CHAPTER 1 – FOUNDATIONS

→ A virus is an infectious agent that can only replicate within a host organism.
→ Protein shell called capsid,
An external membrane called envelope.
→ Possess no organelles, first thing to use in the host is the ribosome.

→ Virome: virus genome

Virion: infectious particles
Viroid: no capsid, but infect plant.
Zoonoses: any disease or infection that is naturally transmissible from vertebrate animals to
humans
Zoonotic: cross-species (HIV, MERS, SARS)

→ Are viruses alive?

→ Human endogenous retroviruses, and elements – 8% of our genome

→ Do we require viruses for our evolution?
In a study, researchers apply big-data analysis to reveal the full extent of viruses' impact
on the evolution of humans and other mammals. Their findings suggest an astonishing 30
percent of all protein adaptations since humans' divergence with chimpanzees have been driven
by viruses.
→ Do viruses have species boundaries?
**swine workers and swine influenza virus infections
At the beginning, viruses are host-specific but if the interaction increases, they evolve in
a way that they can infecting species.

→ Viruses are unique tools to study biology.

A tool for DNA recombinant technologies:
Bacteriophage - Analysis of viral nucleic acids, proteins, and antibodies from ancient human
specimens and in modern populations, tracking ancient human migrations by the viruses they
carried.

→ All of us carry many different viruses throughout our daily lives. Why don’t they make us
sick?

→ How does the discovery of new viruses today differ from 100 years ago?

→ Which host cell function is essential for the reproduction of all viruses? Ribosome.

→ How are viruses classified?

Appearance, nucleic acid genome, hosts they infect…
The Baltimore classification clusters viruses into families depending on their type of genome.
→ Even as our knowledge of viruses continues to increase, it is clear that their reproduction and
survival depend on similar pathways.

→ Our bloodstreams harbor up to 100,000 virus particles per milliliter. In addition to viruses that
can infect us, our intestinal tracts are loaded with myriad plant and insect viruses, as well as
many hundreds of bacterial species that harbor their own constellations of viruses.

→ Do you know the first «virus»? – only about 120 years – 1.5 billion years ago.
→ Beijerinck, in 1898, was the first to call 'virus', the incitant of the tobacco mosaic. He showed
that the incitant was able to migrate in an agar gel, therefore being an infectious soluble agent,
or a 'contagium vivum fluidum' and definitively not a 'contagium fixum' as would be a bacteria.

Vaccination
→ Smallpox – variolation
– taking material directly from the smallpox lesions of an infected individual and scratching it
onto the skin of healthy individuals with a lancet.
– 1 to 2% fatality rate
– 18th century, variolation was perceived as a much better alternative than naturally contracting
natural smallpox, a disease with a fatality rate of 25% in the whole population and 40% in babies
and young children.

→ Edward Jenner
→ Milkmaids variolation protects from previous infections
→ Inoculation with material from cowpox lesions induced only mild symptoms in the recipient
but protected against the far more dangerous disease (infection of others: syphilis and hepatitis)
→ vaccination (vacca = “cow” in Latin)

→ Louis Pasteur
- 1885, he inoculated rabbits with material from the brain of a cow suffering from rabies and then
used aqueous suspensions of dried spinal cords from these animals to infect other rabbits. After
several such passages, the resulting preparations were administered to human subjects, where
they produced mild disease but effective immunity against rabies.

→ poisonous air (miasma) - contagious diseases

Koch’s Postulates – 1890
→ The microorganism must be found in abundance in all organisms suffering from the disease
but should not be found in healthy organisms.
→ The microorganism must be isolated from a diseased organism and grown in pure culture.
→ The cultured microorganism should cause disease when introduced into a healthy organism.
→ The microorganism must be re-isolated from the inoculated, diseased experimental host and
identified as being identical to the original specific causative agent.

