in Egypt still compromises the poultry industry, poses serious hazards to public health and threatens to become potentially pandemic. The control strategies adopted for A/H5N1 in Egyptian poultry using diverse vaccines in commercialized poultry neither eliminated the virus nor did they decrease its evolutionary rate. Several virus clades have evolved, a few of them disappeared and others prevailed. Disparate evolutionary traits in both birds and humans were manifested by accumulation of clade-specific mutations across viral genomes driven by a variety of selection pressures. Viruses in vaccinated poultry populations displayed higher mutation rates at the immunogenic epitopes, promoting viral escape and reducing vaccine efficiency. On the other hand, viruses isolated from humans displayed changes in the receptor binding domain, which increased the viral affinity to bind to human-type glycan receptors. Moreover, viral pathogenicity exhibited several patterns in different hosts. This review aims to provide an overview of the viral evolution, pathogenicity and vaccine efficacy of A/H5N1 in Egypt during the last ten years.

Adel, A., A. S. Arafa, H. A. Hussein, and A. A. El-Sanousi, "Molecular and antigenic traits on hemagglutinin gene of avian influenza H9N2 viruses: Evidence of a new escape mutant in Egypt adapted in quails.", Research in veterinary science, vol. 112, pp. 132-140, 2017 Jun. Abstract

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.

Ali, Z. M., M. A. E. M. Hassan, H. A. Hussein, B. M. Ahmed, and A. A. E. - G. El Sanousi, "Protective efficacy of combined trivalent inactivated ISA 71 oil adjuvant vaccine against avian influenza virus subtypes (H9N2 and H5N1) and Newcastle disease virus.", Veterinary world, vol. 10, issue 10, pp. 1212-1220, 2017 Oct. Abstract

Aim: The objective of the present study was to prepare a trivalent inactivated vaccine of Newcastle disease virus (NDV), H5N1, and H9N2 viruses.

Materials and Methods: Three monovalent and a trivalent vaccines were prepared by emulsifying inactivated NDV (LaSota strain), reassortant H5N1, and H9N2 viruses with Montanide ISA 71 oil adjuvant. Parameters used for evaluation of the efficacy of the prepared vaccines in specific pathogen-free chickens were cellular immunity assays (blastogenesis, interferon gamma, interleukin 1 [IL1], and IL6), humoral immunity by hemagglutination inhibition, protection percentage, and shedding.

Results: A single immunization with trivalent vaccine-enhanced cell-mediated immunity as well as humoral immune response with 90% protection against challenges with highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic (LP) avian influenza H9N2 viruses with 100% protection after challenge with NDV.

Conclusion: Development and evaluation of the trivalent vaccine in the study reported the success in preparation of a potent and efficacious trivalent vaccine which is a promising approach for controlling HPAI H5N1, LP H9N2, and ND viral infections.

Awad, F., R. Chhabra, A. Forrester, J. Chantrey, M. Baylis, S. Lemiere, H. A. Hussein, and K. Ganapathy, "Experimental infection of IS/885/00-like infectious bronchitis virus in specific pathogen free and commercial broiler chicks.", Research in veterinary science, vol. 105, pp. 15-22, 2016 Apr. Abstract

Pathogenesis of an IS/885/00-like (IS/885) strain of variant infectious bronchitis virus (IBV) was examined in one day old specific pathogen free (SPF) and commercial broiler chicks. Chicks were humanely euthanized at 3, 6, 9, 12, 15, 21 and 28 days post infection (dpi) for necropsy examination, and tissues were collected for histopathology and virus detection by reverse transcription polymerase chain reaction (RT-PCR). Respiratory clinical signs and gross lesions consisting of tracheal caseous exudate and plugs, and swollen kidneys (with or without) urate deposits were observed in SPF and broiler chicks. The onset of disease developed more slowly and were of lesser severity in broiler compared to SPF chicks, reflecting the inhibitory effects of the IBV maternal-antibodies in the broiler chicks or genetic/strain susceptibility, or both. Head swelling was observed in one infected broiler chick at 15 dpi and the virus was recovered by RT-PCR and isolation. In the IS/885-infected SPF chicks, cystic oviducts were found in two female chicks. IS/885 was isolated from the cystic fluid. Using ELISA, low to moderate levels of the antibodies to IBV was detected in the SPF compared to broiler infected chicks.

