Virology

A. Introduction
1. Translation of eukaryotic mRNA. The CAP is required for ribosome binding and UTR
(untranslated region). Note: most mRNA encode one protein
2. Eukaryotic ribosome: 80S = 40S + 60S
3. Translation requires various proteins called factors ie. eIF4G
4. Definitions/General
a. endemic: disease is present in the population at a constant level
b. epidemic: sudden outbreak affecting a large # of people
c. pandemic: epidemic over a wide area
d. morbidity: incidents of disease
e. mortality: death from disease
5. Definition/Virology
a. virus: an obligate intracellular parasite
b. virion: an infectious viral particle
c. host cell: cell that replicates the virus
d. host range: species that can be infected by a particular virus
i. Most virus have a narrow host range. ie. smallpox infects humans only
ii. Polio virus infects humans and other primates
iii. A few viruses have a broader host range: rabies virus can infect most
warm blooded animals
e. Tissue tropism: this refers to the type of cell in an organism that can be infected
and replicate a particular virus. Cells must have the correct receptor for viral
attachment and be able to be replicated in the cell. Both affect host range and
tissue tropism.
6. Bacteriophage:
a. Bacteriophage are viruses that infect bacteria. T4 infects E.coli
i. T4 is a virulent phage, this means it has a lytic replicative cycle which
kills the host cell
ii. Structure: It has a dsDNA genome. It has an icosahedral capsid, a central
tube, covered by a helical sheet, and a base plate with tail fibers. The
base plate has a T4 encoded lysozyme attached (this means the T4
genome includes a gene that encodes lysozyme).
iii. Replicative cycle
1. Attachment (adsorption): T4 tail fibers bind to LPS and OmpC on
the surface of E.coli
2. Base plate comes in contact with the cell surface. The sheath
contracts pushing the central tube through the bacterial cell wall.
The T4 lysozyme helps central tube get through the
peptidoglycan.
3. The DNA leaves capsid and enters the bacterial cytoplasm. The
remainder of the phage remains outside the cell.
4. The bacterial cell replicates the viral DNA and synthesizes all the
protein components. They self assemble, forming new virions.
 5. Late in infection, T4 enzyme, holin, forms holes in the bacterial
PM.
6. Unbound T4 lysozyme passes through the holin and opens up
the peptidoglycan. Weakened cell lyses, releasing the newly
synthesized T4 virions.
b. Phage lambda
i. Phage lambda also infects E.coli, it is a temperate phage; this means it
can enter into a state called lysogeny. Lysogeny means that virions are
not being produced, the viral genome is being replicated with the host
chromosome.
ii. Structure: dsDNA genome, icosahedral capsid, non-contractile tail +/-
tail fibers
iii. Lysogenic pathway: attachment is to LamB porin, which is used for
maltose uptake. The genome is inserted into the periplasm through
LamB and then transported into the cytoplasm using a maltose
transporter. The phage DNA is inserted into the bacterial chromosome,
it is now called a prophage. Although some viral proteins are expressed,
the virion components are not. It will remain like this until a signal is
received to enter a lytic cycle.
iv. Lytic pathway: phage lambda can enter the lytic cycle directly or if it is in
lysogenic cycle, stressors like UV light cause the prophage DNA o excise
and enter the lytic cycle.
c. Some temperate phage encode genes that increase the virulence of some bacteria. ie.
the genome of β phage includes the gene that encodes the exotoxin that causes
diphtheria. This phage can lysogenize Corynebacterium diphtheriae, now the
bacteria can cause the disease diphtheria.

