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Chapter 1: Introduction to Virology
VIRUSES CONTAIN:
- Nucleic acid genome (either DNA or RNA)
- Protein coat (capsid)
- In some cases, lipid membrane (envelope)
o Envelope proteins or glycoproteins or peplomeres
VIRUSES
- Smallest & simplest form of life
- Viral genome encodes proteins that enable it to replicate
& transmitted from cell to cell
- Obligatory intracellular parasites (replicates only w/in
living cells)
- Viruses need the ff w/c are provided by living cells:
• Enzyme that synthesize amino acids, nucleotides,
carbohydrates, & lipids
• Enzyme that generate ATP
• Ribosomes, tRNAs, and enzymes in protein synthesis
• Membranes that concentrate cellular macromolecules,
small organic molecules, and inorganic ions
- Only one type of nucleic acid (DNA or RNA)
- Can be either SS or DS; circular or linear
- Only life form that can have RNA as its genome
o Problems w/ RNA as genome:
▪ synthesize messenger RNAs from an RNA
template
▪ replicate their genome RNA
o RNA viruses encode their own RNA-dependent RNA
polymerases to carry out these functions
- Inanimate but they share many attributes of life: ability
to mutate, evolve, and reproduce when they enter cells
- Infects & kill humans, animals, plant, insect, bacteria,
archaea, algae, fungi, and protozoa
- Most abundant form of life
o 1031 bacteriophages in the world
Virion – complete infectious virus particle; very small (20-500
nm)
20 nm : smallest virus; <2000 nucleotides; code for as few as
2 proteins
500 nm : largest virus; 1.2 mil nucleotides; >1200 proteins
Plaque assay - measure virus infectivity
Hemagglutination - cheap and rapid method for detection of
virus particles
The ratio of physical particles to infectious particles is greater
than 1 for many viruses.
VIRUS REPLICATION CYCLE
1. Virion binds to cell surface receptors
2. Virion or viral genome enters the cell; the viral genome is
uncoated
a. Partial disintegration of the virus particle
b. Release (uncoating) of the viral genome within the
cell
3. Early viral genes are expressed
a. viral genome can be used as a template for synthesis
of mRNAs, which in turn synthesize viral proteins
using the enzyme systems of the cell
4. Early viral proteins direct replication of the viral genome
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5. Late viral genes are expressed from newly replicated viral
genomes
6. Late viral proteins package genomes and assemble
progeny virus particles
7. Virions are released from the host cell
- disintegration and reassembly
- must transport its genome into a host cell
Viruses
- host cell provides the virus with all the other
biological molecules
- intact and retain their genomes within their
Unicellular own cellular membranes
Organisms - by growth and division into daughter cells
(chlamydiae - has their own ribosomes and protein
& rickettsiae) machinery
- genes code for enzymes
- Study of gene expression in small DNA viruses led to
identification of promoters for eukaryotic RNA
polymerases
- Research on the replication of bacteriophage and animal
virus DNAs led to understanding the enzymes in cellular
DNA replication
- RNA splicing in eukaryotic cells was discovered by
studying mRNAs of DNA viruses
- Study of cancer-producing viruses led to the isolation of
oncogenes and the understanding that cancer is caused
by their mutation or unregulated expression
BRIEF HISTORY OF VIROLOGY: THE STUDY OF VIRUSES
- virus -“poison” in Latin
- In the last decade of the 19th century, Dimitrii Ivanovski
and Martinus Beijerinck showed that the cause of
tobacco mosaic disease could pass through fine earth or
porcelain filters, which retain bacteria (filterable viruses)
- 1930s – vaccines against influenza & yellow fever
- mid-1930s - Wendell Stanley found that highly purified
tobacco mosaic virus could form crystals. It posed the
question: Are viruses living or inanimate?
- The development of the electron microscope allowed
scientists to see viruses
o tobacco mosaic virus - long rod-shaped virus

o bacteriophages - with their polygonal heads and
tubular tails
o vaccinia virus - one of the largest animal viruses
Bacteriophages
- Max Delbruck, Emory Ellis, and Salvador Luria decided
that bacteriophages) could lead to understanding of the
basic processes of life at a molecular level.
- Helped to found the field of molecular biology in the
1950s and 1960s
- Discovered by Felix d’Herelle
- Can correct defective genes; serve as vectors used for
vaccines; express proteins to kill tumor cells
Tumor Viruses and Nature of Cancer
- DNA tumor viruses led to discovery of viral oncogenes,
whose protein products (tumor antigens) interact with
cell signaling pathways to stimulate cell growth and
division
- RNA tumor viruses led to discovery of reverse
transcriptase, an enzyme that can make a DNA copy of an
RNA molecule, upsetting the one-way central dogma that
“DNA makes RNA makes proteins”
- Oncogenes - normal cellular regulatory genes whose
mutation and/or over-expression can lead to the
development of cancer; protein products are involved in
cellular signaling pathways
DETECTION AND TITRATION OF VIRUSES
- Suckling mice & embryonated chicken eggs are the usual
experimental host
- Intact bacteria diffract visible light and appear cloudy
- Bacteriophages lyse their host cell w/c causes loss in light
diffraction leading to clearing of the bacterial culture
- “Clear lysis” is an indicator of phage replication
1. Plaque assay
- count the number of infectious virus particles in a
suspension with a high degree of precision and
reproducibility; simple and quantitative
- results are in plaque-forming units (PFU) per mL of
suspension.
