The Viruses Part I: Introduction & General Characteristics

Lecture #11Bio3124

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 (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

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 ~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 virusesEnveloped 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 DNAWith double membrane envelope.

Binal symetry: head icosahedron, tail helicalTail 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 peptidoglycanBursts host cell to release progeny phage

Slow releaseFilamentous 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 endocytosisBrought into cell in an endosome

Fuses envelope to plasma membraneReleases 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 CyclesRelease 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 countsdirect counts made with an

electron microscope

indirect countsindirect 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

1:11:21:41:81:161:321:641:1281:5121:10241:20481:4096

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

RationalRational: 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.

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 103.3 X 1066 PFU PFU//mLmL

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