B. Cultivation of viruses
1. Viruses can only be replicated by host cells. ie. bacteriophage can only be replicated in
susceptible species of bacteria.
Method: the phage are mixed with the host bacteria and then added to the surface of
an agar plate. After overnight incubation, the phage can be isolated from the plate.
2. Animal viruses
a. Animal viruses can be replicated in susceptible animal cells grown in-vitro
(outside body) these cells are called cell culture or tissue culture.
b. Tissue culture can be made from tissue removed from an animal and then
separated into cells. These cells can be grown in specialized media. Most cells
will stick to the bottom of a tissue culture flask, these cells will divide a limited
amount of times and then die.
c. Cell lines are tumor cells that can be cultured in-vitro indefinitely. Because of
this they care called inmortal. Many cell lines can be purchased. ie. HeLa cells:
woman had cervical cancer
d. Some viruses cannot be replicated in cell culture. For example Influenza virus is
replicated in embryonated chicken eggs.
3. Plaque Assay
 a. After they’ve been replicated, some viruses lyse their host cell
b. A plaque assay can be used to quantitate lytic viruses
c. Lytic phage
i. The phage is serially diluted
ii. The dilutions are then mixed with the host bacteria added to soft agar
and poured on an agar plate.
iii. As the bacteria grow, they replicate the phage and the phage will lyse
infected host cells.
iv. The newly released phage will infect nearby bacterial cells.
v. Next day, a clear zone called a plaque is seen in the lawn of bacteria.
vi. Plaque are counted and the plaque forming unit (pfu/ml) in the original
sample can be calculated.
d. Lytic animal viruses (the plaque assay is the same general idea)
i. Serial dilutions of the virus are added to tissue culture
ii. Because most tissue culture grows on the bottom of the flask, plaques
can’t be counted.

C. Viral Structure/Genome
Note: this information relates to animal viruses unless stated otherwise
1. Genome
a. Genome refers to all of the genetic material in a cell or virus
b. All cells have a genome made of dsDNA
c. A viral genome will be made of one of the following: dsDNA, ssDNA, dsRNA,
ssRNA.
2. DNA viruses: most DNA viruses have a dsDNA genome. Only one family has a ssDNa
genome.
3. RNA viruses:
a. Most RNA viruses have a ssRNa genome. The RNA is either (+) strand or (-)
strand RNA.
b. (+) strand RNA: it encodes the information for translation. ie. start codon, coding
sequence and stop codon. mRNA is an example of (+) strand RNA.
c. (-) strand RNA: complementary to the (+) strand RNA. Therefore it cannot be
translated.
d. dsRNA is composed of one (+) strand of RNA and one (-) strand. These terms are
used to describe DNA.
e. Retroviridae: this is the only viral family with a diploid genome. Hey have two
copies of the same strand of RNA.
f. All (-) strand RNA viruses have a helical capsid.
g. Some RNA viruses have a segmented (in pieces) genome.

D. Capsid
1. Definition
a. capsid: it is the proteins surrounding the genome
b. nucleocapsid: this refers to the combination of the genome and the capsid.
Note: we will only use this term in reference to HIV.
2. Capsid Structure:
 a. Capsids are composed of multiple copies of one or a few proteins. These
proteins will bind non-covalently to each other in a specific way, forming a
regularly shaped structure.
b. Most viruses have capsids with either helical or icosahedral (20 sided)
symmetry.
3. Helical capsids: the proteins bind directly to the genome and to adjacent protein
subunits. This binding winds the genome into a helix. The overall shape is filamentous.
4. Icosahedral capsids: The number of proteins per side varies in different viruses. A
small virus like polio has 60 copies of 3 different proteins, a total of 180 forming the
capsid well. The overall shape is spherical and encloses genome.
5. Complex Symmetry: ie. T4 bacteriophage and adenovirus

E. Viral Envelope
1. Some viruses have a layer outside their capsid called the envelope. All animal viruses
with helical capsids have one. Some animal viruses with icosahedral capsids have one.
Viruses without an envelope are called naked.
2. Envelope Structure:
a. The envelope is composed of a phospholipid bilayer derived from a host cell
membrane.
b. It has virally encoded glycoproteins inserted into the bilayer, these are often
called spike proteins.
c. spike proteins Structure: They are composed of one or more polypeptides, each
with multiple domains.
i. Internal domain: it is often in contact with other viral proteins
ii. Transmembrane domain: it’s a hydrophobic helix
iii. External domain: it’s larger and glycosylated. It includes the site for
binding to the host cells receptor. It is the major antigenic determinant
of the virus. This is the part recognized and targeted by the adaptive
immune system. It mediates fusion during the entry of some viruses.
d. The overall shape of envelope viruses is either spherical or pleomorphic.

