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Viruses – structure, laboratory detection, b t i h d t ti bacteriophages
• Understand the nature of viruses and how they differ from prokaryotic & eukaryotic cells • Describe the different types of viral genomes • Describe the different types of structure of viruses • Describe how viruses are grown and quantified in the laboratory • Describe the steps involved in virus replication • Describe the one-step growth curve seen in virus replication • Describe the potential outcomes of viral infection of a cell • Understand the principles of virus classification • Understand the nature of bacteriophages • Differentiate virulent and temperate phages • Describe the lytic and lysogenic responses/cycles resulting from bacteriophage infection of bacteria • List the uses of bacteriophage
02 to 0.4 μm • Often measured in nanometres (1000 nm = 1 μm) • Small pox virus (one of largest viruses) 200 nm. poliovirus (one of the smallest) 28 nm.Introduction • Viruses are genetic elements that replicate p y independently of a cell’s chromosomes but not independently of cells themselves • In order to multiply viruses have to get into a cell in which they can replicate – a host cell • Viruses take advantage of the host cell’s metabolic machinery encoded by the host cell’s chromosome • Virus particles vary in size – 0. 3 General properties of viruses Extracellular state • Minute particle containing nucleic acid surrounded by protein & occasionally other macromolecules • In this state virus particle (virion) is metabolically inert • Vi i is the structure b Virion i th t t by which the viral gemome is transferred from the host cell in which it was made to another host cell Intracellular • Once inside a host cell viral nucleic acid released • Viral replication occurs – New copies of genome produced – Viral coat components synthesised • Infection – process that occurs when a virus enters a cell and replicates 4 2 .
Viral genome • Different from prokaryotic and eukaryotic g genomes Viral genome – Mostly have either DNA or RNA – DNA and RNA can be double stranded or single stranded – Some viruses have both DNA and RNA – but at different stages of their reproductive cycle • All viruses use the host cell translational machinery ie viral mRNA must be generated that can be translated on the host cell ribosome 5 6 3 .
Many animal virus particles are surrounded by a lipoprotein envelope 1.Structure of viruses Core of genetic material (nucleic acid) 2. 7 Capsomeres • Subunit structure is important – Economy of genetic information – reduces the number of different proteins genome has to encode if the viral coat is made up of repeating units of a single protein – Allows for construction of the virus particles by a process of self assembly into structures held together by non-covalent bonds as occurs in the process of crystallisation – so no need for enzyme-catalysed reactions for coat assembly – Intracellular release of the viral genome only requires dissociation of non-covalent bonds rather than degradation of a protein coat 8 4 . Protein coat or capsid – Protects viral genes from inactivation by adverse environmental factors – Core + capsid = nucleocapsid – In many viruses important in attachment of viruses to specific receptors on host cells – Composed of a large number of subunits – capsomeres 3.
Icosahedral 2 I h d l symmetry 3.Geometry of capsomeres Icosahedral symmetry 1. Complex structure Papilloma virus Papilloma virus Complex structure – pox virus Helical symmetry 9 Enveloped viruses • Capsid (containing nuclear material) surrounded by a complex membranous envelope • Envelope consists of a lipid bilayer with proteins (usually glycoproteins) imbedded in it – Lipids derived from host cell – Proteins encoded by the virus • Envelope is important in determining host cell specificity & some aspects of cell penetration are affected by it 10 5 . Helical symmetry 2.
pH buffers..Additional components • Some virions carry enzymes that are important in the early stages of the infection process – Some bacteriophages carry lysozyme that makes a small hole in the bacterial cell wall to allow the phage nucleic acid to enter the cell – Some human viruses contain their own nucleic acid polymerases eg reverse transcriptase in retroviruses – Some human viruses (eg influenza viruses) contain neuraminidases – break down glycoproteins and glycolipids and aid release of virus from animal cells 11 Growth and quantification of viruses • Viruses need to be grown in their host cell – animal. salts. antibiotics. plant. i l l t bacteria • Animal viruses – cell culture – cells from animals grown artificially in the laboratory – Often in monolayers on glass or plastic overlaid with suitable liquid media for the cells q to grow • Complex medium – amino acids. 12 6 . pH indicator . b t i …. glucose. vitamins..
Tissue culture flasks • • • • T-flasks Petri dishes coverslips 6 well. 48 or 96 wells 13 14 7 . 12.
others will remain alive only for a short period – primary cell lines. 16 Figure 13.8 8 .Isolation of cells 15 Cell culture • Some cells will grow indefinitely – permanent cell lines.
Cell morphology 3T3 [low] ECs VSM Monocytes y 3T3 [high] Intestinal 17 Embryonated egg inoculation 18 9 .
