ADVANCED VIROLOGY

Course Contents
Chapter one general virology
• Historical Perspective
• General morphology and structure
• Evolution of viruses
• Detailed structure and assembly of animal viruses,
defective-interfering viruses
• Viral Replication
• Classification and Nomenclature of Viruses
• Epidemiology of Viral Diseases
– Virus Transmission
– Mechanisms of Survival of Viruses in Nature
• Incidence of viral diseases related to seasons and animal
management practices
 Chapter two
Systematic virology
DNA viruses
• Adenoviridae

• Herpesviridae

• Papillomaviridae

• Parvoviridae

• Poxviridae

• Asfarviridae
 Chapter three (RNA viruses)
• Arenaviridae

• Birnaviridae

• Bunyaviridae

• Bornaviridae

• Caliciviridae

• Coronaviridae

• Flaviviridae
 RNA Viruses..

• Orthomyxoviridae

• Paramyxoviridae

• Picornaviridae

• Retroviridae

• Rhabdoviridae

• Reoviridae

• Togaviridae
 Practical

• Use of embryonated eggs and cell culture

• Electron Microscopy
 Chapter four
Prions
• Prion protein
• Cellular protein
• Prion Structure
• Prion Replication
• Prions diseases
 Assessment method

• Review assignments- 20%

• Mid exam- 30%
• Final exam- 50%
 Virus

Latin for ‘slimy liquid’ or poison

 Definitions
• Virus particle or virion
– Infectious agent composed of nucleic acid (RNA or DNA), a
protein shell (capsid) and, in some cases, a lipid envelope

• Capsid
– Protein coat that surrounds the viral nucleic acid

– Composed of repeating subunits called capsomeres

– Have either icosahedral or helical symmetry

• Nucleocapsid
– Complete protein-nucleic acid complex
 Definitions..
• Satellite or defective viruses
– Viruses which require a second (helper) virus for replication
• Example: hepatitis delta virus requires hepatitis B
• Viroids
– Small, autonomously replicating molecules
– Single stranded circular RNA, 240-375 residues in length
– Plant pathogens
• Prions
– Not viruses
– Infectious protein molecules responsible for transmissible and
familial spongiform encephalopathies
• e.g., Creutzfeldt-Jakob disease, bovine spongiform encephalopathy
(vCJD in humans)
– Pathogenic prion protein PrPSc formed from normal human
protein, PrPC, through post-translational processing
 Chapter one
General virology
1. Historical perspectives of viruses

• Smallpox was endemic in China by 1000 BC and practice

of variolation (similar to Vaccination) was developed.

• Survivors of smallpox outbreaks were protected from

subsequent infection, the Chinese inhaled the dried crusts
from smallpox lesions like snuff or, in later modifications,
inoculated the pus from a lesion into a scratch on the fore
arm.
 History..
• Introduction of concept of ‘filterable agents’ for plant
pathogens (Mayer, Ivanofsky, Beijerinck in late 1880’s)
• First filterable agent from animals described – foot and
mouth disease virus (Loeffler and Frosch in 1898)
• First human filterable agent described - yellow fever
virus (Reed in 1901)
• Linkage of viruses with cancer (Ellerman, Bang 1908;
Rous 1911)
 History..
• Description of bacteriophages (Twort and D’Herelle in
1915)

• Visualization of viruses by EM and x-ray

crystallography (1939, 1941)

• Development of tissue culture systems (Sanford, Enders,

Gay, Eagle 1948-1955); growth of poliovirus in culture

• Discovery of many agents; explosion in molecular

biology (past 45-50 years)
 History..
New Virus infecting Bacteria:

- Twort (1915) and Felix (1917) were the first to recognize viruses

that infect bacteria, later called bacteriophages (‘eaters of

bacteria’).

- In the 1930s and subsequent decades, pioneering virologists Luria,

Max Delbruck, and others used these viruses as model systems to

investigate many aspects of virology

- These relatively simple agents have since proven to be very

important to our understanding of all types of viruses, including

 CELL CULTURE METHODS

• Began early in the twentieth century with whole-organ cultures,

then progressed to methods involving individual cells

• In 1949, John Enders and his colleagues were able to propagate

poliovirus in primary human cell cultures.

