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The Viruses Part I: Introduction & General Characteristics Lecture #11 Bio3124
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Viruses are ancient many epidemics of viral diseases occurred before anyone understood the nature of their causative agents. measles and smallpox viruses were among the causes for the decline of the Roman Empire Paralytic infection by Poliovirus
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Discovery of Viruses Charles Chamberland (1884) developed porcelain bacterial filters, viruses can pass through Dimitri Ivanowski (1892) demonstrated that causative agent of tobacco mosaic disease passed through bacterial filters thought agent was a toxin Martinus Beijerinck (1898-1900) showed that causative agent of tobacco mosaic disease was still infectious after filtration referred to as filterable agent Loeffler and Frosch (1898-1900) showed that foot-and-mouth disease in cattle was caused by filterable virus
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Discovery of Viruses… Walter Reed (1900) yellow fever caused by filterable virus transmitted by mosquitoes Ellerman and Bang (1908) leukemia in chickens was caused by a virus Peyton Rous (1911) muscle tumors in chickens were caused by a virus Frederick Twort (1915) first to isolate viruses that infect bacteria (bacteriophages or phages) Felix d’Herelle (1917) firmly established the existence of bacteriophages devised plaque assay bacteriophages only reproduce in live bacteria
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What is a Virus? Not living Are intracellular parasites Depends on host metabolism Energy, materials, enzymes Virion: a complete virus particle has a genome DNA or RNA, single- or double-stranded has a protein coat “Capsid” Protects genome Mediates host attachment
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The Structure of Viruses ~10-400 nm in diameter ; too small to be seen with the light microscope Contain a nucleocapsid which is composed of nucleic acid (DNA or RNA) and a protein coat (capsid) some viruses consist only of a nucleocapsid, others have additional components Enveloped vs naked viruses enveloped viruses: surrounded by membrane naked viruses: do not have envelope
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Viral Envelopes and Enzymes Envelope: outer, flexible, membranous layer spikes or peplomers virally encoded proteins, may project from the envelope Neuraminidase releases mature virions from cells Hemagglutinin binds cellular receptor RNA dependent RNA pol Replicates – sense genome Influenza virus
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Capsids large macromolecular structures which serve as protein coat of virus protect viral genetic material and aids in its transfer between host cells made of protein subunits called protomers Protmers form capsomers that arrange symmetrically to form the coat Symmetry in capsid Helical Icosahedral complex
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Filamentous capsids Long tube of protein, with genome inside Tube made up of hundreds of identical protein subunits Tube length reflects size of viral genome Capsid proteins DNA or RNA coiled inside tube Helical Capsids
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Influenza Virus – Enveloped Virus with a Helical Nucleocapsid Helical symmetry Segmented genome 8 RNA genome segments
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Icosahedral Capsids Icosahedral capsids 20 triangular sides Each triangle made up of at least 3 identical capsid proteins Arranged in 2,3 and 5 fold symmetry Many animal viruses
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Viruses with Capsids of Complex Symmetry some viruses do not fit into helical or icosahedral capsids symmetry groups examples are the poxviruses and large bacteriophages Vaccinia virus 200x400x250 nm, enveloped virus DNA With double membrane envelope. Binal symetry: head icosahedron, tail helical Tail fibers and sheath used for binding and pins for injecting genome Phage T4
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Viral Life Cycles All viruses must: 1.Attach to host cell 2.Get viral genome into host cell 3.Replicate genome 4.Make viral proteins 5.Assemble capsids 6.Release progeny viruses from host cell
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Bacteriophage Life Cycles Attach to host cell receptor proteins Inject genome through cell wall to cytoplasm Replicate genome Lytic vs. lysogenic cycle Synthesize capsid proteins Assemble progeny phage Lyse cell wall to release progeny phage “Blows apart” host cell Some phages use slow, non-lytic release
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Bacteriophage Life Cycles Attachment to host cell proteins receptors normally used for bacterial purposes Examples: sugar uptake, iron uptake, conjugation Virus takes advantage of host proteins Injects genome through cell wall to cytoplasm
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Bacteriophage Life Cycles Lytic cycle Phage quickly replicates, kills host cell Generally lytic when host cell conditions are good –Bacteria divide quickly, but phage replicates even faster Or conditions are very bad (e.g., cell damaged) Lysogenic cycle Phage is quiescent May integrate into host cell genome Replicates only when host genome divides Generally lysogenic in moderate cell conditions Phage can reactivate to become lytic, kill host
