The Viruses Part I: Introduction & General Characteristics Lecture #11 Bio3124

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

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 ( )  showed that causative agent of tobacco mosaic disease was still infectious after filtration  referred to as filterable agent  Loeffler and Frosch ( )  showed that foot-and-mouth disease in cattle was caused by filterable virus

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

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

The Structure of Viruses  ~ 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

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

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

 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

Influenza Virus – Enveloped Virus with a Helical Nucleocapsid  Helical symmetry  Segmented genome  8 RNA genome segments

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

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

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

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

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

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

Lambda phage Life Cycle

Lytic and Lysogenic life cycles Animation: Lysis and Lysogeny

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

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

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

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

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

 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

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

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

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

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

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

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)

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

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.

Dilution factor  33 PFU/0.1ml from a dilution of  Thus the titer of the original suspension is? 3.3 X 10 6 PFU/mL Calculation of virus titre:

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