→ Dimitrii Ivanovsky - Beijerinck – tobacco mosaic virus in 1892

- infectious liquid, reproduce in their host organisms, submicroscopic agent
→ foot-and-mouth disease virus in 1898
→ the first ‘filterable agent’ to be discovered in humans was yellow fever virus in 1901
→ Agents passing through filters that retain bacteria came to be called ultrafilterable viruses,
appropriating the term virus from the Latin for “poison.” This term was simplified eventually to
“virus.”

Koch’s Principles Extd.

→ the definitive traits are molecular markers such as genes or full genomes that can uniquely
distinguish samples obtained from diseased subjects from those obtained from matched,
healthy control subjects and,
→ inoculating a healthy individual with a sample from a diseased subject results in transmission
of the disease as well as the molecular markers

Major developments in the technology of virus discovery:

The Pathogen Pyramid

Properties of Viruses
→ Simplicity of structure:
- TEM: 1930s
- Around 100,000-fold magnification
- Morphological classification of viruses
helical, icosahedral, enveloped, and head-
and tail.

Organisms as Hosts:
→ Obligate parasites:
- Plants – tobacco mosaic virus
- Lethal dose
- TCID50
- Plague Assay (1930s, bacteriophages)
- Nobel prize, Dulbecco, plaque assay animal viruses

→ Transmission of yellow fever virus to mice by Max Theiler in 1930, attenuated virus

Bacteriophages
→ Phage therapy
- Multiple phages required
- Safe for microbiome
- So specific
- bacteria have constructed a biofilm composed of a polysaccharide matrix that antibiotics
cannot penetrate.
- Disadvantages?

Classification
1. Nature of the nucleic acid in the virus particle (DNA or RNA)
2. Symmetry of the protein shell (capsid)
3. Presence or absence of a lipid membrane (envelope)
4. Dimensions of the virion and capsid
CHAPTER 2 – THE INFECTIOUS CYCLE

→ Susceptible – allow virus entry.

we do not know whether the virus will replicate itself or not
→ Permissive – support virus reproduction.
we do not know if they have the right receptor or not
→ Resistant – no receptor, no entry
→ If both susceptible & permissive – virus can infect the cell naturally.

Culturing Viruses
→ The first cell lines, such as mouse L929 cells (1948) and HeLa cells (1951)
→ John Enders, Thomas Weller, and Frederick Robbins discovered in 1949 that poliovirus could
multiply in cultured cells (Nobel Prize in 1954)
→ They are also used in vaccine production: for example, infectious attenuated poliovirus
vaccine strains may be propagated in primary monkey kidney cells.
→ We cannot se viruses under light microscope, so we look at host cells to see cytopathic
effect, change shaping effect.
→ No information about quantity.

→ Monolayer and suspension cell cultures

→ Induced pluripotent stem cells: (iPSCs) Adult cells that have been reprogrammed genetically
to an embryonic stem cell-like state.
→ Organoid: A three-dimensional cell culture that is derived from pluripotent stem cells and
composed of multiple cell types, with several phenotypic properties of the tissue it is emulating.
Plaque Assay
→ When the original infected cells release new progeny particles, the gel restricts their spread
to neighboring uninfected cells.
→ If the infected cells are damaged, the plaque can be distinguished from the surrounding
monolayer.
→ Only viruses that cause visible damage of cultured cells can be assayed in this way.

→ Why plaque formation is important? To observe and quantify the virus.

Reproduction

MOI, Multiplicity of Infection

→ the ratio of infectious agents to infection targets
→ the number of infectious viral particles per cell
→ cell culture 106 virus / 16 cell MOI of 1? Not particles!

MOI = (Virus titer X Virus Volume) / Total Cell Number

→ Poisson distribution – P(k) = e-mmk/k!

- P(k) is the fraction of cell infected by k virus particles,
multiplicity of infection, m(MOI), is calculated from the proportion of uninfected cells
k=0
uninfected cells: P(k) = e–10 = 4.5 × 10–5
one particle P(1), k(1)...