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Lambda phage Life Cycle
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Lytic and Lysogenic life cycles Animation: Lysis and Lysogeny
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Use cell components to synthesize capsids Assemble progeny phages Exit from cell Lysis: Makes protein to depolymerize peptidoglycan Bursts host cell to release progeny phage Slow release Filamentous phages can extrude individual progeny through cell envelope Bacteriophage Life Cycles
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Eukaryotic Virus Life Cycles Attachment to host cell receptor Entry into cell Taken up via endocytosis Brought into cell in an endosome Fuses envelope to plasma membrane Releases capsid into cytoplasm
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Eukaryotic Virus Life Cycles Genome replication DNA viruses must go to cell nucleus to use host polymerase Or replicate in cytoplasm with viral polymerase RNA viruses must encode a viral polymerase Host cells cannot read RNA to make more RNA dsRNA and (+)ssRNA genome can be translated (-)ssRNA and retrovirus genomes must be replicated to be translated –Only (+)ssRNA can be used as mRNA
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Eukaryotic Virus Life Cycles All viruses make proteins with host ribosomes Translation occurs in cytoplasm Assembly of new viruses Capsid and genome Assembly may occur in cytoplasm Or in nucleus Capsid proteins must move into nucleus Envelope proteins are inserted in host membrane Plasma membrane or organelle membrane
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Eukaryotic Virus Life Cycles Release of progeny viruses from host cell Lysis of cell, similar to bacteria Budding Virus passes through membrane Membrane lipids surround capsid to form envelope All enveloped viruses bud from a membrane Plasma membrane or organelle membrane
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Infection of a living host (animal or plant) embryonated eggs tissue (cell) cultures monolayers of animal cells plaques localized area of cellular destruction and lysis cytopathic effects microscopic or macroscopic degenerative changes or abnormalities in host cells and tissues The Cultivation of Viruses
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Hosts for Bacterial and Archael Viruses usually cultivated in broth or agar cultures actively growing bacteria broth cultures lose turbidity as viruses reproduce plaques observed on agar cultures
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Virus Assays used to determine quantity of viruses in a sample two types of approaches direct count particles indirect measurement of an observable effect of the virus
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Particle counts direct counts made with an electron microscope indirect counts e.g., hemagglutination assay determines highest dilution of virus that causes red blood cells to clump together virus particles Latex bead
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Indirect Counts: Hemagglutination Test Measures minimal viral quantity needed for agglutination of RBC Relative Concentration. Good for viruses that express hemagglutinin on the envelope; e.g. Influenza virus, paramyxoviruses, adenovirus. Doesn’t distinguish between infectious and non-infectious particles. Simple and Fast. Dilution series of virus is prepared and mixed with chicken RBC in a microtitre plate Hemagglutination is detected by RBC/virus lattice formation that does not sink to the bottom of the wells
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Hemagglutination Titre 1:11:2 1:4 1:81:16 1:32 1:641:128 1:512 1:10241:20481:4096 Titre is 512 HU
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Measuring concentration of infectious units plaque assays dilutions of virus preparation made and plated on lawn of host cells number of plaques counted results expressed as plaque-forming units (PFU)
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Titre of Infectious Viruses: Plaque Assay Infecting cellular monolayers or bacterial lawn with different viral dilutions. Counting the number of plaques from different dilutions Rational Rational: Each plaque is formed when a host cell has been infected by a viral particle
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Plaques assay: virus titre Localized cytopathic effect. Results in death or cell lysis Virions released from the infected cell infect the nearby cells and infection spreads radially Cleared areas (plaques) become visible within uninfected monolyer or bacterial lawn Each plaque represents a focus of infection. Each focus of infection is initiated by an infected cell.
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330 33 3 Dilution factor 33 PFU/0.1ml from a dilution of 10 -4. Thus the titer of the original suspension is? 3.3 X 10 6 PFU/mL Calculation of virus titre:
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Culturing Viruses Viruses grown with host cells as food Viruses bound to host Free virus concentration drops Eclipse period Viruses making proteins, genomes, assembling Rapid rise period Burst of bacteriophage = bacterial lysis Rapid release of eukaryotic viruses
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