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Foundations of Virology,
Classification
8/31/04
1
Viruses in history - I
• Virology as a science is approx. 100 years old - but virus diseases have
been known for millennia
• In Ancient Greece ios = a poisonous substance
• In Latin virus = a poisonous malodorous substance
• Mesopotamian laws concerning rabid dogs date from before 1,000
B.C.E
• Smallpox was endemic in the Ganges river basin by the 5th century
B.C.E
• Hippocrates first attempted to rationalize plagues - concluded they
were caused by small animals in the air too small for human vision
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Viruses in history II
• In 1494 Frascatero advanced the theory that disease was caused by
seminaria, which spontaneously arose from dead material
• Unknowingly, viruses were well characterized by the 16th century due
to striping patterns on tulips
– A case of economic advantage (a “broken’ tulip was worth 3x more than a
Rembrandt masterpiece)
• The birth of microbiology occurred by the invention of the
microscope - notably van Leuwenhoek’ “wee animalcules” first seen
in the 17th century. But how did these microorganisms arise?
• Jacob Henle (1840) was the first to have the idea of a microorganism
too small to be seen by a microscope
• Spontaneous generation of life was finally refuted by Louis Pasteur in
the mid 19th century - disease was not caused by poisonous air
(miasma) by by specific microorganisms. Pasteur’s famous successes
were anthrax (a bacterium) and rabies (a virus)
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Koch’s postulates
• In 1890 Robert Koch explained the following “
•
“The parasite can be encountered in all cases under those conditions
which correspond to the pathological changes and the clinical course of
the disease” - the microbe is always there when there is disease
•
“The pathogen may not occur incidentally as a non-pathogenic parasite
in any other disease” the microbe is never anywhere else - it is specific
•
“The parasite must be isolated and bred in adequate numbers in pure
culture and must be able…. the microbe can be cultured
• To cause the disease anew.” the culture must cause disease in a new host
»-
for bacterial disease
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The germ theory
• Pasteur, Koch and Joseph Lister were the founded of the
germ theory of disease in the 19th century - but at this
point all identified pathogens were bacteria (and fungi)
Semmelweis 1840s Vienna. Childbirth mortality dropped
from 29% to 1% on introduction of hand washing and
chlorine disinfection
• The germ theory placed a study of infectious disease on
secure scientific footing
• However a failure of the existing paradigms led to the
identification of submicroscopic pathogens --- viruses
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The birth of Virology
• Adolf Mayer (1876) took the sap from infected tobacco plants and
transmitted it to healthy plants - note isolation of the germ was not
achieved and Mayer considered his experiment unsuccessful
– A case of (great) economic disadvantage
– Failed Koch’s postulates
Tobacco mosaic
virus
• Dimitri Ivanovski (1892) first noted that the infection was not retained
by a filter (Chamberland filter) - again Ivanovski thought he was
unsuccessful and blamed a cracked filter on his “failure”
• Martinus Beijerinck (1897) achieved the same result, but was less able
to accept defeat and concluded that his “Contagium vividum fluidium” in
order to reproduced itself, must be incorporated into the living protoplasm of
the cell, into whose reproduction it is, so to speak, passively drawn
• Friedrich Loeffler and Paul Frosch (1897) observed that Foot and
Mouth Disease was also filterable - the first animal virus
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Koch’s postulates as modified for
viruses by Rivers (1937)
•
•
•
•
Isolation of the virus from diseased host
Cultivation in host cells
Proof of filterability
Production of a comparable disease in the original
animal host, or a related one
• Re-isolation of the virus
• Detection of a specific immune response to the
virus
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What is a virus? I
• Serial transmission of TMV and FMDV by diluted extracts proved that
the virus was not a toxin.
• Failure of the agents to propagate in solution, and dependence of host
cells, showed they were distinct from bacteria.
• Were they liquids or particles? Biological or chemical?
