Haitham Amer | Cairo University (original) (raw)

Books by Haitham Amer

Infectious laryngotracheitis (ILT) is a respiratory disease that causes significant economic loss... more Infectious laryngotracheitis (ILT) is a respiratory disease that causes significant economic losses in the poultry industry worldwide. In this study, ILT outbreaks were reported on 30 farms located in eight Egyptian governorates between January 2018 and May 2019. Gross examination of diseased chickens revealed congestion and hemorrhage of laryngeal and tracheal mucosa with fibrinohemorrhagic casts and/or caseous material in the lumens. Histopathological examination showed epithelial sloughing, syncytium formation, heterophilic exudation, and development of eosinophilic intranuclear inclusion bodies. Infectious laryngotracheitis virus (ILTV) antigen was detected in the tracheal epithelium, infiltrated inflammatory cells, and syncytial cells, using immunohistochemistry. PCR targeting a portion of the thymidine kinase gene was further utilized to confirm the presence of ILTV DNA. The complete coding sequences of three envelope glycoprotein genes, gG, gD, and gJ, and a partial sequence of the infected cell polypeptide 4 (ICP4) gene from samples representing all of the farms and disease outbreaks were determined. Five prototype strains with unique sequences were chosen for detailed molecular characterization. Sequence comparisons and phylogenetic analysis of the partial ICP4 gene revealed that two strains were chicken embryo origin (CEO)-vaccine-like strains, and three were tissue culture origin (TCO)-vaccine-like strains. Analysis of the gJ gene sequence indicated that all of the strains were CEO vaccine-like strains. It was predicted that the latter three strains were recombinants of CEO-and TCO-vaccine-like strains. In conclusion, immunohistochemistry coupled with multigenomic PCR sequencing proved to be efficient for identification and typing of ILTV strains during disease outbreaks. Both CEO-vaccine-like and recombinant virus strains were circulating in Egypt during the 2018 and 2019 outbreaks.

For further information see our web site at www.rsc.org vi Preface backbone and side-chain placem... more For further information see our web site at www.rsc.org vi Preface backbone and side-chain placement, NMR also provides dynamic (ensemble) information and crystallography provides a 'snapshot' and is often considered static. Solution approaches, such as limited proteolysis combined with mass spectrometry and small-angle scattering approaches (Chapter 3), also provide dynamic information. In cryo-ET and cryo-electron microscopy (cryo-EM) (Chapter 5), macromolecules are frozen in their native state, allowing for discrete selection of dynamic states to be visualized, albeit at lower resolution. Generally, NMR spectroscopy is utilized for small protein molecules that are flexible, X-ray crystallography for medium-sized proteins and complexes that are compact, whereas very large macromolecular assemblages or membranous protein structures are determined by cryo-EM. The largest issue separating cryo-EM and cryo-ET from crystallography, in addition to size and the limitations of crystal formation, is resolution. Cryo-EM has generally been considered a low-resolution technique, giving reconstructions around 15-30 Å , but with advances in sample handling, instrumentation, image processing and model building, near-atomic resolution structures are now being achieved. For cryo-ET the resolution achieveable is still low. In reality, hybrid approaches, combining NMR, X-ray crystallography and cryo-EM, cryo-ET and solution data, are often adopted, which provides a powerful means of filling gaps which can arise in the structural characterization of large macromolecules. For example, in studies where large viruses cannot be crystallized, subcomponents can be crystallized to obtain high-resolution information, which can then be used to interpret the structure at lower resolution obtained by cryo-EM or cryo-ET. Or atomic structures obtained from homologous viral proteins/virus capsids can be used for 3D homology model building. These approaches permit the pseudo-atomic visualization of interaction interfaces between protein-protein subunits, protein-nucleic acids and protein-lipid in virus capsids and also the visualization of virus capsid-host interactions.

Cover Illustration: A model of the small RNA transcriptional silencing complex is shown with the ... more Cover Illustration: A model of the small RNA transcriptional silencing complex is shown with the transcription bubble and RNAPII (dark blue), Argonaute 1 (green), and the antisense strand of the small RNA (red) bound to nascent mRNA (also red). An epigenetic regulatory complex composed of Ezh2, DNMT3a, and HDAC-1 is also shown (red, blue and green), remodeling the histone. (Computer graphics courtesy to Paula J. Morris).

