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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 Jun. 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.

Ahmed, B. M., H. A. Hussein, and A. A. El-Sanousi, "Immunogenicity of DNA Plasmid Expressing HA1 of (A/equine/Egypt/vrlcu/2008 (H3N8)) Equine-1 Influenza virus in Wistar Rat Model.", International Journal of Virology, vol. 10, no. 1, 2014. Abstract
Ahmed, B. M., H. A. Hussein, and A. A. El-Sanousi, Wistar Rat Model, , 2013. Abstract
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.

Bazid, A. I., H. A. Hussein, S. S. Balal, A. A. Elsanousi, and B. M. Ahmed, "Phylogenetic Analysis of Foot and Mouth Disease Virus Type O in Egypt (2009).", International Journal of Virology, vol. 10, no. 1, 2014. Abstract
El-Sebelgy, M. M., B. M. Ahmed, N. S. Ata, and H. A. Hussein, "Molecular detection and characterization of reticuloendotheliosis virus in broiler breeder chickens with visceral tumors in Egypt", International Journal of Veterinary Science and Medicine, vol. 2, no. 1: Elsevier, pp. 21–26, 2014. Abstract
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, no. 12, pp. 1719–1727, 2014. Abstract
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., M. A. Shereen, H. A. Sultan, H. M. Samah, A. A. Nada, A. A. El-Sanousi, and others, "Protective immunity induced by co-injection of mixture of Mannan and B-glucan immunostimulant substances with the inactivated bivalent AI-ND vaccine in broiler chickens.", Veterinary Medical Journal Giza, vol. 57, no. 4: Faculty of Veterinary Medicine. Cairo University, pp. 649–667, 2009. Abstract
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.

Ismail, N. M., H. I. Tawfik, H. A. Hussein, and I. M. Reda, "Immunogenecity D Efficacy of Locally Prepared Montanide-oil Based H5N2 AI Vaccine Containing Flagellin As Immune Enhancer.", International Journal of Virology, vol. 10, no. 1, 2014. Abstract
Kaoud, H. A., H. A. Hussein, A. R. EL-Dahshan, H. S. Kaliefa, and M. A. Rohaim, "Co-circulation of avian influenza viruses in commercial farms, backyards and live market birds in Egypt", International Journal of Veterinary Science and Medicine, vol. 2, no. 2: Elsevier, pp. 114–121, 2014. Abstract
Mawgod, S. A., A. S. Arafa, and H. A. Hussein, "Molecular genotyping of the infectious bursal disease virus (IBDV) isolated from Broiler Flocks in Egypt", International Journal of Veterinary Science and Medicine, vol. 2, no. 1: Elsevier, pp. 46–52, 2014. Abstract
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.

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, no. 3: Wiley Online Library, pp. 240–243, 2013. Abstract
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
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(-3)) than the M1 protein (2.73 × 10(-3)). 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.

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.

Radwan, A. A., H. A. Hussein, E. A. El-Ibiary, M. D. El-Safty, I. M. El-Sabagh, A. H. El-deeb, and others, "Improved quality control protocol for fowl pox virus vaccines based on the use of PCR and sequence analysis to detect REV and ALV as contaminants.", Veterinary Medical Journal Giza, vol. 57, no. 4: Faculty of Veterinary Medicine. Cairo University, pp. 617–629, 2009. Abstract
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.

Selim, K., A. S. Arafa, H. A. Hussein, and A. A. El-Sanousi, "Detection and Molecular Characterization of Infectious Bronchitis virus Isolated from Recent Outbreaks in Chicken Farms in Egypt 2012.", International Journal of Virology, vol. 10, no. 1, 2014. Abstract
Youssef, M. M., H. A. Hussein, I. M. El-Sabagh, E. A. El-Ibiary, M. A. Shalaby, and others, "Molecular detection, isolation and characterization of avian rotavirus from broiler chicken.", Veterinary Medical Journal Giza, vol. 57, no. 4: Faculty of Veterinary Medicine. Cairo University, pp. 573–584, 2009. Abstract