MOI differs based on:

→ Host cell stage
Cell cycle stages – dividing or not
non-dividing like neuron require more virus, dividing - less required to eliminate defective
particles.
→ Characteristic of virus
→ Virus production.
Hemagglutination
→ Hemagglutination is used for the diagnosis of some
enveloped viruses such as influenza viruses.
→ This method relies on the specific feature of some
enveloped viruses that can adsorb to red blood cells
(RBCs). Specifically, hemagglutinin5 (HA), an envelope
glycoprotein of some enveloped viruses, imparts this
property.
→ In the absence of virus particles, RBCs precipitate by
gravity to the bottom of the well, giving rise to a distinct
redcolored dot in a conical shaped well.
→ In the presence of virus particles, RBCs clump together
as a result of interaction between HA proteins of virus
particles and RBC, leading to a lattice formation.

Centrifugation & Differential Centrifugation & Gradient Centrifugation

Virus Neutralization
→ Ab production, monoclonal
cannot neutralize?
→ The end point is defined as the highest dilution of antibody that inhibits the development of
cytopathic effect in cells or virus reproduction in eggs or animals.
→ You should know the virus & specific mAb for that.
→ Monoclonal antibodies tagged w/ fluorescence,
super-resolution microscopy
Chapter 3 – Genome and Genetics

GENOME STRUCTURE
→ Virus particles as naked molecules of nucleic acid
→ Nucleic acid-binding proteins or enzymes are required for the initial phase of reproduction
→ Usually not bound by histones in the virus particle
polyomaviral and papillomaviral genomes are exceptions
→ All viral DNAs become coated with histones shortly after they enter the nucleus
→ Epigenetic factors

→ Secondary and tertiary structures in which nucleotides may engage in long-distance

interactions like priming during DNA synthesis, facilitate translation, genome packaging.

Segmented vs Nonsegmented
→ Split into separate parts
→ Reassortment – they can switch their variants by changing segments
Influenza w/ 8 segments – reassortment contributes flu pandemics
“one gene, one mRNA” wrong – they need mRNAs in the number of segments to infect.

Positive vs Negative Stranded

→ Positive-stranded RNA is just like mRNA
→ Negative-stranded RNA is complementary to mRNA, RNA-dependent RNA polymerase**
(carried in the capsid of all negative-stranded RNA viruses)
→ Most DNA viruses have both a positive and negative strand except parvoviruses
→ Positive strand is being read while negative stranded is ignored.
Size Matter?
→ Mimivirus – protein synthesis system, such as tRNAs and aminoacyl-tRNA synthetases
→ Tupaniviruses – encode all 20 aminoacyl-tRNA synthetases, 70 tRNAs, multiple translation
proteins
→ Tetraselmis virus 1 – encode pyruvate formate-lyase and pyruvate formate-lyase-activating
enzyme, key members of cellular anaerobic respiration and allow energy production when no
oxygen is available

→ An exonuclease encoded in the coronavirus genome is one exception: its present could
explain in large size of these RNAs.
→ Error frequencies for RNA genomes are about 1 misincorporation in 104 or 105 nucleotides
polymerized.

The Baltimore System

→ Viruses are classified according to their conversion to mRNA.
→ The thousands of distinct viruses defined by classical taxonomic methods can be organized
into seven groups, based on the structures of their genomes.
mRNA → Ribosome → Protein
→ Unity in diversity, 7 classes:
RNA viruses to propagate, their RNA genomes must, by definition, encode a nucleic acid
polymerase, RNA-dependent RNA polymerase & Reverse Transcriptase
DNA GENOMES

→ The strategy of having DNA as a viral genome appears at first glance to be the ultimate in
genetic efficiency: the host genetic system is based on DNA, so viral genome replication and
expression could simply emulate the host system. While the replication of viral and cellular DNA
genomes is fundamentally similar, the mechanistic details are varied because viral genomes are
structurally diverse.
RNA GENOMES

→ Cells have no RNA-dependent RNA polymerases that can replicate the genomes of RNA
viruses or make mRNA from RNA templates:
1. RNA virus genomes encode RNA-dependent RNA polymerases that produce RNA from RNA
templates.
2. Reverse transcription of the genome to dsDNA, which can be transcribed by host RNA
polymerase.

