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Virology Research Group (VRG) King Saud University King Saud University Dr. Fahad. N. Alamjhdi Group Leader Associate Professor of Virology Department of Botany and Microbiology College of Science, King Saud University Slide 2 2 Objectives Research Lines MembersFacilities Established Techniques FundingPartnershipAchievements Work in Progress Future Plans Contact Us Virology Research Group (VRG) Main Topics King Saud University Slide 3 3 Virology Research Group (VRG) ObjectivesObjectives King Saud University 1.Conducting basic and applied virological research that has a potential impact on the Saudi community (refer to the main research lines). 2.Development of improved diagnostic tools and vaccine candidates for commercial production (patents). 3.Generating valuable scientific data for publication in respective international journals (ISI cited). 4.Development of a scientific school that delivers skillful scientists in the fields of Virology, Molecular biology and Immunology. Slide 4 4 Virology Research Group (VRG) Research Lines King Saud University Virus Disease Diagnosis: 1.Branch A: Identification and characterization of viruses circulating among the human population in Saudi Arabia. 2.Branch B: Development and validation of improved tools for accurate, rapid and economic diagnosis of virus infections. Slide 5 5 Virology Research Group (VRG) Research Lines King Saud University Control of Virus Infections: 1.Branch A: Developing new vaccine candidates or new strategies for vaccine production. 2.Branch B: Studying the antiviral effect of different bio-molecules (especially those extracted from natural sources) Slide 6 6 Virology Research Group (VRG) Our Team King Saud University Slide 7 7 Virology Research Group (VRG) MembersMembers King Saud University Dr. Fahad N. Almajhdi, Ph.D Principle Scientist and Group Leader Dr. Haitham M. Amer, PhD Associate Scientist and Scientific Coordinator Dr. Zahed Hussein, PhD Assistant Scientist Dr. Ghazanfar Ali, PhD Assistant Scientist Slide 8 8 Virology Research Group (VRG) MembersMembers King Saud University Mr. Moawia E. Hamad, MSc PhD student and Research Assistant Mr. Abdul Latif, MSc PhD student and Research Assistant Mr. Mohamed S. Alshaman, B.Sc MSc student and Research Assistant Mr. Mohamed A. Farrag, B.Sc MSc student and Research Assistant Mr. Ahmed Qadari, MSc PhD student and Research Assistant Mr. Mohamed S. Hodhod, B.Sc MSc student and Research Assistant Mr. Mohamed Arshad, B.Sc MSc student and Research Assistant Slide 9 9 Virology Research Group (VRG) FacilitiesFacilities King Saud University Infrastructure: Laboratory dimensions: Size area: 69 m 2 Length: 9.50 m Width: 7.25 m Basic structures: Central two-side working bench (size area: 7 m 2 ), with cupboards and drawers for storage. Side bench, L-shape (size area: 13 m 2 ). Side shelves on both sides of the laboratory. Supplied with all the required links for electricity, water, gas, vacuum and internet. Slide 10 10 Virology Research Group (VRG) FacilitiesFacilities King Saud University Laboratory Equipment: 1. Sterilization equipment: Autoclave and sterilization oven. 2. Basic lab. equipment: Balances, stirrers, vortices, pH meters, electrical heaters, microscopes, water baths and microtiter pipettes. 3. Storage and transportation vehicles: Ice boxes, refrigerators, freezers, ultra-deep freezers (-80C) and Liquid nitrogen tanks. 4. Molecular biology equipment: Thermal cycler, real-time analyzer, microplate reader and microfuges. Slide 11 11 Virology Research Group (VRG) FacilitiesFacilities King Saud University 5. IN Cell Analyzer Image Analysis System. 6. Egg inoculation equipment: Egg candler and egg incubator. 7. Tissue culture facilities: Laminar air flow, Inverted and fluorescent microscopes, incubators and cooling centrifuge. 