- Titer = # of plaques x dilution factor
1. Bacteria is spread on NA
2. Dilutions of a phage suspension are added
3. Phage binds to a bacterial cell and replicates and then
release its progeny phage particles
4. Progeny phage particles are then taken up by
neighboring cells and further replicated.
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5.
A circular area surrounding the original infected cell
are lysed and seen as a clear plaque against the
cloudy background of the uninfected cells
1.1 Plaque assay using cells cultured in vitro
- For animal viruses, cells of many different tissues are
induced to grow in a monolayer underneath liquid
media.
a. When cells growing in a monolayer are infected,
the progeny virus is released into the medium
and can travel to distant sites, infecting other
cells
b. To restrict diffusion of the progeny virus, the
infected cells are overlaid with melted NA
c. In gelled agar, released virus can infect only
nearby cells on the monolayer, forming a plaque
- Cell monolayers are too thin to diffract light, plaques
are visualized by staining. Dead/lysed cells do not
stain well thus seen as clear, unstained circular areas
on the background of the stained cell monolayer.
- Virus in individual plaques can be isolated by sampling
with a needle or Pasteur pipette, allowing the
“cloning” of progeny virus derived from a single virion
2. Hemagglutination
- RBCs - ideal substrate for assaying viruses because:
o Animal viruses bind to sialic acid residues or other
carbohydrates on cell surface proteins and lipids and
RBCs have carbohydrate-containing receptors
o Visible because of their color
o Isolated easily from the blood of a variety of animals,
are sturdy and can be stored for days or weeks
- Excess amount of viruses w/ receptor binding proteins
bind to the aliquot RBCs surface receptors form a
network called “agglutinated” red blood cells
o Agglutinated RBCs: light pink hemispherical shell in
the bottom of a tube
o Individual RBCs: slide to the bottom & form a
compact dark red pellet
i. Virus suspensions are diluted in twofold steps
ii. Dilutions are added to aliquots of RBCs in a buffer and
mixed
iii. Cells are allowed to settle & then examined
a. the highest dilution that will agglutinate the
aliquot of cells is considered to have 1
hemagglutinating unit (HAU) of virus
- 1 HAU = >105 virus particles
- Sensitive to pH, temperature, and buffer composition
- Some viruses will agglutinate only cells of a particular
mammalian or avian species

- Much less sensitive than plaque assays, but are rapid and
cheap
- Can also be used to detect antibodies because addition of
antibodies will inhibit hemagglutination
3. Electron Microscopy
- virus suspension is mixed with an electron-dense stain
(phosphotungstate or uranyl acetate)
- Negative Staining - stain forms electron-dense pools
around virus particles; virus particles exclude the stain
and therefore show up as light images against a dark
background
- Standard suspensions of tiny latex spheres are added to
the virus suspension to help establish absolute numbers
of virus particles per unit volume
Ratio of physical virus particles to infectious particles
- Measurement of the number of infectious virus particles
by plaque assays and of the number of physical virus
particles in the same virus suspension by electron
microscopy, allows calculation of the ratio of physical
particles to infectious particles
- Reasons for the low infectivity of virus preparations:
o Not all virus particles may be intact. Virus envelopes
are disrupted, rendering the particle non-infectious
or viral surface protein molecules can be denatured
unable to bind to the cell receptor. However, virions
contain many receptor-binding proteins so
denaturation of a few protein molecules ≠ loss of
infectivity
o Some virus particles may contain defective
genomes.