The insect cell culture/baculovirus system has three primary applications: (1) recombinant protei... more The insect cell culture/baculovirus system has three primary applications: (1) recombinant protein synthesis, (2) biopesticide synthesis, and (3) as a model system (e.g., for studying apoptosis). The fundamental techniques involved in these applications are described throughout this book. In this chapter, the most widely techniques are summarized and the reader is directed to detailed information found elsewhere in this book. Furthermore, many useful tips and the author's personal preferences that are rarely published are discussed in this chapter along with quantitative methods to characterize cell growth, baculovirus infection, and metabolism.

ISBN 0 471 91978 0 (World excluding Scandinavia and Finland) ISBN 82 419 0038 4 (Scandinavia and ... more ISBN 0 471 91978 0 (World excluding Scandinavia and Finland) ISBN 82 419 0038 4 (Scandinavia and Finland) Cartoons

Bibliographic Indices. This publication is listed in bibliographic services, including Current Co... more Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® PubMed/MEDLINE Disclaimer. The statements, options and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Papers by Haitham Amer

Molecular and Cellular Probes, 2020

Highly pathogenic avian influenza H5N1 virus causes heavy losses in poultry farms worldwide. Mole... more Highly pathogenic avian influenza H5N1 virus causes heavy losses in poultry farms worldwide. Molecular diagnostic techniques like RT-PCR and real-time RT-PCR are considered the gold standard for identification of H5 influenza viruses in clinical samples. These techniques are hampered by the need of well-equipped laboratories, large space requirement, and relatively long time-to-result. Recombinase polymerase amplification (RPA) assay represents an excellent alternative to PCR since it is more simple, rapid, economic, and portable. Reverse transcription RPA (RT-RPA) assay was recently developed for sensitive and specific detection of H5N1 virus in 6-10 min. To ensure the accuracy of the developed assay, two approaches for using a positive control were evaluated in this study. These approaches included: 1) all-in-one (internal positive control; IPC), 2) two-tubes-per-one-sample (external positive control; EPC). Sigma virus (SIGV) RNA and turkey mitochondrial DNA were tested as positive controls in both approaches. For all-in-one approach, both targets (H5 and IPC) were strongly inhibited. In contrast, very good amplification signals were obtained for the two types of EPC with no effect on the analytical sensitivity and specificity of H5 RT-RPA assay in two-tubes-per-one-sample approach. The performance of EPC-based H5 RT-RPA was further validated using 13 tracheal swabs. The results were compared to real-time RT-PCR and proved superior specificity in detecting H5N1 but not H5N8 viruses. Inclusion of EPC did not affect the aptitude of both assays in terms of sensitivity, specificity and reproducibility. In conclusion, the two-tubes-per-one-sample approach was more reliable to control the false negative results in H5 RT-RPA assay.

Archives of Virology, 2020

Infectious laryngotracheitis (ILT) is a respiratory disease that causes significant economic loss... more Infectious laryngotracheitis (ILT) is a respiratory disease that causes significant economic losses in the poultry industry worldwide. In this study, ILT outbreaks were reported on 30 farms located in eight Egyptian governorates between January 2018 and May 2019. Gross examination of diseased chickens revealed congestion and hemorrhage of laryngeal and tracheal mucosa with fibrinohemorrhagic casts and/or caseous material in the lumens. Histopathological examination showed
epithelial sloughing, syncytium formation, heterophilic exudation, and development of eosinophilic intranuclear inclusion bodies. Infectious laryngotracheitis virus (ILTV) antigen was detected in the tracheal epithelium, infiltrated inflammatory cells, and syncytial cells, using immunohistochemistry. PCR targeting a portion of the thymidine kinase gene was further utilized to confirm the presence of ILTV DNA. The complete coding sequences of three envelope glycoprotein genes, gG,
gD, and gJ, and a partial sequence of the infected cell polypeptide 4 (ICP4) gene from samples representing all of the farms and disease outbreaks were determined. Five prototype strains with unique sequences were chosen for detailed molecular characterization. Sequence comparisons and phylogenetic analysis of the partial ICP4 gene revealed that two strains were chicken embryo origin (CEO)-vaccine-like strains, and three were tissue culture origin (TCO)-vaccine-like strains. Analysis of the gJ gene sequence indicated that all of the strains were CEO vaccine-like strains. It was predicted that the latter three strains were recombinants of CEO- and TCO-vaccine-like strains. In conclusion, immunohistochemistry coupled with multigenomic PCR sequencing proved to be efficient for identification and typing of ILTV strains during disease outbreaks. Both CEO-vaccine-like and recombinant virus strains were circulating in Egypt during the 2018 and 2019 outbreaks.