8. Electrophoresis units: Horizontal and vertical gel electrophoresis tanks, blotting units and power supplies. Slide 12 12 Virology Research Group (VRG) FacilitiesFacilities King Saud University Supplies: Most of the required supplies are available in the lab: A)Tissue culture: Medium, additives, flasks, plates, tubes,etc. B)Molecular biology: DNA/RNA/plasmid extraction kits, Gel recovery/PCR clean up kits, PCR/QPCR reagents and master mixes, ladders,.etc C) Serology and protein analysis: ELISA/HA plates, ELISA kits, buffers, membranes, protein markers,etc. D) Chemicals and glassware: (of considerable relevance ). Slide 13 13 Virology Research Group (VRG) Establishment Techniques King Saud University 1.Basic tissue culture techniques. 2.Virus isolation and propagation (TC and ECEs). 3.Virus titration (Infectivity titration and plaque assay). 4.Virus neutralization assays. 5.Haemagglutination (HA) and haemagglutination inhibition (HI) assays 6.ELISA (different types and formats). 7.Immunofluoresnce Slide 14 14 Virology Research Group (VRG) Establishment Techniques King Saud University 8.SDS-PAGE and Western blot 9.DNA/RNA extraction (cellular, viral, plasmid,..etc) 10.Gel electrophoresis 11.Conventional end point PCR and RT-PCR 12.Quantitative Real-time PCR and RT-PCR 13.Molecular cloning 14.DNA sequence analysis Slide 15 15 Virology Research Group (VRG) FundingFunding King Saud University 2. Research center, College of Science (through funds allocated for the research projects and for supporting graduate students). 3. Projects funded by King Abdul Aziz City for Science and Technology (KACST) and King Saud University (KSU). 1. Center of Excellence in Biotechnology Research (CEBR), project funds. Slide 16 16 Virology Research Group (VRG) PartnershipsPartnerships King Saud University Research on Respiratory Viruses King Khalid University Hospital (KKUH), King Saud University Human Papilloma Virus Research German Cancer Research Center (DKFZ), Heidelberg and University of Constance, Germany Research on Hepatitis Viruses and Interferon Monitoring King Faisal Specialist Hospital and Research Center (KFSH- RC) Research on vaccine Development Drug Delivery Research Group, College of Pharmacy, King Saud University Slide 17 17 Virology Research Group (VRG) On-going Projects & Achievements King Saud University 1.DNA vaccination of cervical cancer patients with rearranged oncogenes of the human pappillomavirus type 16 2.Human parainfluenza viruses in Saudi Arabia: Isolation, identification and sequence analysis. 3.Development of a SYBR-Green based real-time PCR system for detection and quantification of Bovine Coronavirus. 4.Development and validation of molecular diagnostic kits using nano- and recombinant DNA technology for: A- Mutation detection and B- Interferon monitoring. 5.Development of monospecific and multiplex RT- PCR systems for detection of Respiratory Viruses in young children in Riyadh. Research Projects: Slide 18 18 Virology Research Group (VRG) King Saud University 18 A) DNA vaccination of cervical cancer patients with rearranged oncogenes of the human pappillomavirus type 16 Funded by King Saud University In collaboration with the German Cancer Research Center (DKFZ), Heidelberg, and university of Constance, Germany [Two years project; extended for another 1 year]. In this project, different rearranged (shuffled) HPV 16 E6/E7 gene constructs were designed and constructed for development of a potent DNA vaccine candidate. These constructs were tested for immunogenicity in vivo (mice model system) by ELISA (Antibody response) and Elispot (Cellular response). Tumor regression in mice was also evaluated. One construct is highly promising as a vaccine candidate. Currently, the transformation activity and the expression potential of each construct in cell culture is under estimation. On-going Projects & Achievements Slide 19 19 Virology Research Group (VRG) King Saud University B)Human parainfluenza viruses in Saudi Arabia: Isolation, identification and sequence analysis. A work of Master thesis conducted by M.S. Shaman; supported in part by Research Center, College of Science, KSU. [Three years project]. In this project, 180 NPA samples, collected from young children during two successive winter seasons, were screened for human para- influenza viruses 1,2 and 3 using newly optimized RT-PCR