o “Empty” capsids that contain no viral genome can
be made in large numbers. Capsids can be formed in
the absence of the viral genome. Others incorporate
cellular DNA or RNA instead of the viral genome into
the capsid. However, this is prevented by virus
packaging signals
o Cells have antiviral defense mechanisms. Even
though a cell takes up an intact and potentially
infectious virion, it may not produce any progeny
virus
THE VIRUS REPLICATION CYCLE: AN OVERVIEW
- Multiplicity of infection (m.o.i.) - is the number of
infectious virus particles added per susceptible cell. An
m.o.i. of 10 to 100 plaque-forming units per cell is often
used in studies of bacterial/animal viruses
- Single-cycle Approach – simultaneously infecting cells to
study the steps in the growth cycle of viruses
- Latent Phase of infection – infecting virus has disappeared
& no new progeny virus has been made
Analysis of viral macromolecules reveals the detailed
pathways of virus replication
- Important events of virus replication cycle take place
during the latent period w/c are studied by extracting from
the infected cell the macromolecules that constitute viral
genomes, messenger RNAs, and proteins
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- Their interactions w/ cellular macromolecules and
organelles can be studied by:
o Radioactive compounds that are incorporated into viral
or cellular DNA, RNA, protein, lipid, or carbohydrates
o Antibodies against viral or cellular proteins can be used
to detect molecules w/ fluorescent dyes
o Molecular hybridization w/ oligonucleotide probes, or
PCR, is used to detect viral DNA/RNA molecules
o Macromolecules are separated by electrophoresis on
polyacrylamide or agarose gels
o Confocal microscopes allow visualization of horizontal
layers of thickness <1 μm w/in individual cells; its
focused laser light sources, show the localization of viral
macromolecules within the nucleus, or in association
w/ organelles
o Other localization methods use the higher-resolution
electron microscope coupled with specific staining (ex:
colloidal gold particles linked to antibodies)
STEPS IN THE VIRUS REPLICATION CYCLE
1. VIRIONS BIND TO RECEPTORS ON THE CELL SURFACE
- Virus-coded proteins bind to specific proteins,
carbohydrates, or lipids on the cell surface
- Structural surface proteins of viruses bind to
carbohydrate residues found in surface glycoproteins
and glycolipids
- Other viruses bind to cell surface proteins that are found
only on specific cell types (species-specific), limiting the
tropism of the virus to a particular tissue & organism
- Viruses first bind to a non-specific primary receptor
(carbohydrate), and then bind to a specific cell surface
protein that serves as a secondary receptor
2. THE VIRION (OR THE VIRAL GENOME) ENTERS THE CELL
- Bacteriophages and plant have to pass through a rigid cell
wall, as well as the outer cell membrane(s)
o Bacteriophages: specialized tails that drill holes in the
cell wall and membranes and serve as passage for the
DNA genome
o Plant viruses: damage to cell wall by abrasion or a
wound caused by an insect
o Enveloped animal viruses: fuse their lipid envelopes
w/ the cell’s plasma membrane, releasing its viral
capsid and genome into the cell
o Other animal viruses: taken up into the cytoplasm in
vesicles w/c are formed at the plasma membrane.

Vesicles then release the virion or genome into the
cytoplasm OR is transported to the nucleus
- Release happen by disintegration of vesicle membrane or
by fusion of the viral envelope w/ the vesicle membrane
- When the capsid is released into the cell, leads to
uncoating - disintegration of the capsid and release of the
genome
3. EARLY VIRAL GENES ARE EXPRESSED: THE BALTIMORE
CLASSIFICATION OF VIRUSES
- Once in cell, viral genome must direct expression of
“early” proteins to enable genome replication
- Synthesis of early viral proteins from viral messenger
RNAs depend on the chemical nature and strandedness
of the genome
- David Baltimore divided viruses into six (now seven)
groups based on the pathways leading to mRNA and
protein synthesis (Baltimore classification system), this
shows distinct kinds of RNA polymerases needed by
viruses, and indicates if these enzymes are available in
the cell or must be provided by the virus
o Viruses with single-stranded RNA genomes:
▪ Positive/plus strand: “sense” RNA strand, has
coding regions that can be directly translated into
viral proteins
▪ Negative/minus strand: “antisense” strand, w/c
cannot be translated because it does not contain
meaningful coding regions; only its
complementary copy codes for viral proteins.
Seven groups in the Baltimore classification system:
3.1 Viruses with positive-strand RNA genomes
- mRNA directly enters the cell in the form of the genome.
RNA binds to ribosomes and is translated into viral
proteins w/c will then direct replication of the viral
genome
3.2 Viruses with negative-strand RNA genomes
- The virus must supply an enzyme (RNA-dependent RNA
polymerase) capable of synthesizing a complementary
positive-strand copy of the genome, w/c will then serve
as viral mRNA. So, the first step in their replication cycle
is the synthesis of positive-sense viral mRNAs by the RNA-
dependent RNA polymerase, using the negative-sense
genome as template.
