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Foundations of Virology, Classification

8/31/04

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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)

• 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

Semmelweis 1840s Vienna. Childbirth mortality dropped from 29% to 1% on introduction of hand washing and chlorine disinfection

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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

• 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

Tobacco mosaic 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

• 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

T-even phage

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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)

• 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

Poliovirus

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) parasitesobligate (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 ()

• 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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From Principles of Virology Flint et al ASM Press

The size of viruses

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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

20From Principles of Virology Flint et al ASM Press

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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

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DNA viruses

From Principles of Virology Flint et al ASM Press

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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

Informally:

Type e.g. herpes simplex virus 1

Strain e.g. influenza A/PR/8/34 SC16

Virus taxonomy

In biology, binomial names are used. e.g Rattus rattus, Saccharomyces cerevisiaeIn virology, this does not happen:

Tobacco etch potyvirus sounds OKInfluenza A influenzavirus A does not!

Bacteriophage have their own rules

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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 (taxonomically-

speaking)– 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 causepoliovirus, rabies virus

- the type of diseasemurine leukemia virus

- geographic locationsSendai virus, Coxsackie virus

- their discoversEpstein-Barr virus

- how they were originally thought to be contracted

dengue virus (“evil spirit”), influenza virus (the “influence” of bad air)

- combinations of the aboveRous 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.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.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.Making viral genomes. Many viral genomes are copied by the cell’s synthetic machinery in cooperation with viral proteins.

• Forming progeny virions.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.Spread within and between hosts. To ensure survival the virus must propagate itself in new cells.

• Overcoming host defences.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 How to reproduce during its “visit” inside the cell. How to a) copy its genetic information and b) produce mRNA for protein production

• 2 How to spread from one individual to another

• 3 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 virusesanimal plant phage

• 1      Retrovirus (HIV)• 2      Orthomyxovirus (influenza)• 3      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 Poxvirus• 12 Hepadnavirus (hepatitis B)• 13 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

• Chapter 3 of Flint 2nd ed, and appendices

On Thursday class is in LH1