Cells have no RNA-dependent RNA polymerases that can replicate the genomes of RNA viruses
or make mRNA from RNA templates. One solution to this problem is that RNA virus genomes
encode RNA-dependent RNA polymerases that produce RNA from RNA templates. The other
solution, exemplified by retrovirus genomes, is reverse transcription of the genome to dsDNA,
which can be transcribed by host RNA polymerase.
→ Minus-sense or ambisense strategy

After the virus attaches to a cellular receptor, the nucleocapsid, containing the viral RNA
synthetase, is released into the cytoplasm. The viral synthetase first synthesizes mRNAs, which
are translated into the viral proteins required for synthesis of full-length complementary RNAs
(vcRNAs). These vcRNAs are the templates for minus-strand genome RNA synthesis. Throughout
replication, minus-strand genomes and plus-strand vcRNAs are present in nucleocapsids. Viral
mRNAs are also translated into membrane glycoproteins that are transported to the cell plasma
membrane (or in some cases specialized internal membranes). In the final maturation step, the
nucleocapsid buds out through areas of modified membrane to release the enveloped. E.g.
Arenaviridae and Bunyaviridae.

GENETIC ANALYSIS OF VIRUSES

→ Mapping Mutations:
- Recombination mapping results in genetic exchange: under restrictive conditions (high
temperature) by the titer measured under permissive conditions (low temperature).
- Reassortment: mutant and wild-type viruses, the progeny includes reassortants that inherit
RNA segments from either parent.

→ Engineering Mutations into Viral Genomes

- Infectious DNA clone, a dsDNA copy of the viral genome that is carried on a bacterial vector
such as plasmid -transfection
→ DNA viruses: infectivity of deproteinized human adenoviral DNA is between 10 and 100 PFU
per μg. When the genome is isolated by procedures that do not degrade the covalently attached
terminal protein, infectivity is increased by 2 orders of magnitude, probably because this protein
facilitates the assembly of initiation complexes on the viral origins of replication.
→ polyomaviruses, papillomaviruses, and adenoviruses – plasmids
→ herpesviruses and poxviruses – cosmids or BACs
→ Infectious?
→ Poxvirus needs viral DNA-dependent RNA polymerase and transcription proteins.
→ RNA viruses:
→ (+) strand RNA viruses: genomic RNA of retroviruses is copied into dsDNA by reverse
transcriptase
→ Genomic RNA of (–) strand RNA viruses is not infectious, because it can be neither translated
nor copied into (+) strand RNA by host cell RNA polymerases
- When all eight plasmids carrying DNA for each viral RNA segment are introduced into cells,
infectious influenza virus is produced.
- Non segmented:
- Hybridization problem
- When a full-length (+) strand RNA is transfected into cells that have been engineered to
synthesize the vesicular stomatitis virus nucleocapsid protein, phosphoprotein, and
polymerase, the (+) strand RNA is copied into (–) strand RNAs. These RNAs initiate an infectious
cycle, leading to the production of new virus particles.
- dsRNA viruses: reovirus cloned DNA copies of the 10 RNA segments of the viral genome.

→ Types of Mutation – deletion, insertion, substitution (nonsense & missense).

Chapter 4 – Structure

→ Watson & Crick – 1956

- spherical or rod-like shape
- genetic economy

→Viruses need to protect their genome and entry into host, important for structure.

→ Viral proteins permit regular and repetitive interactions among them, causing self-assembly
of viral capsid formation.

→ Symmetry:
- helical (rod-like) or spherical (platonic polyhedral)

→ Identical bonding with neighbor

→ Noncovalent bonding, reversible

→ Virus-like capsid; self-assemble, HBV and HPV viruses made in yeast:

They are using in vaccine production,
you can change the size of VLC’s only by arranging protein amount.

PROTEIN COAT
→ Irreversibly by a break in the nucleic acid or by mutation during passage through hostile
environments.
- proteolytic and nucleolytic enzymes; extremes of pH, humidity, or temperature; and various
forms of natural radiation.
- Poliovirus (Picornaviruses) sensitive to dryness but survive longer in water. They are
even resistant to very strong detergents.
- Dried onto a solid surface, human rotavirus loses <20% of its infectivity in 30 days at
room temperature.