F. Other
1. Some enveloped viruses have a layer of proteins bound to the inner surface of the
envelope. They bind to the internal domain of spike proteins and to phospholipids.
ie. matrix proteins: they are found in some negative strand RNA viruses and
retroviruses. The matrix links the capsid to the envelope.
2. Some virions contains virally encoded enzymes. These usually catalyze reactions
unique to viruses and are required before transcription and translation of the viral
genome.
3. Some viruses contain host cell macromolecules (in addition to lipid bilayer of
envelope)
ie. retroviruses have host tRNA inside their capsid

G. Classification of animal viruses

 1. Classical system groups viruses based on type of genome, the capsid symmetry +/-
envelope. This system is used to produce latinized names ending in viridae
(underlined), genus names ending in virus (underlined) and species names that can
also end in virus but are not underlined.
2. Baltimore classification is based on how mRNA is synthesized.

H. Animal Virus Replication Overview

1. List of Steps:
a. Attachment
b. Entry and uncoating
c. Replication of virion components
d. Exit
e. +/- maturation
2. Attachment
a. There is a chance collision of the virion and the host cell. The larger the number
of virions, the greater the chance of infection.
b. Surface molecules on the virion will bind to specific receptors on the host cell.
These receptors have a function for the host cell. Many are members of the Ig
superfamily. ie. Rhinovirus causes colds; they use ICAM as receptor.
c. Receptors can be identified using MAb
3. Entry and uncoating
Note: uncoating is a general term for the release of the genome from the virion
a. Naked virus: after attachment the virion is taken up by endocytosis, the
genome will be released from the endosome.
b. Envelope virus:
i. Endocytosis: after attachment and endocytosis, the envelope fuses with
the endosomal membrane and the contents are released into the
cytoplasm. The genome is released.
ii. Fusion: envelope virus fuses with the PM releasing the capsid into the
cytoplasm were it will be uncoated
4. Replication of viral components
a. mRNA synthesis depends on the genome type.
b. genome synthesis also varies with the type of viral genome
c. mRNA and/or genome synthesis may require virally encoded enzymes if the
host cells don’t have these enzymes. Polymerase names refer to the type of
nucleic acid used as a template and to the type of nucleic acid being
synthesized.
ie. DNA dependant RNA polymerase (DDRP)
RNA dependant RNA polymerase (RDRP)
d. Spike proteins: are synthesised by ribosomes on the RER and inserted into the
PM (using the endomembrane system)
5. Assembly: the capsid proteins self assemble, enclosing the genome
6. Exit:
a. Naked viruses accumulate in the cell’s cytoplasm. They cause the cell to lyse,
which release the viral particles.
 b. Envelope viruses: if the virus does not have matrix proteins, the capsid will still
bind to the PM and spike proteins. If the virus has matrix proteins, they will
bind to the inner surface of the PM/spike proteins. The capsid can then bind to
the matrix proteins. In both cases, the virus exits by budding.
7. Maturation: some viruses require an extra step called maturation to become
infectious. In this step, specific viral proteins are cleaved by a viral protease. Once
mature, it is then infectious and can be called a virion.
8. The one step growth curve of adenovirus
a. Adenovirus is a dsDNA, naked (no envelope), lytic, animal virus.
b. In a one step growth curve method, the virus is added to the host cell culture in
a small volume and allowed to attach. The culture is then rinsed to remove any
unattached virions, this synchronizes the replicative cycle.
c. At set time points, samples of the culture’s supernatant (fluid) are tested for
extracellular virions, using a plaque assay. At the same time points, the cells
are lysed and tested for intracellular virions.
d. Results: during the eclipse period, once uncoating has occurred, there are no
virions. At the end of the latent period, cells begin to lyse and virions begin to
appear extracellularly. Between the end of eclipse and latent period, virions
begin to appear.