3.3 Viruses with double-stranded RNA genomes
- The cell does not produce an enzyme that can generate
an mRNA by transcribing one of the RNA strands in the
double-stranded genome
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- Theoretically, it is possible that the dsRNA could be
denatured w/in the cell, allowing the positive-strand RNA
to be translated but is unlikely and does not occur
- Virus provides a RNA-dependent RNA polymerase w/c
can recognize the dsRNA genome and transcribe its
negative strand into positive-strand mRNAs
3.4 Viruses with double-stranded DNA genomes
- Transcribes their genomes into mRNAs, using the cell-
provided DNA-dependent RNA polymerase
- Since cellular mRNAs are synthesized in the same way, all
cells contain such enzyme
- Some larger DNA viruses bring their own RNA polymerase
or make additional RNA polymerase later in the cycle to
transcribe a subset of their genes
3.5 Viruses with single-stranded DNA genomes
- Convert their ssDNA into a dsDNA, as there are no known
enzymes that will transcribe ssDNA directly into RNA
- Conversion to dsDNA is done by a cellular DNA
polymerase
- The dsDNA is then transcribed by a cellular RNA
polymerase to generate viral mRNAs
- Either of the complementary DNA strands can be
packaged in virions. Most viruses package only one, but
some viruses package both, making two virions identical
except for the polarity of their ssDNA genomes
- In all cases, the ssDNA is converted into a dsDNA before
being transcribed, so the polarity is of little importance
3.6 Retroviruses have a single-stranded, positive-sense RNA
genome
- Diff pathway different from other positive-strand RNA
viruses
- Packages a virus-coded reverse transcriptase (an RNA
dependent DNA polymerase) within the virion, and use
this to produce a dsDNA copy of their RNA genome after
cell entry
- Once the ds DNA is made, it is transcribed into viral
mRNAs by host cell RNA polymerase II
3.7 Hepadnaviruses
- Best representative of other categories in the system
- Package a circular DNA genome that is partly double-
stranded, with a single-stranded gap on one strand
- A special case of #5 category because like those DNA
viruses, their genomes are made fully double-stranded
by cellular DNA polymerases after entering the cell &
then transcribed into mRNAs by cellular RNA
polymerase
- But, like retroviruses and unlike other DNA viruses,
hepadnaviruses code for a reverse transcriptase used to
synthesize the partially ssDNA genome packaged in
virions
- This ssDNA is made by reverse transcribing a genome-
length RNA molecule that is one of the transcription
products of the fully dsDNA
4. EARLY VIRAL PROTEINS DIRECT REPLICATION OF
VIRAL GENOMES
- Once early viral proteins are made, they promote
replication of the viral genome
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- All RNA viruses (except retroviruses) must synthesize - Many enveloped viruses are assembled by budding at
their own RNA-dependent RNA polymerase to replicate the plasma membrane, and released w/ little or no
their genomes effect on the host cell while others bud at internal cell
- Early proteins of DNA viruses induce production of membranes or form their envelopes de novo; many of
cellular enzymes involved in the synthesis of DNA and of these viruses use cellular transport vesicles to reach the
its building blocks, deoxyribonucleoside triphosphates cell membrane and to exit the cell
w/c is achieved by interaction of early viral proteins w/
cellular signaling pathways that direct the cell to enter
the DNA synthesis (S) phase
- Small DNA viruses use host cell DNA polymerases to
replicate their genomes & larger DNA viruses code for
their own DNA polymerases
5. LATE MESSENGER RNAS ARE MADE FROM NEWLY
REPLICATED GENOMES
- Viruses synthesize a distinct set of “late” mRNAs after
genome replication has begun
- Many templates may be abundant for late mRNA
synthesis thus mRNAs can also be abundant
6. LATE VIRAL PROTEINS PACKAGE VIRAL GENOMES
AND ASSEMBLE VIRIONS
- Most abundant viral proteins made in an infected cell
are structural proteins used to package viral genomes
and assemble the capsid
- Simplest capsids: one protein (either closed shell or
helical tube) w/in which the viral genome is packaged
- Some viruses make scaffolding proteins - involved in
virion assembly but are subsequently discarded and not
part of the mature virion
- Enveloped viruses code for glycoproteins that are
inserted into lipid membranes and that direct formation
of the viral envelope by a process called budding
7. PROGENY VIRIONS ARE RELEASED FROM THE
HOST CELL
- Virions leave the cell to find and infect new host cells
- Viruses that infect unicellular organisms kills & and lyse
the host cell
- Many viruses code for specialized late proteins that lead
to cell death. But some bacteria and archaea viruses are
released by extrusion from the cell membrane & do not
kill their host cells
- For viruses that infect multicellular organisms, they can
spread from one cell to another w/in an organism, or
move from one organism to another; virions in these
two pathways are not necessarily identical
o Viruses of higher plants spread from cell to cell
through the plasmodesmata but insects that feed on
plant juices carry out spread from plant to plant
- Insect viruses spread as individual virions from cell to
cell w/in the host organism, but wrap one or more
virions in a specialized protein coat that is released
when the insect dies w/c can then be ingested by other
insects in where it starts another cycle
- Some viruses accumulate w/in host cells and are
released only on cell death
- Certain viral proteins function to retard cell death,
prolonging the period of virus replication, and other viral
proteins can kill the host cell, leading to virus release

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