Journal of Medical Virology, 2020

Lower respiratory tract infections caused by Human orthopneumovirus are still a threat to the ped... more Lower respiratory tract infections caused by Human orthopneumovirus are still a threat to the pediatric population worldwide. To date, the molecular epidemiology of the virus in Saudi Arabia has not been adequately charted. In this study, a total of 205 nasopharyngeal aspirate samples were collected from hospitalized children with lower respiratory tract symptoms during the winter seasons of 2014/15 and 2015/ 16. Human orthopneumovirus was detected in 89 (43.4%) samples, of which 56 (27.3%) were positive for type A and 33 (16.1%) were positive for type B viruses. The fragment that spans the two hypervariable regions (HVR1 and HVR2) of the G gene of Human orthopneumovirus A was amplified and sequenced. Sequence and phylogenetic analyses have revealed a genotype shift from NA1 to ON-1, which was prevalent during the winter seasons of 2007/08 and 2008/09. Based on the intergenotypic p-distance values, ON-1 was reclassified as a subgenotype of the most predominant genotype GA2. Three conserved N-glycosylation sites were observed in the HVR2 of Saudi ON-1 strains. The presence of a 23 amino acid duplicated region in ON-1 strains resulted in a higher number of O-glycosylation sites as compared to other genotypes. The data presented in this report outlined the replacement of NA1 and NA2 subgenotypes in Saudi Arabia with ON-1 within 7 to 8 years. The continuous evolution of Human orthopneumovirus through point mutations and nucleotide duplication may explain its ability to cause recurrent infections. K E Y W O R D S lower respiratory tract infections, molecular epidemiology, ON-1, phylogenetic analysis, RSV

International Journal of Pharmcology, Phytochemistry and Ethnomedicine, 2019

Hybridomas that secreted antibodies against aflatoxin B1 for multiple uses were prepared using a ... more Hybridomas that secreted antibodies against aflatoxin B1 for multiple uses were prepared using a unique immunization schedule. Aflatoxin B1-BSA conjugate was used for immunization of Balb/c mice. Spleen cells were harvested from the hyper immunized mice to be fused with myeloma cell line (P3NS1) using polyethylene glycol 3000, 50% concentration as a fusogenic agent. The produced hybridomas were selected using HAT selective medium that was replaced by complete HT medium. From the 10 th day after fusion, wells that contain colonies of hybridomas covering 30% or greater of the wells surface were screened for production of monoclonal antibodies against aflatoxin B1 using ELISA. 21 hybridomas were found to be reactive to aflatoxin B1. All were found to belong to IgG2a isotype except one was found to belong to IgM isotype. The prepared monoclonal antibodies and their application to immunoassays represent a useful and rapid quantitative measurement with high affinity and low detection limits in order to purify environmentally occurring levels of this carcinogen specially in areas at high risk for liver cancer.

Molecular and Cellular Probes, 2020

Archives of Virology, 2020

Infectious laryngotracheitis (ILT) is a respiratory disease that causes significant economic loss... more Infectious laryngotracheitis (ILT) is a respiratory disease that causes significant economic losses in the poultry industry worldwide. In this study, ILT outbreaks were reported on 30 farms located in eight Egyptian governorates between January 2018 and May 2019. Gross examination of diseased chickens revealed congestion and hemorrhage of laryngeal and tracheal mucosa with fibrinohemorrhagic casts and/or caseous material in the lumens. Histopathological examination showed
epithelial sloughing, syncytium formation, heterophilic exudation, and development of eosinophilic intranuclear inclusion bodies. Infectious laryngotracheitis virus (ILTV) antigen was detected in the tracheal epithelium, infiltrated inflammatory cells, and syncytial cells, using immunohistochemistry. PCR targeting a portion of the thymidine kinase gene was further utilized to confirm the presence of ILTV DNA. The complete coding sequences of three envelope glycoprotein genes, gG,
gD, and gJ, and a partial sequence of the infected cell polypeptide 4 (ICP4) gene from samples representing all of the farms and disease outbreaks were determined. Five prototype strains with unique sequences were chosen for detailed molecular characterization. Sequence comparisons and phylogenetic analysis of the partial ICP4 gene revealed that two strains were chicken embryo origin (CEO)-vaccine-like strains, and three were tissue culture origin (TCO)-vaccine-like strains. Analysis of the gJ gene sequence indicated that all of the strains were CEO vaccine-like strains. It was predicted that the latter three strains were recombinants of CEO- and TCO-vaccine-like strains. In conclusion, immunohistochemistry coupled with multigenomic PCR sequencing proved to be efficient for identification and typing of ILTV strains during disease outbreaks. Both CEO-vaccine-like and recombinant virus strains were circulating in Egypt during the 2018 and 2019 outbreaks.