procedures. The entire sequence of HN gene of 2 selected HPIV-3 and one HPIV-2 strains was retrieved using overlapping sets of primers. Sequence analysis enabled allocation of unique mutations and identification of the phylogenetic relationship with the international strains. Virus isolation and propagation in cell culture is proceeding. On-going Projects & Achievements Slide 20 20 Virology Research Group (VRG) King Saud University C) Development of a SYBR-Green based real-time PCR system for detection and quantification of Bovine Coronavirus. In this project, an optimized system for detection and quantification of BCV using SYBR green based real-time PCR assay was developed. Different variables like MgCl 2 and primer concentrations, annealing temperature and template volume were evaluated and chosen. Testing the assays specificity, sensitivity, reproducibility and linearity proved its competence and efficiency. Quantification standards were constructed and utilized for generation of standard curve for use in absolute quantification of BCV in clinical samples. On-going Projects & Achievements Slide 21 21 Virology Research Group (VRG) King Saud University D) Development and validation of molecular diagnostic kits using nano- and recombinant DNA technology for: A- Mutation detection and B- Interferon monitoring. Funded by Center of Excellence in Biotechnology Research, King Saud University, in collaboration with King Faisal Specialist Hospital and Research Center (KFSH-RC), Riyadh, SA. The aim of this project (our group) is to develop a newly modified, simple and high-throughput bioassay for interferon in form of genetically engineered cell line responsive and selective to type-I IFN. Until now, different IFN stimulated promoters in the human genome was assessed by microarray and bioinformatics; strong and specific reporter constructs was prepared and transfected in different cell lines; and stable clones that express the promoter-reporter system were established. On-going Projects & Achievements Slide 22 22 Virology Research Group (VRG) King Saud University E) Development of monospecific and multiplex RT-PCR systems for detection of Respiratory Viruses in young children in Riyadh. Funded by Research Center, College of Science, King Saud University, in collaboration with King Khalid University Hospital, Riyadh, SA. In this project, different RT-PCR systems were developed for detection and typing of human respiratory viruses like RSV, influenza A and B, parainfluenza and metapneumo viruses. These systems were exploited for screening more than 200 nasopharyngeal aspirate samples collected from young children at KKUH, Riyadh. 70 RSV, 10 HPIV and 9 influenza B positive samples were identified. Isolation and characterization of these virus strains ongoing. On-going Projects & Achievements Slide 23 23 Virology Research Group (VRG) Selected Publications King Saud University 1. Amer, H.M. and Almajhdi, F.N. Development of a SYBR-Green based real-time PCR system for detection and quantification of Bovine Coronavirus. Mol. Cell. Prob., In Press. [ISI Journal Impact Factor 1.899]. 2. Fahad Al-Majhdi. The identification of the first isolate of influenza B Virus using a duplex RT-PCR DNA sequencing in Saudi Arabia (B/Riyadh/01/2007), 2010. African J. Microbiol. Res., 4 (9): 697-703. [ISI Journal Impact Factor 0.409]. 3. Amer, H.M.; Elzahed, H.M.; Yousef, A.A.; Badawy, A.A. and Elabiare, E.A., 2011. An Optimized PCR Assay Identifies Possible Contamination of Avian Virus Vaccines by Chicken Anemia Virus. J. Vet. Diagn. Invest., 23 (1): In Press [ISI Journal Impact factor 1.546]. 4. Fahad Al-Majhdi, 2009. A report on the association of influenza B Virus with respiratory tract infection of hospitalized children in Saudi Arabia. Saudi J. Biol. Sci., 16 (2): 109-111. 6. Fahad Al-Majhdi, A. Al-Jarallah, M. Elaeed, A. Latif, L. Gissmann and Haitham M. Amer, 2009. Prevalence of Respiratory Syncytial Virus Infection in Riyadh During the Winter Season 2007-2008 and Different Risk Factors Impact. Inter. J. Virology, 5 (4): 154-163. 