Quantification of viruses • Quantify by measuring their effect on host cells ll • Virus infectious unit – smallest unit that causes a detectable effect • Bacteriophage – plaque assay (lysis of bacterial cells on an agar plate) • Animal viruses – cell monolayers overlaid with agar – plaque assay 19 Bacteriophage plaque assay – lytic bacterophage 20 10 .
Penetration (injection) of the virion or i nucleic acid i its l i id into the cell h ll 3. Attachment – adsorption of the virion to a susceptible host cell 2. Release of mature virions from the cell Virus replication is a one-step growth curve One step growth curve of virus replication 22 11 . Assembly of structural subunits (and membrane components in enveloped viruses) and packaging of nucleic acid into new virus particles 5.Animal virus plaque assay 21 Virus replication 1. Synthesis of nucleic acid & protein • Early phase – virus redirects cell metabolism to synthesise new viral nucleic acid and proteins • Late phase – structural proteins that are subunits of the viral coat are synthesised 4.
species • Increased phylogenetic studies contributing to this – Baltimore scheme (see next lecture) – based on how the viral nucleic acid replicates 24 12 . Survival of the infected cell in a dramatically altered or transformed state eg transformation of a normal cell to one having the properties of a cancerous cell 23 Viral hosts and taxonomy • Viruses can be classified on the basis of the y hosts they infect – Animal viruses – Plant viruses – Bacterial viruses (bacteriophages) • Provide a useful model system for studying molecular biology and genetics of virus reproduction • There is a formal system of viral taxonomy that organises viruses within hierarchical taxon levels – Order. Elimination of the virus from the cell and infection aborted without any recognisable effect on the cells 3. family. genus. Multiplication of the virus and destruction of the host cell 2. Survival of the infected cell in an unchanged state – but it continues to carry the virus in a latent state 4.Virus-host cell interactions Four main scenarios 1.
Bacteriophages • Bacteriophages are viruses that infect bacteria • Important in study of virology as the mechanisms of viral replication were first worked out using bacteriophage (phage) • All species of bacteria probably susceptible to phage • Phages are highly host specific – Eg Staph aureus phage will not attack a Staph epidermidis bacterium – Many phages strain specific – eg different phages attack bovine strains of Staph aureus from those that attack human strains 25 Phage • Most phage are tadpole-shaped – Heads .contain n cleic acid nucleic – Tails – attach the virus to the host cell • There are some simple icosahedral and helically symmetrical phages • Phages vary in size • Majority of phages ds DNA – some small icosahedral & helical phages have ss DNA or RNA 26 13 .
28 14 .27 Virus Structure Bacteriophage T4.
Virulent phage • As a result of infection of a bacterium with phage. Temperate phage • After infection the viral genome does not take over the host cell cellular activity. the cell survives. phage particles replicate in the cell and the cell is lysed as the particles are released – lytic response 2.Phages Phages classified on the basis of the response they p produce in the host cell 1. viral DNA is incorporated into host cell genome (prophage) • Cells carrying viral genes in this way – lysogenic • Lysogenic infections can proceed to lytic infections 29 Replication of virulent bacteriophage 30 15 .
32 16 .some strains 4 or 5 prophages • Sometimes prophage can pick up some bacterial chromosomal genes when they move out of the chromosome → potential to spread bacterial genes from one bacterium to another (transduction) • Some phages carry virulence factors eg the β-phage of Corynebacterium diphtheriae encodes for diphtheria toxin ie strains without the prophage are non-toxigenic. UV light) y p leads to mass lysis & production of lots of phage – ie a lytic response/cycle – process called induction • • 31 Lysogeny • When a lysogenic cell is exposed to the same type of phage as it carries the activity of the invading viral genes are repressed by the same mechanisms that are keeping the prophage dormant • Lysogeny is generally a very stable state but cells can lose their prophage – “cured” cells are once more susceptible to infection with that phage • Lysogeny is a common phenomenon – most bacteria carry one or more prophages eg Staph aureus .Lysogeny • • Infection of bacterial cells with temperate bacteriophage Integration of the prophage into the bacterial chromosome ensures that each daughter cell will receive a set of viral genes each cell division In normally growing cells the host cells generally keep the prophage in a dormant state – but in a few cells multiplication of the virus and lysis of the cells will occur Exposure of lysogenic cultures to certain chemical & physical agents (eg H2O2. mitomycin C.
12 Use of phages • Studies on viral replication • Molecular biology – transduction – transfer of genes from one bacterium to another.Lytic and lysogenic cycles 33 Figure 13. Pseudomonas aeruginosa-infected burns • Treatment of ready-to-eat food products to remove pathogens – eg Listeria monocytogenes. E coli O157 34 17 . transfer of virulence factors • Typing of bacteria – strains of the same species of bacteria can be differentiated by sensitivity to sets of phages – Staphylococcus aureus – Salmonella spp • Ph Phage therapy t t t infections – eg MRSA th to treat i f ti MRSA.
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