Journal of Medical Virology, 2020

International Journal of Pharmcology, Phytochemistry and Ethnomedicine, 2019

Archives of Virology, 2019

Acute lower respiratory tract infection is a major health problem that affects more than 15% of t... more Acute lower respiratory tract infection is a major health problem that affects more than 15% of the total population of Saudi Arabia each year. Epidemiological studies conducted over the last three decades have indicated that viruses are responsible for the majority of these infections. The epidemiology of respiratory viruses in Saudi Arabia is proposed to be affected mainly by the presence and mobility of large numbers of foreign workers and the gathering of millions of Muslims in Mecca during the Hajj and Umrah seasons. Knowledge concerning the epidemiology, circulation pattern, and evolutionary kinetics of respiratory viruses in Saudi Arabia are scant, with the available literature being inconsistent. This review summarizes the available data on the epidemiology and evolution of respiratory viruses. The demographic features associated with Middle East respiratory syndrome-related coronavirus infections are specifically analyzed for a better understanding of the epidemiology of this virus. The data support the view that continuous entry and exit of pilgrims and foreign workers with different ethnicities and socioeconomic backgrounds in Saudi Arabia is the most likely vehicle for global dissemination of respiratory viruses and for the emergence of new viruses (or virus variants) capable of greater dissemination.

Animal Health Research Reviews, 2018

Coronaviruses (CoVs) produce a wide spectrum of disease syndromes in different mammalian and avia... more Coronaviruses (CoVs) produce a wide spectrum of disease syndromes in different mammalian and avian host species. These viruses are well-recognized for their ability to change tissue tropism, to hurdle the interspecies barriers and to adapt ecological variations. It is predicted that the inherent genetic diversity of CoVs caused by accumulation of point mutations and high frequency of homologous recombination is the principal determinant of these compe-tences. Several CoVs (e.g. Severe acute respiratory syndrome-CoV, Middle East respiratory syndrome-CoV) have been recorded to cross the interspecies barrier, inducing different disease conditions in variable animal hosts. Bovine CoV (BCoV) is a primary cause of gastro-enteritis and respiratory disease in cattle calves, winter dysentery in lactating cows and shipping fever pneumonia in feedlot cattle. Although it has long been known as a restrictive cattle pathogen, CoVs that are closely related to BCoV have been recognized in dogs, humans and in other ruminant species. Biologic, antigenic and genetic analyses of the so-called 'bovine-like CoVs' proposed classification of these viruses as host-range variants rather than distinct virus species. In this review, the different bovine-like CoVs that have been identified in domesticated ruminants (water buffalo, sheep, goat, dromedary camel, llama and alpaca) and wild ruminants (deer, wild cattle, antelopes, giraffes and wild goats) are discussed in terms of epidemiology, transmission and virus characteristics. The presented data denote the importance of these viruses in the persistence of BCoV in nature, spread to new geographical zones, and continuous emergence of disease epidemics in cattle farms.

Acta virologica, 2018

Development of potent vaccine for human respiratory syncytial virus (HRSV) that confers better pr... more Development of potent vaccine for human respiratory syncytial virus (HRSV) that confers better protection than natural infection remains a global challenge. Vaccination with naked DNA is considered successful approach for the control of many viral diseases. In this study, the potential of DNA vaccination using full-length attachment gene of HRSV type A Saudi strain cloned in pcDNA3.1 + vector (pcDNA/GA) was evaluated in BALB/c mice. The expression efficiency of pcDNA/GA was first confirmed in HEp-2 cells on RNA and protein levels. Mice immunization with either pcDNA/GA or the positive control formalin-inactivated vaccine (FI-RSV) has generated significant serum antibody concentration in ELISA (7.31±0.418 and 9.76±0.006 µg/ml, respectively) with superior neutralizing activity. Similarly, both immunogens evoked robust HRSV-specific CD8+ T-cell response in ELISPOT assay compared to mice immunized with pcDNA3.1 + vector or saline (negative controls). Challenge of the immunized mice with the wild-type HRSV did not provoke clinical symptoms or mortality in any mice group. On the 7 th day post-challenge, mice were euthanized and lungs were extirpated for evaluation of viral load, histopathological changes and cytokine profile. A significant diminish in the viral load and histology score were concluded in lungs of pcDNA/GA immunized mice compared to those immunized with FI-RSV and negative controls. The pulmonary cytokine profile of pcDNA/GA immunized mice displayed notable upregulation of Th1-associated cytokines while that of FI-RSV immunized mice exhibited high levels of Th2-associated cytokines. In conclusion, the DNA vaccine candidate pcDNA/GA has proven prominent efficacy and safety in mouse model, which encourages further evaluation in clinical trials.