• This achievement regarded as the ‘Golden Age of Virology’ and led

to the identification and isolation during the1950s and 1960s of

many viruses and their association with human diseases.

 Plaque Assay

• Renato Dulbecco in 1952 first to quantify accurately animal viruses using a plaque assay.

• In this technique, dilutions of the virus are used to infect a cultured cell monolayer,

Counting the number of plaques directly determines the number of infectious virus particles

applied to the plate.

• The same Plaque technique can also be used biologically to clone a virus (i.e., isolate a pure

form from a mixture of types).

• This technique had been in use for some time to quantify the number of infectious virus

particles in bacteriophage suspensions applied to confluent ‘lawns’ of bacterial cells on

agar plates, but its application to viruses of eukaryotes enabled rapid advances in the study

of virus replication to be made.

• Plaque assays largely replaced earlier endpoint dilution techniques, such as the tissue

culture infectious dose (TCID50) assay.

• Monoclonal Antibodies (MA) Technology
In 1975, George Kohler and C. Milstein isolated the first MA from
clones of cells selected in vitro to produce an antibody of a single
specificity directed against a particular antigenic target.
• This enabled virologists to look not only at the whole virus, but at
specific regions—epitopes—of individual virus antigens as well
function of individual virus proteins.
• MA produced by immunization of an animal with an antigen that
usually contains a complex mixture of epitopes.
• Recently, in vitro molecular techniques have been developed to
speed up the selection of monoclonal antibodies
 Ultrastructural & Physical Testing

- Physical measurements of virus particles began in 1930s with the earliest determinations

of their proportions by filtration through colloidal membranes of various pore sizes.

• The first electron micrograph of virus (TMV) published in 1939.

• Over subsequent years, techniques were developed that allowed the direct examination of

viruses at magnifications of over 100,000 times.

• The two fundamental types of electron microscope are the transmission electron

microscope (TEM) and the scanning electron microscope (SEM) - Studies of the

sedimentation properties of viruses in ultracentrifuges in the 1960s in obtaining purified

and highly concentrated preparations of many different viruses, free of contamination

from host cell components that can be subjected to chemical analysis.

• The relative density of particles, measured in solutions of sucrose or CsClvirus

 Molecular Biology Technology

• The term ‘molecular biology’ has taken on the new and different meaning of ‘genetic

engineering’ or ‘genetic manipulation.’

• These techniques for manipulating nucleic acids in vitro (outside living cells or organisms)

• This powerful new technology has revolutionized virology and has shifted the focus of

attention away from the virus particle onto the virus genome.

• Initially, any investigation of a virus genome will usually include questions about the

following:

- Composition—DNA or RNA, single-stranded or double-stranded, linear or circular

- Size and number of segments

- Terminal structures

- Nucleotide sequence
 Basic characteristics of Viruses
 Although viruses are very heterogeneous, there is a unity of
structure, basically protein and nucleic acid (RNA or DNA).
 They are reproduced by replication.

 Size: viruses are "filterable" agents.

 Obligate (genetic) parasites- dependent on host cell genetic

material.
 Virus genome is either DNA or RNA not both.
 Characteristics..
• Virion

– complete virus particle

– consists of 1 molecule of DNA or RNA enclosed in coat of

protein

– may have additional layers

– cannot reproduce independent of living cells nor carry out

cell division but can exist extracellularly

 Virions infect all cell types

• Bacterial viruses called bacteriophages

(phages)
• Few archaeal viruses
• Most are eukaryotic viruses
– plants, animals, protists, and fungi
• Classified into families based on
– genome structure, life cycle, morphology,
genetic relatedness
 Bacteriophages
• lso known informally as a phage

• is a virus infects and replicates within bacteria and archaea.

• Their genomes may encode as few as four genes (e.g. MS2) and as

many as hundreds of genes.

• Phages replicate within the bacterium following the injection of their

genome into its cytoplasm.

• Bacteriophages are ubiquitous viruses, found wherever bacteria exist.

• It is estimated more than 1031 bacteriophages on the planet, more than

every other organism on Earth, including bacteria, combined.