Azab, A. A., A. Arafa, A. Selim, M. K. Hassan, A. I. Bazid, A. H. Sultan, H. A. Hussein, and E. M. Abdelwhab, "Pathogenicity of the Egyptian A/H5N1 avian influenza viruses in chickens.", Microbial pathogenesis, vol. 110, pp. 471-476, 2017 Sep. Abstract

Long-term circulation of highly pathogenic avian influenza H5N1 viruses of clade 2.2.1 in Egyptian poultry since February 2006 resulted in the evolution of two distinct clades: represents antigenic-drift variants isolated from vaccinated poultry and that caused the newest upsurge in birds and humans in 2014/2015. In the present study, nine isolates were collected from chickens, ducks and turkeys representing the commercial and backyard sectors during the period 2009-2015. The subtyping was confirmed by hemagglutination inhibition (HI) test, RT-qPCR and sequence analysis. The Mean Death Time (MDT) and Intravenous Pathogenicity Index (IVPI) for all isolates were determined. Sequence analysis of the HA gene sequences of these viruses revealed that two viruses belonged to clade and the rest were clade Antigenic characterisation of the viruses supported the results of the phylogenetic analysis. The MDT of the isolates ranged from 18 to 72 h and the IVPI values ranged from 2.3 to 2.9; viruses of the clade were less virulent than those of the clade. In addition, clade-specific polymorphism in the HA cleavage site was observed. These findings indicate the high and variable pathogenicity of H5N1 viruses of different clades and host-origin in Egypt. The upsurge of outbreaks in poultry in 2014/2015 was probably not due to a shift in virulence from earlier viruses.

Gadalla, M. R., A. H. El-Deeb, M. M. Emara, and H. A. Hussein, "Insect cell surface expression of hemagglutinin (HA) of Egyptian H5N1 avian influenza virus under transcriptional control of whispovirus immediate early-1 promoter.", Journal of microbiology and biotechnology, vol. 24, issue 12, pp. 1719-27, 2014 Dec 28. Abstract

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

Hussein, H. A., E. Frost, B. Talbot, M. Shalaby, E. Cornaglia, and Y. EL-Azhary, "Comparison of polymerase chain reaction and monoclonal antibodies for G-typing of group A bovine rotavirus directly from fecal material.", Veterinary microbiology, vol. 51, issue 1-2, pp. 11-7, 1996 Jul. Abstract

A reverse transcriptase and polymerase chain reaction (RT-PCR)-based assay for G-typing of bovine rotaviruses (BRV) was compared with a monoclonal antibody-based immunoassay (MAbs-ELISA) in the characterization of BRV field strains obtained from calves in different regions of Quebec between 1992 and 1994. The strains were analysed for two G types (G6 and G10) which are the most predominate BRV field strains. Fecal samples positive for BRV by polyacrylamide gel electrophoresis (n = 74) were typed by both methods revealing 77% correlation. RT-PCR detected 10 more G6 and 2 more G10 serotypes than MAbs-ELISA. Nine of the 12 discrepant samples could be cultivated and were confirmed as G6 (8) or G10 (1) by both methods. RT-PCR was able to efficiently detect artificial mixes of G6 and G10 and detected two mixed field infections. Four additional infections considered as mixed by MAbs-ELISA and as only G6 by RT-PCR were possibly MAbs-ELISA cross-reactions. RT-PCR provided a very sensitive method for typing BRV field isolates.