Journal of Molecular Microbiology and Biotechnology, 2016

Human metapneumovirus (HMPV) is an important cause of respiratory tract illness in children. Two ... more Human metapneumovirus (HMPV) is an important cause of respiratory tract illness in children. Two HMPV subgroups, A and B, and four genotypes, A1, A2, B1 and B2, have been identified. Concurrent circulation of the different genotypes in yearly epidemics has been recorded globally, but not in Saudi Arabia. The current report was designed to study HMPV epidemiology in Saudi children and to analyze
the genetic diversity and circulation patterns. Nasopharyngeal aspirates (n = 174) were collected from hospitalized children in Riyadh (2008–2009). The screening of samples using real-time RT-PCR identified 19 HMPV strains. The majority of the strains belonged to subgroup B, while all strains of subgroup A were members of genotype A2. In 2008, only
subgroup B was recognized, whereas in 2009 both subgroups were identified to be cocirculating at similar rates. The full-length attachment (G) gene and a partial sequence of the fusion (F) gene of positive samples were sequenced. The G gene showed a high degree of genetic diversity and exhibited a variable number of positively selected sites in different lineages. In contrast, the F gene demonstrated an extensive genetic stability with a higher tendency toward purifying selection. This is the first report on HMPV genotype circulation in Saudi Arabia; however, the exact circulation kinetics requires further retrospective and prospective study.

The development of safe and potent vaccines for human respiratory syncytial virus (HRSV) is still... more The development of safe and potent vaccines for human respiratory syncytial virus (HRSV) is still a challenge for researchers worldwide. DNA-based immunization is currently a promising approach that has been used to generate human vaccines for different age groups. In this study, novel HRSV DNA vaccine candidates were generated and preclinically tested in BALB/c mice. Three different versions of the codonoptimized HRSV fusion (F) gene were individually cloned into the pPOE vector. The new recombinant vectors either express full-length (pPOE-F), secretory (pPOE-TF), or M2 82-90 linked (pPOE-FM2) forms of the F protein. Distinctive expression of the F protein was identified in HEp-2 cells transfected with the different recombinant vectors using ELISA and immunofluorescence. Mice immunization verified the potential for recombinant vectors to elicit significant levels of neutralizing antibodies and CD8 C T-cell lymphocytes. pPOE-TF showed higher levels of gene expression in cell culture and better induction of the humoral and cellular immune responses. Following virus challenge, mice that had been immunized with the recombinant vectors were able to control virus replication and displayed lower inflammation compared with mice immunized with empty pPOE vector or formalin-inactivated HRSV vaccine. Moreover, pulmonary cytokine profiles of mice immunized with the 3 recombinant vectors were similar to those of the mock infected group. In conclusion, recombinant pPOE vectors are promising HRSV vaccine candidates in terms of their safety, immunogenicity and protective efficiency. These data encourage further evaluation in phase I clinical trials.

Respiratory tract infections are a principal cause of illness and mortality in children worldwide... more Respiratory tract infections are a principal cause of illness and mortality in children worldwide and mostly caused by viruses. In this study, the epidemiology of 11 respiratory RNA viruses was investigated in a cohort of hospitalized children at a tertiary referral center in Riyadh from February 2008 to March 2009 using conventional and real-time monoplex RT-PCR assays. Among 174 nasopharyngeal aspirates, respiratory syncytial virus (RSV) was detected in 39 samples (22.41%), influenza A virus in 34 (19.54%), metapneumovirus (MPV) in 19 (10.92%), coronaviruses in 14 (8.05%), and parainfluenza viruses (PIVs) in 11 (6.32%). RSV, PIVs and coronaviruses were most prevalent in infants less than 6 months old, MPV and influenza A virus were more prominent in children aged 7-24 and 25-60 months, respectively. The majority of the viruses were identified during winter with two peaks observed in March 2008 and January 2009. The presented data warrants further investigation to understand the epidemiology of respiratory viruses in Saudi Arabia on spatial and temporal basis. This article is protected by copyright. All rights reserved.