 1.2. General morphology and Structure
• Virion size range is ~10–400 nm in diameter and most
viruses must be viewed with an electron microscope
• All virions 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
– Envelopes
 Morphology..
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
• Capsids are helical, icosahedral, or complex
• procapsids
Morphology..
Morphology..
Morphology..
 Capsid..

• Functions
– Protect the genome
– Deliver the genome
– Interact with the host
Viral envelopes..
Morphology..
 Viral Envelope Proteins

• are viral encoded, may project from the envelope surface

as spikes or peplomers

– involved in viral attachment to host cell

• e.g., hemagglutinin of influenza virus

– used for identification of virus

– may have enzymatic or other activity

• e.g., neuraminidase of influenza virus

– may play a role in nucleic acid replication

 Virion Enzymes
• It was first erroneously thought that all virions
lacked enzymes
• Now accepted that a variety of virions have
enzymes
• some are associated with the envelope or
capsid but most are within the capsid
 Viral enzymes..
Integrase
• IN is a 288–amino-acid, 32-kDa viral enzyme that mediates the
linkage of double-stranded viral DNA into the host cell genome.
• Integration occurs after the translocation of a large complex
derived from the viral core from the cytoplasm into the nucleus.
• IN is part of this complex and catalyzes the cleavage of viral DNA
and ligation to host cell DNA.
• A large central acidic domain of IN is highly conserved
in retroviruses and retrotransposons.
• Once integrated, the provirus can be considered for most purposes
to be a stable genetic element remaining for the life of the cell
and, through cellular replicatio1n, for the life of the individual.
• Integrase is required for viral replication and integrase inhibitors,
such as raltegravir, are one of the most recent classes of
antiretroviral drugs entering clinical use
 Viral Genome
• Diverse nature of genomes
• A virus may have single or double stranded
DNA or RNA
• The length of the nucleic acid also varies
from virus to virus
• Genomes can be segmented or circular
 1.4. Evolution viruses
Three classical hypotheses

• Viruses are ancient. Studies at the molecular level revealed r/ships b/n

viruses infecting organisms from each of the three domains of life,

suggesting viral proteins that pre-date the divergence of life and thus

infecting the last universal common ancestor.

• This indicates that some viruses emerged early in the evolution of

life, and that they have probably arisen multiple times.

• It has been suggested new groups of viruses have repeatedly emerged

at all stages of evolution, often through the displacement of ancestral

structural and genome replication genes.

 Evolution..
• Virus-first hypothesis: Viruses evolved from complex molecules of protein and nucleic acid before cells first

appeared on earth.

• By this hypothesis, viruses contributed to the rise of cellular life.

• This is supported by the idea that all viral genomes encode proteins that do not have cellular homologs. The

virus-first hypothesis has been dismissed by some scientists because it violates the definition of viruses, in

that they require a host cell to replicate.

• Reduction hypothesis (degeneracy hypothesis): Viruses were once small cells that parasitized larger cells.

• This is supported by the discovery of giant viruses with similar genetic material to parasitic bacteria.

However, the hypothesis does not explain why even the smallest of cellular parasites do not resemble viruses

in any way.

• Escape hypothesis (vagrancy hypothesis): Some viruses evolved from bits of DNA or RNA that "escaped"

from the genes of larger organisms.

• This doesn't explain the structures that are unique to viruses and are not seen anywhere in cells. It also does

not explain the complex capsids and other structures of virus particles.

• Virologists are in the process of re-evaluating these hypotheses .

 Evolution..
• Later hypotheses

• Coevolution hypothesis (Bubble Theory): At the beginning of life, a community of early replicons

(pieces of genetic information capable of self-replication) existed in proximity to a food source

such as a hot spring or hydrothermal vent.

• Therefore, evolutionary pressure could push replicons along two paths of development: merging

with a vesicle, giving rise to cells; and entering the vesicle, using its resources, multiplying and

leaving for another vesicle, giving rise to viruses.

• Chimeric-origins hypothesis: Based on the analyses of the evolution of the replicative and

structural modules of viruses, a chimeric scenario for the origin of viruses was proposed in 2019.

• According to this hypothesis, the replication modules of viruses originated from the primordial

genetic pool, although the long course of their subsequent evolution involved many

displacements by replicative genes from their cellular hosts.