Hussein, H. A., B. M. Ahmed, S. M. Aly, A. H. El-deeb, A. A. El-Sanousi, M. A. Rohaim, A. A. Arafa, and M. R. Gadalla, "Protective efficacy of a prime-boost protocol using H5-DNA plasmid as prime and inactivated H5N2 vaccine as the booster against the Egyptian avian influenza challenge virus.", Acta virologica, vol. 60, issue 3, pp. 307-15, 2016. Abstract

In this study, a recombinant DNA plasmid was constructed, encoding for HA1 of a selected Egyptian H5N1 virus (isolated during the 2012 outbreaks). In the immunization and challenge experiments, SPF chickens received 1 or 2 doses of H5-DNA plasmid prime, and boosted with the inactivated H5N2 vaccine. Haemagglutination inhibition (HI) titers, protection levels, and the magnitude of virus shedding were compared to that of the chickens that received either DNA plasmid or inactivated H5N2 vaccine alone. H5N1 virus A/chicken/Egypt/128s/2012 (H5N1) highly pathogenic avian influenza (HPAI) clade 2.2.1/C was used for the challenge. Chickens immunized with 1 or 2 doses of H5-DNA vaccine failed to overcome the challenge with 0% and 10% protection, respectively. Quantitative real-time reverse transcription-PCR revealed virus shedding of 2.2 x 104 PCR copies/ml 3 days post challenge (dpc) in the only surviving bird from the group that received 2 doses of plasmid. However, chickens immunized with 1 or 2 doses of H5-DNA plasmid as prime and inactivated H5N2 vaccine as booster, showed 80% protection after challenge, with a viral shedding of 1.2 x 104 PCR copies/ml (1 dose) and 1.6 x 104 PCR copies/ml (2 doses) 3 dpc. The surviving birds in both groups did not shed the virus at 5 and 7 dpc. In H5N2-vaccinated chickens, protection levels were 70% with relatively high virus shedding (1.8 x 104 PCR copies/ml) 3 dpc. HI titers were protective to the surviving chickens. This study reports the efficacy of H5-DNA plasmid to augment reduction in viral shedding and to provide better protection when applied in a prime-boost program with the inactivated AI vaccine.

Hussein, H. A., A. V. Parwani, B. I. Rosen, A. Lucchelli, and L. J. Saif, "Detection of rotavirus serotypes G1, G2, G3, and G11 in feces of diarrheic calves by using polymerase chain reaction-derived cDNA probes.", Journal of clinical microbiology, vol. 31, issue 9, pp. 2491-6, 1993 Sep. Abstract

On the basis of antigenic variability in the VP7 outer capsid glycoprotein, at least 14 G serotypes exist for group A rotaviruses. Serotypic diversity exists among bovine rotaviruses (BRV), with serotypes G1, G6, G8, and G10 reported for cattle. Although G1 and G8 rotaviruses were originally described for humans, the recent isolation of G6 and G10 rotaviruses from humans further emphasizes the serotypic similarity between human and bovine rotaviruses and the possible zoonotic potential of rotaviruses. Results of our previous studies have indicated that more than 24% of BRV-positive field samples from diarrheic calves were nonreactive with cDNA probes or monoclonal antibodies to serotypes G6, G8, and G10. In this study, cDNA probes were prepared by polymerase chain reaction amplification of the hyperdivergent regions of the VP7 genes (nucleotides 51 to 392) from human (G1, G2, and G3) and porcine (G4, G5, and G11) rotaviruses. These probes were used in a dot blot hybridization assay to further characterize the G types of 59 BRV strains (fecal samples from diarrheic calves in Ohio, Nebraska, Washington, and South Dakota) that were nonreactive with cDNA probes to G6, G8, and G10. Rotaviruses belonging to serotypes G1 (n = 7), G2 (n = 1), G3 (n = 2), and G11 (n = 3) were identified among the BRV field samples. The BRV associated with these G types accounted for 22% of the samples tested; the other 78% of these samples remained untypeable with these probes. To our knowledge, this is the first report in the United States of the identification among BRV isolates of rotavirus serotypes G1, G2, G3, and G11.