• Early work on showed that TMV behaved like a protein
(electrophoresis and raising of antibodies) and
• Eventually TMV was crystallized in 1935 by Wendell Stanley -- rods
of constant diameter in hexagonal arrays - viruses can be analyzed
according the the laws of chemistry, as well as biology
• and was seen by electron microscopy
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Bacteriophages
• Bacterial viruses first identified in 1915 by Frederick
Twort and in 1917 by Felix d’Herrelle - given the name
bacteriophage (phage = greek for eating)
• The modern era began with Max Delbrück, (a physicist)
who promoted the genetic nature of phage T-even phage
• In 1939 Ellis and Delbrück designed the one-step growth
curve and defined the latent period of infection
• In 1941 they were joined by Salvador Luria (a geneticist)
to form the Cold Spring Harbor phage group (WWII-1975)
- pioneers of modern molecular biology
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2 classic experiments
• Hershey and Chase - T2 bacteriophage - 1952 the Waring blender experiment
• Fraenkel-Conrat and Singer - Tobacco Mosaic
Virus (TMV) - 1957
– These experiments (along with many others) laid
the foundation of our understanding of nucleic acid
as the genetic material of life
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Hershey/Chase experiment
T2 phage grown in E. coli and labeled with either :
- 35S
(as sulphate) - to label protein or
- 32P (as
phosphorous) - to label DNA
Phage were allowed to attach and infect, and then
put into the Waring blender
The shear force of the blender stripped away the
phage components attached to the surface, but did
not affect components that had penetrated the E.
coli
When E. coli was centrifuged, 75% of the 35S was
removed from the cells, whereas only 15% of the
32P was removed
i.e the DNA (and not the protein) is carried into
the cell and it the carrier of viral heredity
From Introduction to Modern Virology, 5th ed
Dimmock et al. Blackwell
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Fraenkel-Conrat/Singer experiment
Already known that TMV particles can be dissociated, and
reassembled into infectious particles
Also known that TMV (as with other viruses) can exist as
different strains (i.e different symptoms in the host)
Different strains were dissociated and protein and RNA
isolated. The RNA of one strain was then reassociated
with the protein of another ( and vice versa)
The “hybrid’ particles were then inoculated into plants, and
the disease outcome matched the RNA and not the protein
i.e RNA (and not protein) is the genetic material
Later proven by the finding that purified RNA is capable
of initiating infection (under special circumstances)
From Introduction to Modern Virology, 5th ed
Dimmock et al. Blackwell
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The development of animal virology
• The first human virus was identified in 1901 (yellow fever) by Walter
Reed and colleagues
• But a study of animal and human viruses was very slow due to the lack
of an experimental system - need for single cells
• Use of embryonated eggs by the 1930s were of great value
• In the period 1948-55 animal virology finally became a laboratory
science by the development of cell or tissue culture by Renato
Dulbecco - plaque assay (1953)
Poliovirus
• Other notable highlights of animal virology in the late 20th century
include:
– the breakdown of the central dogma of molecular biology by the
finding of reverse transcriptase in retroviruses (Howard Temin
and David Baltimore)
– the discovery of oncogenes, fundamental knowledge of gene regulation,
transcription/translation, restriction mapping (Nathans expt.), DNA
cloning
SV40
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What is a virus ? II
• The fundamental characteristic is their
absolute dependence on a living host
organism for reproduction - they are
obligate (intracellular) parasites
• They are small -- usually in the nanometer
range - hence they are filterable and visible
only by the electron microscope
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Diphtheria
• Corynebacterium diphtheriae (a bacteria) was
originally identified as the causative agent of
diphtheria, according to Koch’s postulates
• Now known that disease per se caused by a
bacterial toxin
• However, all virulent strains are lysogenic with a
phage (b)
• The lysogenic phage is responsible for toxin
production
• i.e the virus causes the disease
• A fundamental breakdown of Koch’s postulates
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The size of viruses
From Principles of Virology Flint et al ASM Press
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What is a virus ? III
• A virus is a very small, infectious, obligate intracellular (molecular)
parasite
• The virus genome comprises either DNA or RNA
• Within an appropriate host cell the viral genome is replicated and
directs the synthesis, by cellular systems, of other viral components
• Progeny virions are formed by de novo assembly from newly
synthesized components within the host cell
• A progeny virion assembled during the infectious cycle is the vehicle
for transmission of the viral genome to the next host cell or organism,
where its disassembly leads to the beginning of the next infectious
cycle
But viruses don’t actually “do” anything
(see Box 1.5 in Flint)
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What is a virus ? IV
• A virus is an elementary biosystem that possesses some of
the properties of living systems such a s having a genome
and being able to adapt to changing environments
• However, viruses cannot capture or store free energy and
they are not functionally active outside their host cells.