• By contrast, the genes encoding major structural proteins evolved from functionally diverse host

proteins throughout the evolution of the virosphere. This scenario is distinct from each of the

three traditional scenarios but combines features of the Virus-first and Escape hypotheses.
 Evolution..
• Viruses have been able to continue their infectious existence due to evolution.
• Their rapid mutation rates and natural selection has given viruses the advantage to
continue to spread. One way that viruses have been able to spread is with the evolution
of virus transmission. The virus can find a new host through:
• Droplet transmission- passed on through body fluids (sneezing on someone)
– An example is the influenza virus
• Airborne transmission- passed on through the air (brought in by breathing)
– An example would be how viral meningitis is passed on
• Vector transmission- picked up by a carrier and brought to a new host
– An example is viral encephalitis
• Waterborne transmission- leaving a host, infecting the water, and being consumed in a
new host
– Poliovirus is an example for this
• Sit-and-wait-transmission- the virus is living outside a host for long periods of time
– The smallpox virus is also an example for this
• There are some ideas behind the idea that virulence, or the harm that the virus does on
its host, depends on a few factors. These factors also have an effect on how the level of
virulence will change over time.
• Viruses that transmit through vertical transmission will evolve to have lower levels of
virulence. Viruses that transmit through horizontal transmission will usually evolve to
have a higher virulence.
 Evolution..
• Generally Viral genomes are considered as one
of the most rapidly evolving organisms in
biology due to
– short replication time and
– a large amount of the offspring’s released from the
infected host cell.
 1.5. Viral replication
General Features of Virus Replication

• Virologists describe the formation of viruses during the infection process in

target host cells as viral replication.

• Viral populations do not grow through cell division, b/s they are acellular.

Instead, they use the machinery and metabolism of a host cell to produce

multiple copies of themselves, and they assemble in the cell.

• The life cycle of viruses differs b/n species but there are six basic stages in

the life cycle of viruses: attachment, penetration (viral entry), uncoating,

replication, and lysis.

• Some viruses undergo a lysogenic cycle where the viral genome is

incorporated by genetic recombination into a specific place in the host’s

Replication..
 Replication..
Attachment (Adsorption)
• Specific receptor attachment
• Receptor determines host preference
– may be specific tissue (tropism)
– may be more than one host
– may be more than one receptor
– may be in lipid rafts providing entry of virus
 Replication..
• Viral Entry and Uncoating

• Entire genome or nucleocapsid

• Varies between naked or enveloped virus
• Three methods used
– fusion of the viral envelope with host
membrane; nucleocapsid enters
– endocytosis in vesicle; endosome aids in viral
uncoating
– injection of nucleic acid
Replication..
 Replication..
Synthesis Stage
• Genome dictates the events
• ds DNA typical flow
• RNA viruses
– virus must carry in or synthesize the proteins
necessary to complete synthesis
• Stages may occur, e.g., early and late
Replication..
Replication..
Replication..
 Viral DNA replication

• Viral DNA genomes have limited coding capacity and therefore

harness cellular factors to facilitate replication of their genomes
and generate progeny virions.

• The replicative life cycles of many DNA viruses have been shown
to engage components of the host DNA damage and repair
machinery.

• Viruses have evolved numerous strategies to navigate the cellular

DNA damage response.

• By hijacking and manipulating cellular replication and repair

processes,
 Viral Replication Centers
• Cellular DNA replication takes place at discrete nuclear foci
where replication proteins localize.
• In contrast, viral DNA replication occurs within distinct virus-
induced structures that have been referred to as viral replication
compartments or centers (VRCs) (93–96).
• Both viral and cellular proteins concentrate at these VRCs and
aid viral gene transcription and DNA replication.
• Among the cellular factors recruited to promote virus infection
are proteins involved in cellular DNA replication and repair.
• In contrast, some potentially detrimental host proteins are
actively sequestered away from sites of viral DNA synthesis.
• The challenge of defining host proteins that are harnessed or
inactivated by DNA viruses has been facilitated by proteomic
approaches that identify proteins associated with replicating
viral genomes.
 Viral DNA Replication..
(+/-) double-stranded DNA
• To replicate the viral genome, DNA-dependent DNA
polymerase enzymes copy both the (+) and (-) DNA strands
producing dsDNA viral genomes.
• To produce viral mRNA molecules, DNA-dependent RNA
polymerase enzymes copy the (-) DNA strand into (+) viral
mRNA.
• The (+) viral mRNA then translated into viral proteins by host
cell ribosomes. Examples include most bacteriophages,
Papovaviruses, Adenoviruses, and Herpesviruses.
Viral DNA Replication..
 Viral DNA Replication..