Hussein, H. A., M. M. Youssef, A. Osman, E. A. El-Ebiary, and M. A. Shalaby, "Immunopathogenesis of attenuated strain of chicken infectious anemia virus in one-day-old specific-pathogen-free chicks.", The Egyptian journal of immunology, vol. 10, issue 1, pp. 89-102, 2003. Abstract

In the present study, the immunopathogenicity of chicken anemia virus (CAV) vaccinal strain was studied in one-day-old specific-pathogen-free (SPF) chicks. Hematocrit values, histopathological changes in haemopioetic and lymphoid organs, ELISA for CAV antibodies and PCR for CAV genome were used as testing assays for the study. Vaccinated chicks showed signs of anemia, lower hematocrit values and histopathological lesions in liver in the form of hepatocytes swelling to Centro lobular necrosis and apoptosis. Histopathology change in spleen (depletion of lymphocytes and apoptosis) and thymus (depletion of thymocytes and apoptosis) together with variable degrees of seroconversion rate were observed along the 10 weeks of the experiment indicating 2 waves of immune response in vaccinated chicks compared to the control non-vaccinated group. Detection of CAV-DNA in the liver of vaccinated chicks indicated the presence of the virus, when the antibody levels were decreased in some chicks. There was a consistent correlation between the 4 parameters used. It is concluded that the attenuated CAV vaccine strain induces anemia and lesions in the lymphoid organs. The histopathology and PCR are useful tools for evaluation and quality assurance of CAV vaccines.

Hussein, H. A., E. H. Frost, S. Deslandes, B. Talbot, and Y. Elazhary, "Restriction endonucleases whose sites are predictable from the amino acid sequence offer an improved strategy for typing bovine rotaviruses.", Molecular and cellular probes, vol. 11, issue 5, pp. 355-61, 1997 Oct. Abstract

Variation in the third base of a codon hampers genotypic characterization, particularly of RNA viruses. Some restriction endonucleases, however, have a recognition site with a variable base at the third position and will always cleave when a certain amino acid pair occurs (such as glycine-proline for Sau96I and glutamic or aspartic acid followed by serine usually for HinfI). We developed a restriction fragment length polymorphism (RFLP) procedure based on these enzymes for P-typing bovine group A rotaviruses (BRV). Employing this procedure 20 BRV local strains, isolated in tissue culture as well as the original faecal sample, could be typed in one of three patterns. More variability was observed when restriction endonucleases were employed whose cleavage sites cannot be predicted from the amino acid sequence (TaqI and Tsp509I). These RFLP results agreed with the PCR-VP4 typing assay, neutralization tests, and nucleotide sequence analysis. RFLP with Sau96I and HinfI provided quick and objective P-typing of strains and could detect multiple genotypes in the same sample.

Khalifa, M. E., A. H. El-Deeb, S. M. Zeidan, H. A. Hussein, and H. I. Abu-El-Naga, "Enhanced protection against FMDV in cattle after prime- boost vaccination based on mucosal and inactivated FMD vaccine.", Veterinary microbiology, vol. 210, pp. 1-7, 2017 Oct. Abstract

Improved immunization and control strategies and platforms are greatly needed for foot and mouth disease virus (FMDV) and mucosal vaccines propose an effective strategy for the control FMDV by blocking viral entry. In this study, several immunization strategies, using two FMDV vaccine formulations, including Montanide ISA 206 oil-based FMD inactivated vaccine and Montanide IMS 1313 VG N PR-based concentrated semi-purified FMD mucosal vaccine, were applied. Results of intranasal immunization with the prepared FMD mucosal vaccine, given once or twice, induced IgA levels in both nasal and salivary secretions besides a high response of lymphocyte proliferation with protection levels reaching 20% and 40%, respectively, in a challenge trial in cattle. Immunization with Montanide 206 inactivated FMD vaccine was capable of inducing 80% protection whereas prime-boost strategy based on the administration of mucosal vaccine followed by inactivated vaccine appeared to be the most potent strategy by achieving 100% protection against an FMDV challenge. Indeed, the study reports the efficacy of the prepared IMS 1313 FMD mucosal vaccine and the possible use of this vaccine in the context of different vaccination strategies to control FMDV.