Saudi journal of gastroenterology : official journal of the Saudi Gastroenterology Association, Jan 16, 2015

Hepatitis B virus (HBV) continues to be one of the most important viral pathogens in humans. Surf... more Hepatitis B virus (HBV) continues to be one of the most important viral pathogens in humans. Surface (S) protein is the major HBV antigen that mediates virus attachment and entry and determines the virus subtype. Mutations in S gene, particularly in the "a" determinant, can influence virus detection by ELISA and may generate escape mutants. Since no records have documented the S gene mutations in HBV strains circulating in Saudi Arabia, the current study was designed to study sequence variation of S gene in strains circulating in Saudi Arabia and its correlation with clinical and risk factors. A total of 123 HBV-infected patients were recruited for this study. Clinical and biochemical parameters, serological markers, and viral load were determined in all patients. The entire S gene sequence of samples with viral load exceeding 2000 IU/mL was retrieved and exploited in sequence and phylogenetic analysis. A total of 48 mutations (21 unique) were recorded in viral strains in ...

Journal of Virology, 2015

Cytokines are a group of small secreted proteins that mediate a diverse range of immune and nonim... more Cytokines are a group of small secreted proteins that mediate a diverse range of immune and nonimmune responses to inflammatory and microbial stimuli. Only a few of these cytokines mount an antiviral response, including type I, II, and III interferons (IFNs). During viral infections and under inflammatory conditions, a number of cytokines and chemokines are coproduced with IFN; however, no systematic study exists on the interactions of the cytokine repertoire with the IFN response. Here, we performed the largest cytokine and chemokine screen (the human cytokinome, with >240 members) to investigate their modulation of type I and type II IFN responses in a cell line model. We evaluated the cytokine activities in both IFN-stimulated response element (ISRE) and IFN-␥ activation sequence (GAS) reporter systems. Several cytokine clusters that augment either or both ISRE-and GAS-mediated responses to IFNs were derived from the screen. We identified novel modulators of IFN responsebetacellulin (BTC), interleukin 11 (IL-11), and IL-17F-that caused time-dependent induction of the IFN response. The ability to induce endogenous IFN-␤ and IFN-stimulated genes varies among these cytokines and was largely dependent on Stat1, as assessed by Stat1 mutant fibroblasts. Certain cytokines appear to augment the IFN-␤ response through the NF-B pathway. , which activates a cascade of biochemical events that aim to control the virus life cycle. In our work, we examined more than 200 cytokines, soluble mediators produced within the body as a result of infection, for the ability to enhance IFN action. We identified enhanced interactions with specific IFNs and cytokines. We also revealed that betacellulin, IL-17, and IL-11 cytokines have the novel property of enhancing the antiviral action of IFN against several viruses. These results demonstrate that the human genome codes for previously unknown proteins with unrelated functions that can augment the innate immunity to viruses. Knowing these interactions not only helps our understanding of immunity to viruses and emerging diseases, but can also lead to devising possible new therapeutics by enhancing the mediator of antiviral action itself, IFN.

Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16) is the causative ev... more Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16) is the causative event for the development of cervical cancer and other malignant tumors of the anogenital tract and of the head and neck. Despite many attempts to develop therapeutic vaccines no candidate has entered late clinical trials. An interesting approach is a DNA based vaccine encompassing the nucleotide sequence of the E6 and E7 viral oncoproteins. Because both proteins are consistently expressed in HPV infected cells they represent excellent targets for immune therapy. Here we report the development of 8 DNA vaccine candidates consisting of differently rearranged HPV-16 E6 and E7 sequences within one molecule providing all naturally occurring epitopes but supposedly lacking transforming activity. The HPV sequences were fused to the J-domain and the SV40 enhancer in order to increase immune responses. We demonstrate that one out of the 8 vaccine candidates induces very strong cellular E6- and E7- specific
cellular immune responses in mice and, as shown in regression experiments, efficiently controls growth of HPV 16 positive syngeneic tumors. This data demonstrates the potential of this vaccine candidate to control persistent HPV 16 infection that may lead to malignant disease. It also suggests that different
sequence rearrangements influence the immunogenecity by an as yet unknown mechanism.