single-stranded DNA

• To replicate the viral genome, DNA-dependent DNA polymerase

enzymes copy the (+) DNA strand of the genome producing a

dsDNA intermediate.

• DNA-dependent DNA polymerase enzymes then copy the (-) DNA

strand into ss (+) DNA genomes.

• To produce viral mRNA molecules, DNA-dependent RNA

polymerase enzymes copy the (-) DNA strand into (+) viral mRNA.

• The (+) viral mRNA can then be translated into viral proteins by
Viral DNA Replication..
 Replication of double stranded RNA viruses

(+/-) double-stranded RNA

• To replicate the viral genome, RNA-dependent RNA polymerase

enzymes copy both the (+) RNA and (-) RNA strands of the
genome producing a dsRNA genomes.

• To produce viral mRNA molecules, RNA-dependent RNA

polymerase enzymes copy the (-) RNA strand into (+) viral
mRNA.

• The (+) viral mRNA can then be translated into viral proteins by
host cell ribosomes.

• Reoviruses are an example.

Replication of double stranded RNA Viruses..
 Replication of single stranded RNA Viruses..
(-) RNA

• To replicate the viral genome, RNA-dependent RNA polymerase

enzymes copy the (-) RNA genome producing ss (+) RNA.

• RNA-dependent RNA polymerase enzymes then copy the (+)

RNA strands producing ss (-) RNA viral genome.

• The (+) mRNA strands also function as viral mRNA and can then

be translated into viral proteins by host cell ribosomes.

• Examples include Orthomyxoviruses, Paramyxoviruses,

Rhabdoviruses.
 Viral RNA riplication..

(+) RNA Retroviruses

• To replicate the viral genome, reverse transcriptase enzymes (RNA-dependent DNA

polymerases) copy the (+) RNA genome producing ss (-) DNA strands.

• DNA-dependent DNA polymerase enzymes then copy the (-) DNA strands to produce a

dsDNA intermediate.

• DNA-dependent RNA polymerase enzymes then copy the (-) DNA strands to produce

ss (+) RNA genomes.

• To produce viral mRNA molecules, DNA-dependent RNA polymerase copy the (-)

DNA strand into (+) viral mRNA.

• The (+) viral mRNA can then be translated into viral proteins by host cell ribosomes.

• Retroviruses, such as HIV-1, HIV-2, and HTLV-1 are examples.

Viral RNA riplication..
 Types of Viral Infections
• Infections in Bacteria and Archaea
• Infections in eukaryotic cells
• Viruses and cancer
 Bacterial and Archaeal Viral
Infections
• Virulent phage – one reproductive choice

– multiplies immediately upon entry

– lyses bacterial host cell

• Temperate phages have two reproductive options

– reproduce lytically as virulent phages do

– remain within host cell without destroying it

• many temperate phages integrate their genome into

host genome (becoming a ‘prophage’ in a ‘lysogenic

bacterium’) in a relationship called lysogeny

 Lysogenic Conversion
• Temperate phage changes phenotype of its host
– bacteria become immune to superinfection
– phage may express pathogenic toxin or enzyme
• Two advantages to lysogeny for virus
– phage remains viable but may not replicate
– multiplicity of infection ensures survival of host cell
• Under appropriate conditions infected bacteria will
lyse and release phage particles
Bacterial/phage infections
 Archaeal Viruses
• May be lytic or temperate
• Most discovered so far are temperate by
unknown mechanisms
 Infection in Eukaryotic Cells
• Cytocidal infection results in cell death
through lysis
• Persistent infections may last years
• Cytopathic effects (CPEs)
– degenerative changes
– abnormalities
• Transformation to malignant cell
 Viruses and Cancer