Milani, A., A. Fusaro, F. Bonfante, G. Zamperin, A. Salviato, M. Mancin, E. Mastrorilli, J. Hughes, H. A. Hussein, M. Hassan, et al., "Vaccine immune pressure influences viral population complexity of avian influenza virus during infection.", Veterinary microbiology, vol. 203, pp. 88-94, 2017 May. Abstract

Vaccines are useful tools to control influenza A virus infection in poultry, but they need to be periodically reformulated to guarantee appropriate protection from infection and to limit viral replication and circulation, which could favour the emergence of new variants. In this study, a deep sequencing approach was used to characterize and follow the evolution of the hemagglutinin of the H5N1 highly pathogenic avian influenza viral population in infected animals vaccinated with two vaccines conferring different protection levels. Results from this preliminary investigation suggested that the evolution of the viral population, as well as the abundance and heterogeneity of minority variants could be influenced by the immune pressure conferred by vaccination.

Monne, I., H. A. Hussein, A. Fusaro, V. Valastro, M. M. Hamoud, R. A. Khalefa, S. N. Dardir, M. I. Radwan, I. Capua, and G. Cattoli, "H9N2 influenza A virus circulates in H5N1 endemically infected poultry population in Egypt.", Influenza and other respiratory viruses, vol. 7, issue 3, pp. 240-3, 2013 May. Abstract

We describe the identification and characterization of the H9N2 influenza subtype reported in Egyptian broiler and broiler breeder farms for the first time. Circulation of this subtype in a highly pathogenic H5N1 influenza virus endemic population provides an opportunity for genetic reassortment and emergence of novel viruses.

Mosaad, Z., A. S. Arafa, H. A. Hussein, and M. A. Shalaby, "Mutation signature in neuraminidase gene of avian influenza H9N2/G1 in Egypt.", Virusdisease, vol. 28, issue 2, pp. 164-173, 2017 Jun. Abstract

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.

Naggar, H. E. M., M. S. Madkour, and H. A. Hussein, "Preparation of mucosal nanoparticles and polymer-based inactivated vaccine for Newcastle disease and H9N2 AI viruses.", Veterinary world, vol. 10, issue 2, pp. 187-193, 2017 Feb. Abstract

AIM: To develop a mucosal inactivated vaccines for Newcastle disease (ND) and H9N2 viruses to protect against these viruses at sites of infections through mucosal immunity.

MATERIALS AND METHODS: In this study, we prepared two new formulations for mucosal bivalent inactivated vaccine formulations for Newcastle and Avian Influenza (H9N2) based on the use of nanoparticles and polymer adjuvants. The prepared vaccines were delivered via intranasal and spray routes of administration in specific pathogen-free chickens. Cell-mediated and humoral immune response was measured as well as challenge trial was carried out. In addition, ISA71 water in oil was also evaluated.

RESULTS: Our results showed that the use of spray route as vaccination delivery method of polymer and nanoparticles Montanide™ adjuvants revealed that it enhanced the cell mediated immune response as indicated by phagocytic activity, gamma interferon and interleukin 6 responses and induced protection against challenge with Newcastle and Avian Influenza (H9N2) viruses.

CONCLUSION: The results of this study demonstrate the potentiality of polymer compared to nanoparticles adjuvantes when used via spray route. Mass application of such vaccines will add value to improve the vaccination strategies against ND virus and Avian influenza viruses.

Naguib, M. M., N. Hagag, A. A. El-Sanousi, H. A. Hussein, and A. - S. Arafa, "The matrix gene of influenza A H5N1 in Egypt, 2006-2016: molecular insights and distribution of amantadine-resistant variants.", Virus genes, vol. 52, issue 6, pp. 872-876, 2016 Dec. Abstract

