Download Viruses - Chap 13 partI

Biol 3400
Tortora - Chap 13
Virology
What is a virus?
How do viruses differ from cellular microorganisms?
1. Acellular
o consist of a particle consisting of a nucleic acid surrounded by a protein coat.
o In extracellular form a virus particle or virion is metabolically inert
o A variety of sizes and shapes – 20 nm (Poliovirus is 28 nm in diameter or the size
of a ribosome) to 1000 nm (Smallpox virus is about 200 nm in diameter; Ebola
virus can be up to 1000nm in length)
2. Viral Genomes can be used to classify viruses
o viral genome - RNA or DNA; ss or ds
 linear (T4, T7) or circular dsDNA (SV40)
 linear ssDNA (X174)
 linear ssRNA (Q) or dsRNA (6)
 May have one or more nucleic acid molecules (segmented – Reoviruses 10 dsRNA molecules or influenze virus – 8 ssRNA molecules)
 One group uses both RNA and DNA but at different stages of their life
cycle
 This variety of form of the hereditary molecule is unique to viruses
suggests that different viruses evolved from different host cells possibly as
genetic extensions
 Genomes often contain unusual nitrogenous bases – T-even phages of E.
coli contain 5-hydroxymethylcytosine
 Size ranges from approximately 4 kb (Q) to 670 kbp (bacteriophage G)
3. Protein coat or capsid
o Single or several types of protein - protein subunits (protomers) that are formed
into larger assemblies or structural subunits called capsomers. Information
necessary for proper folding and aggregation is contained within the structure of
the proteins so that they are capable of self-assembly
o nucleocapsid = capsid and genome
o Helical (rods) or isometric symmetry (spherical or icosahedral – 20 triangular
faces and 12 corners), binal = helical and isometric (e.g., T-even bacteriophage)
or complex (pox viruses)
o The capsid morphology is an important taxonomic criterion
o Viral particle may also contain one or more virus-specific enzymes (e.g.,
lysozyme, RNA-dependent RNA polymerase, reverse transcriptase,
neuraminidases) that are important in the infection and replication processes
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Biol 3400
Tortora - Chap 13
4. Enveloped viruses - some viruses are further surrounded by a membrane envelope
o membrane (phospholipids, proteins and carbohydrates) layer surrounding the
capsid
o membrane does not function like the membrane of a living organism
o proteins are typically virus-specific and coded by the virus genome. May be
important in attachment of the virion or release of the virion from the host cell
o may function in initial attachment or protection of host immune system
o acquisition of envelope is part of maturation process - acquired after assembly of
nucleocapsid - e.g., upon leaving cell
5. Require a host to replicate - independent activity
o Viruses can be classified according to hosts (i.e., animal, plant, bacteria,…)
o Host range is generally very specific
I.
Viral Taxonomy
 Much less satisfactory state than that of either bacteria or eukaryotic microorganisms
 International Committee for Taxonomy of Viruses – 1971 – 2000 virus species placed in 3
orders, 73 families, 9 subfamilies and 287 genera.
 Definition of families - greatest weight placed upon
i) nucleic acid type
ii) nucleic acid strandedness
iii) the sense of ssRNA genomes
iv) presence or absence of an envelope
v) symmetry of capsid
vi) dimensions of the virion and capside
orders end in virales; family names end in viridae, subfamily, in virinae; and genus and
species in virus
e.g., poxviruses – family Poxviridae; subfamily Chorodopoxvirinae (poxviruses of
vertebrates); Genus Orthopoxvirus – variola major (smallpox), vaccinia and cowpox
Detailed List of Taxonomic characteristics
1 . Nature of host
2. Nucleic acid characteristics
o DNA or RNA (never both)
o ss or ds
o molecular weight
o segmentation and number of pieces of nucleic acid (RNA viruses)
o sense of the strand in ssRNA
3. Capsid symmetry – helical, isometric, binal
4. Presence of envelope and ether sensitivity
5. Diameter of virion
6. Number of capsomers in isometric viruses
7. Immunological properties
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Biol 3400
Tortora - Chap 13
8. Gene number and genomic map
9. Intracellular location of replication
10. The presence or absence of a DNA intermediate (ssRNA viruses) and the presence of
reverse transcriptase
11. Type of virus release
12. Disease caused and/or special clinical features – method of transmission
II.
Viral Cultivation and Purification
 Require host cells for cultivation – inoculate suitable hosts or use tissue culture
 Some plant viruses are difficult to work with because they require the whole plant
Viruses are
 large relative to proteins
 more stable than normal cell components
 have surface proteins.
These properties allow the use of techniques designed for isolation of proteins and organelles
including
 Differential and density gradient centrifugation
 Precipitation of viruses (e.g., ammonium sulfate, polyethylene glycol)
 Denaturation of contaminants (e.g., chloroform)
 Enzymatic digestion of cellular constituents
Viral Assays
How do you quantify the number of virions in a suspension?
i) Direct count with an electron microscope (virus containing sample is mixed with known
concentration of small latex beads and sprayed on a coated specimen grid) – not easy
ii) Indirect count
a) Hemagglutination assay – many viruses can bind to the surface of red blood cells – if the
ratio of virions to RBC is large enough then they agglutinate. The hemagglutination titre
is the highest dilution of virus that still causes hemagglutination
b) Quantify number of infectious units by measuring effects on hosts
 A virus infectious unit is the smallest unit that causes a detectable effect when added to a
susceptible host – as few as one virion
 The number of infectious units per volume is referred to as the titre
 Plaque assay – when a virus initiates infection on a layer or lawn of host cells growing out
on a flat surface, a zone of lysis or zone of growth inhibition may occur that results in a
clear area (plaque) in the lawn of growing cells. It is assumed that each plaque originated
from replication events that began with one virion. Viral titres are expressed as plaqueforming units (PFU).
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Biol 3400
Tortora - Chap 13
 Plating efficiency - counts made by plaque assays are always lower than counts made with
electron microscope. Plating efficiency with bacteriophage is usually > 50% but with
some animal viruses may be <1%
 The plaque procedure may be used to prepare pure viral strains
 Cell cultures may also be used to titre virus – infection with viruses leads to cellular
deterioration - known as the cytopathic effect
iii) Whole animal methods – some viruses do not cause recognizable effects in cell culture and
yet kill animals.
 Quantification in these systems relies on preparing serial dilutions of unknown samples
and injecting these into several sensitive animals.
 After a suitable incubation period, the number of live and dead animals at each dilution is
determined and an end point dilution is calculated. This may be the dilution that kills 50%
of the animals – Lethal Dose killing 50% of the animals = LD50
III.
Viral Replication
A.
Requirements
1. Host cell (a cell within which a virus replicates) must be permissive and virus compatible
with host cell
Host range
 Permissive or compatible host cells permit viral replication
 Nonpermissive host cells do not permit viral replication
 Host range may be broad (a number of species) or narrow (one cell type of a single
species)
 e.g., bacteria, archaea, fungi, protozoa, plants and animals are infected by viruses
2. Host cell does not degrade virus
3. Viral genome contains necessary information for modifying host metabolism
4. Virus must be able to use the host metabolic activities to produce new virus particles
 Viral genome achieves control of cell’s metabolic activities – viral protein synthesis
dominates over host cell protein synthesis
How? - Host transcription is inhibited or
Viral mRNA’s are translated more efficiently than host cell proteins

Virus uses host’s ribosomes, ATP and reduced coenzymes for viral progeny (virion)
production
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