7. Hussain Z, Jung SH, Ryu DK and Ryu WS, 2009. Genetic dissection of naturally occurring basal core promoter mutations of hepatitis B virus reveals the silent phenotype in the overlapping X gene. J of General Virology, 90 (9): 2272-2281 [ISI Journal Impact Factor 2.97] 8. Jeong-Ki, Kim., AL-Majhdi, F., Kumar, Shanmukhappa and Sanjay, Kapil. 2006. Defining the Cellular Target(s) of Porcine Reproductive and Respiratory Syndrome Virus Blocking Monoclonal Antibody 7G10. J. Virology. 80: 689- 696. [ISI Journal Impact Factor 5.15]. Slide 24 24 Virology Research Group (VRG) GenBank Submission King Saud University Accession number: GU135839 Influenza B virus (B/Riyadh/01/2007) segment 8 nuclear export protein (NEP) and nonstructural protein (NS1) mRNAs, partial cds Accession number: HM460888 Human Parainfluenza virus type 2, strain Riyadh 105/2009, haemagglutinin- neuramindase gene, complete cds Accession number: HM460886 Accession number: HM460886 Human Parainfluenza virus type 3, strain Riyadh 11/2008, haemagglutinin- neuramindase gene, complete cds Accession number: HM460887 Human Parainfluenza virus type 3, strain Riyadh 149/2009, haemagglutinin- neuramindase gene, complete cds Under processing in GenBank Human Respiratory Syncytial Virus, Genotypes A and B (2008 and 2009), Attachment and Fusion glycoproteins, complete cds Slide 25 25 Virology Research Group (VRG) Work in Progress King Saud University 1.Cell-based screening of antiviral and anti- proliferative activities of selected medicinal plants. 2.Isolation, antigenic and genomic characterization of respiratory syncytial and influenza viruses (Saudi strains). 3.Studying the expression potential and transformation activity of the rearranged oncogene constructs of human papilloma virus 16. 4.Molecular Genetic Bio-Markers Associated with the Development of Viral Hepatitis in Saudi Patients Slide 26 26 Virology Research Group (VRG) Future Plans King Saud University Laboratory structure: A. Construction: Nine cupboards will be designed, fabricated and hanged at the right side of the laboratory. B. Equipment: Tissue culture basic equipment (Laminar flow, CO2 incubator and inverted microscope). Electrophoresis facilities (horizontal gel, vertical gel and blotting systems, UV trans-illunminator). Storage vehicles (Ultradeep freezer, ordinary freezer and refrigerator). Slide 27 27 Virology Research Group (VRG) Future Plans King Saud University Scientific Publications: Two articles are expected to be publish in ISI peer reviewed journals during the next few months. 1.Alshaman, M.S.; Amer, H.M and Almajhdi, F.N. The first Human Parainfluenza Virus type 3 isolates in Saudi Arabia: Sequence analysis of haemagglutinin- neuramindase (HN) gene. 2.Al-Ahmadi, W.; Al-Haj, L.; Almajhdi, F.; Amer, H. and Khabar, K. GFP reporter system with transcriptional sequence heterogeneity for monitoring viral induced interferon response. Slide 28 28 Virology Research Group (VRG) Future Plans King Saud University Research projects: 1.Development of Respiratory Viral Particulate Subunit Vaccine (A project proposal has been submitted to CEBR). 2.Isolation, Characterization and Sequence Analysis of Human Respiratory Syncytial Viruses in Saudi Arabia (A project proposal has been submitted to CEBR). 3.Phylogenetic analysis of respiratory viruses circulating in the human population of Riyadh (A project proposal will be submitted during the next few months). Slide 29 29 Virology Research Group (VRG) Future Plans King Saud University 29 Techniques to be establish: Hybridoma technology. Baculovirus expression vector system. Virus purification (Cesium chloride and sucrose gradient). Cell transfection procedures. Slide 30 30 Virology Research Group (VRG) Contact US King Saud University Address: Lab.2A82, 2 nd Floor, College of Science, Botany and Microbiology Department, Building 5, King Saud University Tel.: Tel.: +966-1-4677879 / +966-1-4677778 Fax: Fax: +966-1-4675833, +966-1-4674253 E-mail: E-mail: vrl_cos_ksu@yahoo.com P.O.Box 2455, Riyadh 11451, Saudi Arabia Slide 31 4/23/201531 31 Virology Research Group (VRG) End of Presentation King Saud University Thank You!