Large-scale sequence analysis of Matrix (M) gene and its coding proteins M1 and M2 was performed for 274 highly pathogenic avian influenza viruses H5N1 circulated in Egypt from 2006 to 2016. The aim is to study the amantadine-resistant markers distribution and to estimate the evolutionary rate. 246 viruses were obtained from the Global Initiative on Sharing All Influenza Data base, and 28 additional viruses were sequenced. Maximum clade credibility (MCC) phylogenetic tree revealed that the M gene has evolved into two different lineages. Estimated Evolutionary analysis showed that the M2 protein possessed higher evolutionary rates (3.45 × 10) than the M1 protein (2.73 × 10). M gene encoding proteins revealed significant markers described to be associated with host tropism and increase in virulence: V15I, N30D, and T121A in M1 and L55F in M2 protein. Site analysis focusing attention on the temporal and host distribution of the amantadine-resistant markers was carried out and showed that vast majority of the M2 amantadine-resistant variants of clade (n = 90) is N31 marker, in addition to G27 (n = 7), A27 (n = 5), I27 (n = 1), and S30 (n = 1). In 2010-2011, amantadine resistant frequency increased considerably resembling more than half of the resistant variants. Notably, all viruses of clade possessed amantadine-resistant marker. However, almost all current circulating viruses in Egypt of clade from 2014 to 2016 did not carry any amantadine-resistant markers.

Naguib, M. M., A. Graaf, A. Fortin, C. Luttermann, U. Wernery, N. Amarin, H. A. Hussein, H. Sultan, B. Al Adhadh, M. K. Hassan, et al., "Novel real-time PCR-based patho- and phylotyping of potentially zoonotic avian influenza A subtype H5 viruses at risk of incursion into Europe in 2017.", Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin, vol. 22, issue 1, 2017 Jan 05. Abstract
Parwani, A. V., H. A. Hussein, B. I. Rosen, A. Lucchelli, L. Navarro, and L. J. Saif, "Characterization of field strains of group A bovine rotaviruses by using polymerase chain reaction-generated G and P type-specific cDNA probes.", Journal of clinical microbiology, vol. 31, issue 8, pp. 2010-5, 1993 Aug. Abstract

Dot and Northern blot hybridization assays were used to analyze field strains of group A bovine rotaviruses (BRVs) by using nucleic acid probes representing P and G type specificities. The probes were prepared by polymerase chain reaction amplification of hyperdivergent regions of the cloned VP4 (nucleotides 211 to 686) and VP7 (nucleotides 51 to 392) genes from four serotypically distinct (in P or G types) strains of rotaviruses: NCDV (G6, P1), IND (G6, P5), 69M (G8, P10), and Cr (G10, P11). The P and G type cDNA probes were radiolabeled with [32P]dCTP and hybridized with RNA extracted from reference cell culture-passaged rotavirus strains or the field samples. The field samples were obtained from young diarrheic calves from Ohio, Nebraska, Washington State, and Canada. The cDNA probes were specific for their respective G or P types on the basis of analysis of known P and G type reference strains. The G typing analysis of 102 field samples revealed that 36.3% (37 of 102) were G6, 2.9% (3 of 102) were G8, 12.7% (13 of 102) were G10, and 23.5% (24 of 102) were untypeable. The P typing results for 93 samples indicated that 2.2% (2 of 93) were P1 (NCDV-like), 20.4% (19 of 93) were P5 (UK-like), 9.3% (10 of 93) were P11 (B223-like), and 40.8% (38 of 93) were untypeable. This is the first report of the identification among BRV strains in North America of a G type other than G6 or G10. Our report further confirms that G6, P5 rotaviruses are predominant among the BRV field strains that we examined, and the P types of these strains differ from that of the BRV vaccine strain used in the United States (G6, P1). The large number of untypeable G (23.5%) and P (40.8%) types suggests that other or new P and G types exist among BRV field strains.

Rohaim, M. A., R. F. El Naggar, A. M. Helal, H. A. Hussein, and M. Munir, "Reverse spillover of avian viral vaccine strains from domesticated poultry to wild birds.", Vaccine, vol. 35, issue 28, pp. 3523-3527, 2017 06 16. Abstract

Transmission of viruses from the commercial poultry to wild birds is an emerging paradigm of livestock-wildlife interface. Here, we report the identification and isolation of vaccine strains of avian paramyxovirus serotype 1 (APMV1) and avian coronaviruses (ACoV) from different wild bird species across eight Egyptian governorates between January 2014 and December 2015. Surveillance of avian respiratory viruses in free-ranging wild birds (n=297) identified three species that harboured or excreted APMV1 and ACoVs. Genetic characterization and phylogenetic analysis of recovered viruses revealed a close association with the most widely utilized vaccine strains in the country. These results highlight the potential spillover of vaccine-viruses probably due to extensive use of live-attenuated vaccines in the commercial poultry, and close interaction between domesticated and wild bird populations. Further exploring the full spectrum of vaccine-derived viral vaccine strains in wild birds might help to assess the emergence of future wild-birds origin viruses.