• Tumor

– growth or lump of tissue;

– benign tumors remain in place

• Neoplasia

– abnormal new cell growth and reproduction due to loss of regulation

• Anaplasia

– reversion to a more primitive or less

differentiated state

• Metastasis

– spread of cancerous cells throughout body

Viruses and Cancer..
 Possible Mechanisms by Which Viruses Cause Cancer

• Viral proteins bind host cell tumor suppressor

proteins
• Carry oncogene into cell and insert it into host
genome
• Altered cell regulation
• Insertion of promoter or enhancer next to
cellular oncogene
 Cultivation of viruses
• Requires innoculation of appropriate living
hosts
Hosts for Bacterial and Archael Viruses

• Usually cultivated in broth or agar cultures of

suitable, young, actively growing bacteria
• Broth cultures lose turbidity as viruses
reproduce
• Plaques observed on agar cultures
 Viruses persist in two stages

 dormant phase--extracellular; this phase is

neither "alive nor dead" rather should be
described as functionally active or inactive.
 Vegetative phase--intracellular. One should
note that the nucleic acid of the virus, in some
cases, is infectious.
Comparison between Viruses and other microorganisms
 1.6. Viruses Classification
• Older based on
– Host, target organ or vector
• Modern based on
– Type of viral nucleic acid
• RNA or DNA
• Single stranded (SS) or double stranded (DS)
• Replication strategy
– Capsid symmety
• Icosahedral or helical
– Presence or absence of lipid envelope
• Governed by International Committee on
Taxonomy of Viruses
 Polythetic properties for classification
• Virion morphology

• Nature of genome in virion

• Presence or absence of lipid membrane (envelope)

• Genome organization and replication

• Antigenic properties

• Biological properties
Baltimore system
 1.7. Epidemiology of Viral Diseases

Virus Transmission

• Disease and disease transmission an enormous variety of

organisms exist, including some which can survive and
even develop in the body of people or animals.
• If the organism can cause infection, it is an infectious agent.

• infectious agents which cause infection and illness are

called pathogens.
• Diseases caused by pathogens, or the toxins they produce,
are communicable or infectious diseases.
 Transmission..

• To be able to persist or live on, pathogens must

be able
– to leave an infected host, survive transmission in
the environment,
– enter a susceptible host, and develop, multiply in
the newly infected host.
– The transmission of pathogens from current to
future host follows a repeating cycle.
• This cycle can be simple, with a direct
transmission from current to future host, or
complex, where transmission occurs through
(multiple) intermediate hosts or vectors.
 Transmission..

• The cycle is called the transmission cycle of disease, or

transmission cycle.
• The transmission cycle has different elements: „
– The pathogen: the organism causing the infection „
– The host: the infected person or animal ‘carrying’ the pathogen „
– The exit: the method the pathogen uses to leave the body of the
host
– The entry: the method the pathogen uses to enter the body of the
susceptible person or animal „ „
– Transmission: how the pathogen is transferred from host to
susceptible animal, which can include developmental stages in the
environment, in intermediate hosts, or in vectors
– The environment: the environment in which transmission of the
pathogen takes place. „
– The susceptible person/animal: the potential future host who is
 Viruses which contain ds DNA genome

 Poxviridae
 Iridoviridae
 Asfarviridae
 Herpesviridae
 Adenoviridae
 Polyomaviridae
 Papillomaviridae
Viruses which contain ds DNA genome with
an RNA intermidiate

 Hepadinaviridae

Viruses which contain ss DNA genome:

 Circoviridae
 Parvoviridae
Viruses which contain ds RNA genome
 Reoviridae

 Birnaviridae

Viruses which contain Nss RNA genome

 Paramyxoviridae

 Rhabdoviridae

 Filoviridae

 Boraviridae
Viruses which contain Nss RNA genome contd’
 Orthomyxoviridae

 Bunyaviridae

 Arenaviridae

Viruses which contain ss RNA genome with a

DNA intermidiate
 Retroviridae
Viruses which contain ss RNA genome:
 Coronaviridae
 Arteriviridae
 Picornaviridae
 Caliciviridae
 Astroviridae
 Togaviridae
 Flaviviridae