MVI: Molecular Virology and Immunology Lecture 2 PDF

MVI: Molecular Virology and Immunology Course Coordinator: Dr George Krashias *: [email protected] (: 22392648 26-09-2024 Department of...

MVI: Molecular Virology and Immunology Course Coordinator: Dr George Krashias *: [email protected] (: 22392648 26-09-2024 Department of 1 Molecular Virology Taxonomy of Viruses Outline Virus taxonomy Methodologies for virus culture Methodologies for virus detection Department of 2 Molecular Virology Examples of the origins of virus names Adenovirus Coronavirus Epstein-Barr virus Coxsackie HSV Department of 3 Molecular Virology Virus taxonomy: brief history Virology is around 125 years old 1900 Viruses classified according to their ability to pass through filters Use measurable properties for classification Animal viruses causing liver pathology were grouped together as hepatitis viruses 1930 Viruses classified based on their physical characteristics 1960 Numerous data available for describing different viruses Collective and organized effort need to classify and name viruses 1966 creation of International Committee on Taxonomy of Viruses (ICTV) Department of 4 Molecular Virology Virus taxonomy: the ICTV system The system currently being used is characterized by a: Nonsystematic Polythetical Hierarchical Study groups of Groups viruses by Viruses are hierarchical virologists within the ICTV comparing simultaneously placed in the following consider numerous numerous properties of levels: characteristics of a virus individual viruses Order (-virales) and make as rational an Family (-viridae) assignment to a group as Subfamily (-virinae) possible Genus (-virus) Species Current ICTV list: - 72 orders - 264 families - 182 subfamilies - 2818 genera - 11273 species Department of 5 Molecular Virology Virus taxonomy: the ICTV system Department of 6 Molecular Virology Virus taxonomy: criteria for virus classification ?? ? Physical, chemical properties ? Virion MW Genome properties Lipids, carbohydrates, protein content Number, size and ?? ? functional activities of structural and non structural proteins Description and Antigen and biologic determination of the properties functional activities of Properties related to Natural host range viral proteins (ie RT etc) viral genome and life Tissue tropism cycle Mode of transmission in Genome nature organization Geographic distribution Replication strategy Site of virion assembly Department of 7 Molecular Virology Virus taxonomy: the Baltimore classification Viruses are classified depending on their type of genetic material: (ie DNA, RNA, etc) and their method of replication The Baltimore classification system divides viruses in 7 categories Department of 8 Molecular Virology Virus taxonomy: the Baltimore classification Department of 9 Molecular Virology Some methods for studying virus Viruses can be studied in different culture systems Culture Limitation Advantages system Expensive Variation between individuals requires large numbers Natural Animal infection Ethical considerations Simple and safe Primary cells: derived from an organ or tissue, remain differentiated, but do not survive for long Uniform population Cell lines: diploid, express some of the proteins of primary Cell cells, can replicate in culture for a limited period Good for biochemical Continuous cell lines: aneuploid, express some of the studies proteins of primary cells, but immortal Department of 10 Molecular Virology Cell cultures Cells in culture are kept in isotonic solution Contain a mixture of salts Na, K, Ca, Mg Supplemented with serum Provides nutrients for cells All components must be sterile and used under aseptic conditions Department of 11 Molecular Virology Cell culture hoods in virology Biosafety level 2 culture hood (BSL-2) Study viruses that cause mild disease in humans or are difficult to transmit via aerosol in a lab Example: EBV CING Biosafety level 3 culture hood (BSL-3) Study viruses that may cause serious or even lethal disease in humans after inhalation or infection Examples: HIV Currently build at the CING Department of 12 Molecular Virology Cell culture hoods in virology Biosafety level 4 culture hood (BSL-4) Study viruses that pose a high individual risk of aerosol-transmitted infections and are known to cause severe disease in humans Completely isolated, used by highly trained individuals that operate while wearing a positive pressure suit Examples?? Department of 13 Molecular Virology Investigating viral infections in cell cultures The usual way of detecting the presence of virus in an infected cell is by the pathology that it causes Cytopathic effect (CPE) A virus or a group of related viruses changes the morphology of the cell in a characteristic way Can be recognized by inspecting the cell culture under the microscope CPE provides a quick and easy check on the progress of infection Influenza Human respiratory syncytial virus (HRSV) CPE Chick embryo cells Monkey cells Department of 14 Molecular Virology Identification of viruses: biological assays Electron Antibody microscopy assays Plaque PCR assay Department of 15 Molecular Virology Identification of viruses: electron microscopy Dimensions of viruses are below the resolving power of visible light Visualization requires the shorter wavelengths available Department of 16 Molecular Virology Identification of viruses: electron microscopy EM accelerates electrons to high energy and magnetically focuses them High energy gives electron a short wavelength Viruses are stained or coated with a heavy metal Magnification: 2-50 million times Department of 17 Molecular Virology Identification of viruses: electron microscopy Counting of virions with the EM Viruses particles can be visualized------ can be counted Counting of particles is simple, in theory at least Virus particle number can be utilized to estimate the number of virus particles present in the original stock solution Number of virus particles counted and the volume of the solution visualized 10 particles in 1 μL How many virus particles/mL??? Total number of virus particles in 10mL of virus culture? EM caveat: can not distinguish between biologically active and inactive virus particles Department of 18 Molecular Virology Identification of viruses: plaque assay The plaque assay is based simply on the ability of a single infectious virus particle to give rise to a macroscopic area of cytopathology The plaque assay can be used to quantify virus as follows: Target: identify a dilution of virus that yields 30-300 plaques on a single plate Resulting value: titer (pfu/mL) 10 µL 3 plaques 0 plaques 30 plaques Department of 19 Molecular Virology Identification of viruses: hemagglutination assay The hemagglutination assay presents an indirect way for identifying viral particles Relies on the ability of a virus to agglutinate cells Basic components View from side View from top Department of 20 V Molecular Virology Identification of viruses: ELISA Enzyme-linked immunosorbent assay Used to detect antibodies against a specific virus present in a sample A known Antibody is bound and the remainder washed away Department of 21 V Molecular Virology Identification of viruses: Polymerase chain reaction (PCR) Relies on the detection of viruses via detection of viral genomes Amplifies a specific region within the genetic material of a virus particle Pre-requisites for PCR Department of 22 V Molecular Virology  Questions? Department of 23 V Molecular Virology