Many types of naturally-growing plants are used traditionally for the treatment of different type... more Many types of naturally-growing plants are used traditionally for the treatment of different types of cancers and infectious diseases. In this report, the cytotoxic activity of 30 medicinal plants, commonly used in folk medicine in Saudi Arabia and Indonesia, was evaluated in vitro using Vero and HEp-2 cell lines. Plants were randomly chosen and harvested from different districts of both countries based on ethnobotanical information and subsequently extracted by methanol. Serial two-fold dilutions of each extract, starting from the concentration of 1000 µg/ml, were incubated with Vero and HEp-2 cells for 72 hours. The cytotoxic effect of different extracts was in vitro characterized by identification of cellular alterations microscopically and cellular viability colormetrically. The plant extracts were classified according to the minimal toxic concentration and 50% cytotoxicity concentration indexes into three groups: highly cytotoxic (8-31 µg/ml), moderately cyototoxic (32-499 µg/ml) and low cyototoxic (500-1000 µg/ml). The results showed that the extracts of Juniperus phoenicea and Calotropis procera were highly cytotoxic on both cell lines to the minimal concentration of 1 µg/ml and may be well-considered as potential candidates for anticancer research. Two more extracts (Datura inoxia and Citrullus colocynthis) produced significant cytotoxicity to the minimum concentration of 16 µg/ml, with selective powerful activity of Citrullus colocynthis on HEp-2 cells. The other extracts showed lower degrees of cytotoxicity and may be utilized for testing as antiviral agents using cell culture models.

Virus Genes, Apr 2014

The genetic variability and circulation pattern of human respiratory syncytial virus group B (HRS... more The genetic variability and circulation pattern of human respiratory syncytial virus group B (HRSV-B) strains, identified in Riyadh during the winters of 2008 and 2009, were evaluated by partial sequencing of the attachment (G) protein gene. The second hypervariable region (HVR-2) of G gene was amplified by RT-PCR, sequenced and compared to representatives of different HRSV-B genotypes. Sequence and phylogenetic analysis revealed that all Saudi strains belonged to the genotype BA, which is characterized by 60-nucleotide duplication at HVR-2. Only strains of 2008 were clustered with subgroup BA-IV, while those isolated at 2009 were clustered among the most recent subgroups (particularly BA-X and CB-B). Amino acid sequence analysis demonstrated 18 amino acid substitutions in Saudi HRSV-B strains; among which five are specific for individual strains. Furthermore, two potential N-glycosylation sites at residues 230 and 296 were identified for all Saudi strains, and an additional site at amino acid 273 was found only in Riyadh 28/2008 strain. O-glycosylation was predicted in 42-43 sites, where the majority (no = 38) are highly conserved among Saudi strains. The average ratio between non-synonymous and synonymous mutations (x) implied stabilizing selection pressure on G protein, with evidences of positive selection on certain Saudi strains. This report provides preliminary data on the circulation pattern and molecular characteristics of HRSV-B strains circulating in Saudi Arabia.

Journal of Medical Virology, Oct 2013

Influenza viruses are known as continuing threats to human public health every year worldwide. ... more Influenza viruses are known as continuing
threats to human public health every year
worldwide. Evolutionary dynamics of influenza
B viruses in humans are in a unique progression
having two lineages; B/Yam and B/Vic-like
viruses, which are circulating simultaneously
worldwide. There is a considerable lack of data
on influenza B viruses circulating in Saudi
Arabia. During the winter-spring season of
2010–2011, 80 nasopharyngeal aspirates were
collected from hospitalized patients with flu--
like symptoms in Riyadh. Screening of samples
by one-step RT-PCR identified three (3.8%)
influenza B viruses. Sequencing of hemagglutinin
(HA) and neuraminidase (NA) genes was
performed to analyze influenza B viruses circulating
in Riyadh as compared to the globally
circulating strains. Several common and six
unique amino acid substitutions were observed
for both HA and NA genes of influenza B Saudi
strains. Three unique substitutions (T182A,
D196N, and K254R) were identified in HA gene
of the B/Yam-like Riyadh strains. In NA gene, a
unique common substitution (D53G) was
found in all Riyadh strains, while two unique
substitutions (L38P, G233R) were recognized
only in B/Vic-like Riyadh strains. Riyadh strains
were also found to contain N-glycosylation site
in HA gene of both B/Vic and B/Yam lineages
at positions 197–199 (NET) and 196–198 (NNK/
DNK), respectively. The significance of these
mutations on the antigenicity of both lineages
is discussed herein. The unique changes observed
in HA and NA genes of influenza B
Riyadh strains support strongly the need for
continuous surveillance and monitoring of new
evolving strains that might pose threat to the
Saudi community.