Saad, M. D., H. A. Hussein, M. M. Bashandy, H. H. Kamel, K. C. Earhart, D. J. Fryauff, M. Younan, and A. H. Mohamed, "Hepatitis E virus infection in work horses in Egypt.", Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, vol. 7, issue 3, pp. 368-73, 2007 Jun. Abstract

Hepatitis E virus (HEV) is an important cause of hepatitis among young Egyptian adults with high seroprevalence rates seen in both rural areas of the Nile Delta and in suburban Cairo. Because natural antibodies to HEV have been detected in animals and zoonotic transmission is postulated, we surveyed work horses in Cairo for evidence of HEV exposure and viremia. Sera from 200 Cairo work horses were tested by ELISA for the presence of IgG anti-HEV antibody revealed a seropositivity of 13%. Among 100 samples processed for detection of viral genome by means of nested polymerase chain reaction (N-PCR), 4% were positive and indicative of viremia. Viremic animals were less than 1 year old. Relative to PCR-negative horses, PCR-positive animals demonstrated significant elevation of AST (p=0.03). Phylogenetic analysis of a 253-bp fragment, in the ORF-1,2,3 overlap region of the HEV genome from the viremic animals showed that three of these viral strains to be identical, and closely related (97-100% nucleotide identity) to two human isolates from Egypt, and distant (78-96%) from 16 other HEV isolates from human and animals and shared 99.6% NI with the fourth strain. The consensus sequence of the four strains was origin obtained elsewhere. These data indicated that horses acquire HEV infection and suggest that cross-species transmission may occur. Whether horses play a role in the transmission of HEV needs further investigation.

Saad, A. M., A. Samy, M. A. Soliman, A. Arafa, A. Zanaty, M. K. Hassan, A. H. Sultan, A. I. Bazid, and A. H. Hussein, "Genotypic and pathogenic characterization of genotype VII Newcastle disease viruses isolated from commercial farms in Egypt and evaluation of heterologous antibody responses.", Archives of virology, vol. 162, issue 7, pp. 1985-1994, 2017 Jul. Abstract

Newcastle disease viruses (NDV) represent a major threat to poultry production worldwide. Recently in Egypt NDV circulated extensively, even in vaccinated farms. In the present study samples were collected from sixteen vaccinated broiler farms in animals exhibiting the typical gross lesions of NDV. Virus isolation and pathogenicity studies for positive samples were carried out in accordance to reference procedures and phylogenetic analysis was carried out based on partial sequences of the Fusion gene. Furthermore, in vivo investigation of the ability of heterologous antibody, induced by commercially available lentogenic strain-based vaccines, to efficiently reduce viral shedding was examined. Results revealed that all the sixteen farms were positive for the presence of NDV. Out of these fifteen were confirmed to due to velogenic viruses, based on a main death time (MDT) ≤ 48 hours and partial sequencing of the F gene that showed the presence of a polybasic amino acid motif. However, three patterns in the cleavage site of these velogenic viruses were identified in the present study. Phylogenetic analysis revealed that all fifteen isolates were clustered with class II genotype VIIb while the remaining isolate (B81) was class II genotype II. Results of the in vivo study revealed that adequate heterologous antibody levels, induced by the proposed vaccination program, sufficiently protected birds from morbidity and mortality. However, virus shedding was quantitatively affected in relation to the time of challenge after vaccination. Altogether, with an absence of vaccines able to induce homologous antibody to the presently circulating viruses, higher antibody levels, which depend on efficient and timely implementation of the vaccination program, are considered as highly important in relation to the reduction of virus shedding.