• A virus has both intrinsic properties (e.g. its size) and
extrinsic properties (e.g. its host)
• Viruses are not living organisms; however they can be
considered to lead a “borrowed” life
It is important to discriminate between the
entity called a virus and the single, discrete
virus particle or virion
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Virus Classification I
- the Baltimore classification
• All viruses must produce mRNA, or (+) sense RNA
• A complementary strand of nucleic acid is (–) sense
• The Baltimore classification has + RNA as its central point
• Its principles are fundamental to an understanding of virus
classification and genome replication, but it is rarely used
as a classification system in its own right
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From Principles of Virology Flint et al ASM Press20
Virus classification II the Classical system
• This is a based on three principles -
– 1) that we are classifying the virus itself,
not the host
– 2) the nucleic acid genome
– 3) the shared physical properties of the infectious agent
(e.g capsid symmetry, dimensions, lipid envelope)
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Virus classification III the genomic system
• More recently a precise ordering of viruses
within and between families is possible
based on DNA/RNA sequence
• By the year 2000 there were over 4000
viruses of plants, animals and bacteria - in
71 families, 9 subfamilies and 164 genera
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RNA viruses
From Principles of Virology Flint et al ASM Press
23
DNA viruses
From Principles of
Virology Flint et al
ASM Press
24
Virus taxonomy
Order
virales
e.g Mononegavirales
Family
viridae
e.g. Orthomyxoviridae
Herpesviridae
Subfamily
virinae
e.g.
Alphaherpesvirinae
Genus
e.g. influenzavirusA
Simplexvirus
Species
e.g. influenza A virus
human herpesvirus1
Type
e.g.
herpes simplex virus 1
Strain
e.g. influenza A/PR/8/34
SC16
Informally:
In biology, binomial names are used. e.g Rattus rattus, Saccharomyces cerevisiae
In virology, this does not happen:
Tobacco etch potyvirus sounds OK
Bacteriophage have their
own rules
Influenza A influenzavirus A does not!
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The species concept in virus taxonomy
• How different is different enough to be something else?
• “Species” is the universally accepted term for the lowest
taxonomic clustering of living organisms
• Taxonomy now ratified by the International Committee on
Taxomony of Viruses (ICTV)
• Plant viruses are especially problematic (taxonomicallyspeaking)
– The Potyviridae - filamentous particles, 650-900 nm, +ve sense
RNA, polyprotein
– 6 genera with initially very confusing biological properties, can now
be classified based on sequence
– The animal Picornaviridae can be equally challenging
RNA viruses especially are not a single molecular species, but must be
viewed as a dynamic population consisting of thousands of viral
mutants that are always present in a viral clone
This population is often referred to as a viral quasi-species
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What is a virus species?
• “a polythetic class of viruses that constitute
a replicating lineage and occupy a particular
ecological niche” - as defined by ICTV in
1991
A polythetic class is defined as a class whose
members always have several properties in
common, although no single attribute is present in
all of its members
-- allows for some degree of “fuzziness”
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Classification based on serology
• A classification based on Diagnostic
Virology
– Infectious bronchitis virus (IBV) of chickens
- a coronavirus
• Three predominant virus “types” in US
• Massachusetts, Arkansas and Delaware
• No cross-protection (from antibodies) between these
serotypes
– i.e. significant antigenic differences, but perhaps very little
genetic or biological difference between these viruses
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How are viruses named?