Bovine coronavirus (BCoV) is an economically significant cause of calf scours and winter dysenter... more Bovine coronavirus (BCoV) is an economically significant cause of calf scours and winter dysentery of adult cattle, and may induce respiratory tract infections in cattle of all ages. Early diagnosis of BCoV helps to diminish its burden on the dairy and beef industry. Real-time RT-PCR assay for the detection of BCoV has been described, but it is relatively expensive, requires well-equipped laboratories and is not suitable for on-site screening. A novel assay, using reverse transcription recombinase polymerase amplification (RT-RPA), for the detection of BCoV is developed. The BCoV RT-RPA was rapid (10-20 min) and has an analytical sensitivity of 19 molecules. No cross-reactivity with other viruses causing bovine gastrointestinal and/or respiratory infections was observed. The assay performance on clinical samples was validated by testing 16 fecal and 14 nasal swab specimens and compared to real-time RT-PCR. Both assays provided comparable results. The RT-RPA assay was significantly more rapid than the real-time RT-PCR assay. The BCoV RT-RPA constitutes a suitable accurate, sensitive and rapid alternative to the common measures used for BCoV diagnosis. In addition, the use of a portable fluorescence reading device extends its application potential to use in the field and point-of-care diagnosis.

Human respiratory syncytial virus (HRSV) is a frequent cause of hospitalization and mortality in ... more Human respiratory syncytial virus (HRSV) is a frequent cause of hospitalization and mortality in children worldwide. The molecular epidemiology and circulation pattern of HRSV in Saudi Arabia is mostly uncharted. In the current study, the genetic variability and phylogenetic relationships of HRSV type A strains circulating in Riyadh Province were explored. Nasopharyngeal aspirates were collected from hospitalized children with acute respiratory symptoms during the winter-spring seasons of 2007/08 and 2008/09. Among 175 samples analyzed, 39 (22.3 %) were positive for HRSV by one-step RT-PCR (59 % type A and 41 % type B). Propagation of positive samples in HEp-2 cells permitted the recovery of the first Saudi HRSV isolates. Genetic variability among Saudi HRSV-A strains was evaluated by sequence analysis of the complete attachment (G) protein gene. The nucleotide sequence was compared to representatives of the previously identified HRSV-A genotypes. Sequence and phylogenetic analysis showed that the strains examined in this study were very closely related at both the nucleotide and amino acid level, and all of them are clustered in the GA2 genotype (and mostly belonged to the NA-1 subtype). A total of 23 mutation sites, 14 of which resulted in an amino acid change, were recorded only in Saudi strains. This is the first report on genetic diversity of HRSV-A strains in Saudi Arabia. Further analysis of strains on a geographical and temporal basis is needed to fully understand HRSV-A circulation patterns in Saudi Arabia.

Virology Journal, Dec 22, 2012

Background: Although human parainfluenza type 2 (HPIV-2) virus is an important respiratory pathog... more Background: Although human parainfluenza type 2 (HPIV-2) virus is an important respiratory pathogen, a little is known about strains circulating in Saudi Arabia. Findings: Among 180 nasopharyngeal aspirates collected from suspected cases in Riyadh, only one sample (0.56%) was confirmed HPIV-2 positive by nested RT-PCR. The sample that was designated Riyadh 105/2009 was used for sequencing and phylogenetic analysis of the most variable virus gene; the haemagglutinin-neuramindase (HN). Comparison of HN gene of Riyadh 105/2009 strain and the relevant sequences available in GenBank revealed a strong relationship with Oklahoma-94-2009 strain. Phylogenetic analysis indicated four different clusters of HPIV-2 strains (G1-4). Twenty-three amino acid substitutions were recorded for Riyadh 105/2009, from which four are unique. The majority of substitutions (n=18) had changed their amino acids characteristics. By analyzing the effect of the recorded substitutions on the protein function using SIFT program, only two located at positions 360 and 571 were predicted to be deleterious. Conclusions: The presented changes of Riyadh 105/2009 strain may possess potential effect on the protein structure and/or function level. This is the first report that describes partial characterization of Saudi HPIV-2 strain.

Journal of medical Virology, Jan 1, 2012