I. General Patterns of Infection

1. Outcome depends on the immune system of the host and on the viral species
2. Acute Infections
a. Example: Rhinovirus and Influenza virus
b. Period before disease symptoms occur is called incubation period. This is 1 to 2
days for these viruses. It can be several weeks for other viruses. This means that
by the time the person feels sick, they may have passed the virus to others.
c. These illnesses are cleared by the adaptive immune system in about a week to
10 days.
3. Persistent infections
a. Occur because the adaptive immune system cannot clear the infection for a
long time or sometimes ever.
b. Persistent infections include latent persistent, slow persistent and
transforming persistent.
c. Latent persistent infection example: Herpes Simplex Virus. The first acute
infection is followed by a period without virion production, virions will be
produced during periods of reactivation that occur with or without symptoms.
d. Slow persistent infection example: HIV virions are produced during the
primary acute infection. During this time the patient may have flu-like
symptoms. Then the adaptive immune system usually reduces the number of
virions. The patient may not have symptoms again, until years later. Without
treatment, the outcome is fail.
e. Transforming infections
i. Some DNA viruses and some retroviruses can cause tumours. Note:
cells that grow abnormally and have an altered morphology, are
transformed.
 ii. A benign tumor is a clone of cells that forms a localized mass. ie. some
of the Papillomaviridae can cause many benign tumours called warts.
iii. A malignant tumour, or cancer is a tumour that continues to grow and
cause damage to organs and tissues. If some of the cells enter the blood
or lymph, they can cause a secondary tumour or metastasis. ie. HPV

J. Human Papillomavirus
1. Classification: member of the Papillomaviridae, the genus us alpha papilloma virus.
2. Structure: dsDNA genome and an icosahedral capsid, naked.
3. There are about 100 different serotypes of HPV. About ½ of these cause mucosal
infection in the throat and genitals. Most of these infections will be cleared by the
immune system.
4. Different serotypes can cause different diseases.
Note: serotypes are sometimes referred as types.
Example: HPV types 16 and 18 cause about 70% of cervical cancer and 80% of anal
cancer.
Example: HPV 6 and 11 do not cause cancer, they produce 80% of genital warts.
5. 1 vaccine is called Gardasil, it is a quadrivalent vaccine, this means it contains antigen
from HPV types 6, 11, 16 and 18. Serotypes are express separately in yeast. The proteins
are purified and mixed to make the vaccine.
Note: vaccinated women still need to get a pap test due to different serotypes.
Polio Virus
A. Classification
1. Family: Picornaviridae
2. Genus: Enterovirus
3. Species: Polio virus

B. Pathogenesis
1. It is spread by the oral, fecal route
2. The virus first replicates in mucosal tissue on the throat and the intestines virions are
shed in the saliva and feces.
3. Virions then enter the blood, this is called viremia. 99% of the patients recover
completely.
4. In the remaining 1% the virus infects the brain, causing meningitis, and the spinal cord,
which can cause paralysis. More serious disease is associated with the viral serotype
and the alleles of the patient’s MHC.
C. Structure
1. It is naked (no envelope)
2. The capsid: icosahedral
a. It has icosahedral symmetry
i. It is composed of 60 copies of VP1, VP2, VP3, VP4
ii. VP 1, 2, 3 form the surface of the capsid. There is a total of 180 subunits.
iii. VP4 is non-covalently attached to the inner surface of the capsid
b. The primary structure of VP 1, 2 and 3 differ but all of them form a structure
called a beta barrel jelly roll. It is wedge shaped with the beta sheet forming
 one wall, and second beta sheet forming a second wall and floor. This shape
facilitates interaction b/w the subunits forming a rigid structure.
3. The genome
a. Positive strand RNA: it does not have a CAP like mRNA, it does have a 3’ poly-A
tail.
b. Covalently attached to the 5’ end is a protein called VPg
c. At both the 5’ and 3’ ends there is an untranslated terminal region (UTR)

D. Replicative Cycle
1. Attachment
a. The capsid surface is uneven, it has plateaus and canyons

Influenza Virus A
A. Introduction
1. Classification
Family: Orthomyxoviridae
Genera: Influenzavirus A
Species: Influenza virus A
2. Influenza Virus A
a. It has subtypes or serotypes based on antigenic differences of the two spike
proteins (HA) and (NA).
b. These subtypes can also differ in their virulence
c. It causes yearly epidemics
d. It can cause pandemics
e. It can infect humans, other mammals and birds (host range)
3. Nomenclature
The names reflect differences in the strains.