• Based on:
- the disease they cause
poliovirus, rabies virus
- the type of disease
murine leukemia virus
- geographic locations
Sendai virus, Coxsackie virus
- their discovers
Epstein-Barr virus
- how they were originally thought to be contracted
dengue virus (“evil spirit”), influenza virus (the “influence” of bad air)
- combinations of the above
Rous Sarcoma virus
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Sub-viral agents
• Satellites
–
–
–
–
–
Contain nucleic acid
Depend on co-infection with a helper virus
May be encapsidated (satellite virus)
Mostly in plants, can be human e.g. hepatitis delta virus
If nucleic acid only = virusoid
• Viroids
– Unencapsidated, small circular ssRNA molecules that replicate
autonomously
– Only in plants, e.g. potato spindle tuber viroid
– Depend on host cell polII for replication, no protein or mRNA
• Prions
– No nucleic acid
– Infectious protein e.g. BSE
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Unifying principles
• All viruses package their genomes inside a particle that
mediates transmission of the viral genome from host to host
• The viral genome contains the information for initiating and
completing an infectious cycle within a susceptible,
permissive cell. An infectious cycle includes attachment,
and entry of the particle, decoding of genome information,
translation of viral mRNA by host ribosomes, genome
replication, and assembly and release of particles containing
the genome
• All viruses are able to establish themselves in a host
population so that virus survival is ensured
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Strategies for virus survival
•
Finding and getting into a host cell. As viruses are obligate parasites they must
find the right type of cell for their replication, they must invade that cell and get their
genome to the site of replication.
•
Making virus protein. All viruses are parasites of translation. The virus must make
mRNA (unless it has a + sense RNA genome already). Strategies must exist to
synthesize mRNA.
•
Making viral genomes. Many viral genomes are copied by the cell’s synthetic
machinery in cooperation with viral proteins.
•
Forming progeny virions. The virus genome, capsid (and envelope) proteins must
be transported through the cell to the assembly site, and the correct information for
assembly must be pre-programmed.
•
Spread within and between hosts. To ensure survival the virus must propagate
itself in new cells.
•
Overcoming host defences.The host defends itself against “nonself”. Viruses have
evolved ways to fight back.
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Three problems every virus must solve
• 1
• 2
• 3
How to reproduce during its “visit”
inside the cell. How to a) copy its genetic
information and b) produce mRNA for protein
production
How to spread from one individual to
another
How to evade the host defenses. This
need not be complete.
• Viral diseases are the (usually unintended)
consequences of the way each virus has
chosen to solve these three problems.
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Our top 13 viruses
animal plant phage
• 1
• 2
• 3
•
•
•
•
•
•
•
Retrovirus (HIV)
Orthomyxovirus (influenza)
Picornavirus superfamily
(poliovirus, potyvirus)
4 Adenovirus
5
Herpesvirus (HSV1)
6 Tobacco mosaic virus
7 T-even phage
8
Polyomavirus (SV40)
9
Rhabdovirus (VSV)
10 Reovirus (Rotavirus)
• 11
• 12
• 13
Poxvirus
Hepadnavirus (hepatitis B)
Alphavirus (Semliki Forest, Sindbis)
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Reading assignments
• Chapter 1 of Flint
• Chapter 1 of Fields Virology “The Origins of
Virology” (for history)
•
“The Greatest Benefit to Mankind” Porter, Norton&Co
– An excellent history of medicine
• Lysogeny
• For Thursday – Chapter 2 of Flint
On
Thursday
class is in
LH1
• Chapter 3 of Flint 2nd ed, and appendices
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