→ Capsid proteins have sensors to make parts achieve self-assembly.

→ Functions of virion proteins:

1. Protection of the genome
- Assembly of a stable protein shell
- Specific recognition and packaging of the nucleic acid genome
- Interaction with host cell membranes to form the envelope
2. Delivery of the genome
- Binding to external receptors of the host cell
- Transmission of signals that induce uncoating of the genome
- Induction of fusion with host cell membranes
- Interaction with internal components of the infected cell to direct transport of the
genome to the appropriate site.
3. Other functions
- Interaction with cellular components for transport to intracellular sites of assembly
- Interaction with cellular components to ensure an efficient infectious cycle.
→ Metastability

→ The protective function of virus particles

depends on stable intermolecular interactions
among their components during assembly, egress
from the virus-producing cell, and transmission.

→ These interactions must be reversed readily

during entry and uncoating in a new host cell.
Energy is used during disassembly.

How do viruses achieve metastability?

ENVELOPE
→ Derived from cellular membranes, into which viral glycoproteins have been inserted.
→ Impermeable to many molecules and block entry of chemicals or enzymes in aqueous
solution.
→ Sensitive to pH dryness and disinfectants.
→ No cell lysis required, can be released like exosomes.
→ Difficult to detect by immune cells.
→ Glycoproteins present.
→ Integral membrane proteins:
Ectodomain – attachment, antigenic sites, function
Internal domain – assembly
Oligomeric – spikes

*Hemagglutinin protein – influenza

*E-dimer – flavivirus

Why they require envelope?

CAPSID/NUCLEOCAPSID

→ A small number of proteins -repeated- independent size of virus

→ Size of genome and capsid complexity have no correlation
→ Optimization of regular protein-protein interactions
→ Helical or icosahedral symmetry.

→ Non-covalent bonds, if covalent – hard to break, metastability.

→ Icosahedral symmetry – 20 faces, equilateral triangle, closed shell with identical 60 subunits
(to make closed shell self-assemble, it requires 60 subunits), 5,3,2, fold axes of symmetry.

→ Quasiequivalent – more than 60, 1 more addition not identical

→ Triangulation number – number of faces

→ Large viruses have more complex symmetry: Adenovirus – fibers, Reoviruses – two shell inner
and outer, Bacteriophages – tail, Herpes simplex virus – capsid portal.
CHAPTER 5 – ATTACHMENT AND ENTRY

→ Susceptible – producing the receptors required for virus entry

→ Permissive – able to support virus reproduction when the viral genome is introduced, has no
receptor but viruses can replicate.

→ Collision of viruses – they may around to find host

→ Obligate parasite, virus: need to enter the host!
- cannot pass passively, receptor binding
- have a shield for nucleic acids, uncoating

Receptor:
1. Initiating conformational changes
-prime fusion
-uncoating -by directing the virus particle into endocytic pathways, where fusion and uncoating
may be triggered by low pH or by the action of proteases.
-RNA viruses - cytoplasm
-DNA viruses – nucleus

→ Random collision between a virus particle and a cell:

Steps:
1. adhere
2. attach receptor
1985 – sialic acid - influenza
fungi – no extracellular phase
plant – mechanical damage, due to cell damage
3. transfer genome

→ Receptors have another roles in out body – viruses use them as receptors

→ late endosome – pH decrease – capsid disintegrates

→ first receptor found – sialic acid
→ collision of viruses – they may around to find host
→ Different receptors – same receptors
1. Adenovirus and Coxsackievirus B3
2. Swine herpesvirus, pseudovaries virus and human poliovirus
3. Same family different receptors: rhinoviruses (3) and retroviruses (16)

Identification of Receptors for Virus Particles

1. Monoclonal antibody production
2. Cell transfection with DNA
Virus-Receptor Interactions
1. Nonenveloped Virus Receptor Binding
– one or more of the viral capsid proteins
Canyons, fibers,
surface loops that include amino acid sequence motifs
recognized by their integrin receptors
2. Enveloped
– oligomeric integral membrane glycoproteins that have been
incorporated into the cell-derived membranes of virus particles.

Alternatives:
→ Often passage of viruses in cells in culture selects variants that bind heparan sulfate.
→ Infection of cells with foot-and-mouth disease virus type A12 requires integrin αvβ3.
→ However, the receptor for the O strain of this virus, which has been extensively passaged in
cells in culture, is not integrin αvβ3 but cell surface heparan sulfate.
→ On the other hand, the type A12 strain cannot infect cells that lack integrin αvβ3, even if
heparan sulfate is present.
→ Enveloped – oligomeric integral membrane glycoproteins that have been incorporated into
the cell-derived membranes of virus particles.
Chapter 6 – Synthesis of RNA from RNA Templates

→ 1936 – TMV crystals contain 5% RNA

→ 1952 – Hershey-Chase experiments showing that DNA is the genetic material
→ 1956 – Frankel-Conrat & Singer experiment TMV RNA is genetic material
→ 1960 – RNA replication with Poliovirus experiment

• Human viruses do not have have RNA-dependent RNA polymerase.

RNA → mRNA: Reovirus, Rotavirus

-RNA → mRNA: Influenza virus, VSV
+RNA → -RNA: Poliovirus
Stages:
1. Virus Infection
2. Cell Extract
3. ATP, UTP, GTP, CTP
4. Measure RNA synthesis

** RNA polymerase vs poliovirus = correlation

→ Discovered RNA dependent RNA polymerase in (-) strand viruses

w/o it can be degraded
→ (+) strand RNA genomes (poliovirus, flavivirus): no RdRp, naked except coronavirus (long
genome?) and retrovirus coated
→ dsRNA genomes: RdRp, naked, far from ribosome

Rules for viral RNA synthesis

→ RNA genome – end to end copy
→ Viral mRNA – translated efficiently (poliovirus directly like mRNA)
→ Specific sites for initiation and termination of replication
→ Start de novo (like DpRp) or with a primer
→ Proteins from cell or virus
→ RNA synthesized by template-directed stepwise incorporation of NTPs, elongated in 5’-3’
direction.
→ There is some non-templated synthesis
TEMPLATE SPECIFITY

→ Affinity of the RdRp for the initiating nucleotide:

- Bovine viral diarrhea virus and bacteriophage ϕ6 prefer 3′-terminal C
- Reovirus RdRP prefers a G at the second position of thetemplate RNA
- RNA pseudoknot: An RNA secondary structure formedwhen a single-stranded loop region base
pairs with acomplementary sequence outside the loop
- Protein-protein interactions
- Purified polymerases often lack template specificity

→ dsRNA – unwinding by helicases exceptbacteriophage ϕ6 RNA (polymerase activity resolve)

- Structural vs non-structural proteins

→ dsRNA:
A distinctive feature of the infectious cycle of double-stranded RNA
viruses is the production of mRNAs and genomic RNAs from distinct
templates in different viral particles. Because the viral genomes are
double stranded, they cannot be translated. Therefore, the first step in
infection is the production of mRNAs from each viral RNA segment by
the virion associated RdRP (Fig. 6.24). Reoviral mRNAs carry 5′ cap
structures but lack 3′ poly(A) sequences.
Origin of Diversity in RNA Virus Genomes

• mutations can be excreted out of capsid but do not replicate.

→ Misincorporation of Nucleotides – No proofreading. RNA virus populations are

quasispecies, or mixtures of many different genome sequences.

→ Segment Reassortment and RNA Recombination

→ Reassortment is the exchange of entire RNA molecules between genetically related
viruses with segmented genomes.
→ Recombination is the exchange of nucleotide sequences among different genomic
RNA molecules. 10 to 20% of polioviral genomic RNA molecules recombine in a single growth
cycle.

→ RNA Editing - Viral mRNAs can be edited either by insertion of a non-templated nucleotide
during synthesis or by alteration of the base after synthesis.