Lecture 2.

Cultivation, Purification and

Enumeration of Viruses
Burst concept and its implication in virus replication
•According to Delbrück, one bacterial cell usually makes hundreds of progeny
virus particles.
•The yield from one cell is one viral generation; it is called the burst because
viruses literally burst from the infected cell.
•Under carefully controlled laboratory conditions, most cells make, on
average, about the same number of bacteriophages per cell.
•A burst occurs for viruses that kill the cell after infection, namely, the
cytopathic viruses.
•However, some viruses do not kill their host cells, and virus particles are
produced as long as the cell is alive. Examples include filamentous
bacteriophages, some retroviruses, and hepatitis viruses.
•A number of parameters limit the number of particles produced per cell,
such as metabolic resources, the number of sites for replication in the cell,
the regulation of release of virus particles, and host defenses.
•Larger cells (e.g., eukaryotic cells) produce more virus particles per cell:
yields of 1,000 to 10,000 virions per eukaryotic cell.
The average yield of infectious virus per cell
(the burst size) can be calculated from the formula,
 The One-Step Growth Cycle
•One-step growth analysis can be used to study the single-cell life
cycle of viruses from the work on bacteriophages by Emory Ellis and
Delbrück.
•Virus particles were added to a culture of rapidly growing E. coli
cells. These particles adsorbed quickly to the cells.
•The infected culture was then diluted, preventing further
adsorption of unbound particles.
•This simple dilution step is the key to the experiment: it reduces
further binding of virus to cells and effectively synchronizes the
infection.
•Samples of the diluted culture were then taken every few minutes
and analyzed for the number of infectious bacteriophages.
•When the results were plotted, several key observations emerged.
•The results were not resemble the growth curves of bacteria or
cultured cells.
 The One-Step Growth Cycle
•After a short lag, bacterial cell growth becomes exponential (i.e.,
each progeny cell is capable of dividing) and follows a straight
line (Fig. 2.18A).
•Exponential growth continues until the nutrients in the medium are
exhausted.
•In contrast, numbers of new viruses do not increase in a linear
fashion from the start of the infection (Fig. 2.18B, left).
•There is an initial lag, followed by a rapid increase in virus
production, which then plateaus.
•This single cycle of virus reproduction produces the “burst” of virus
progeny.
•If the experiment is repeated, so that only a few cells are initially
infected, the graph looks different (Fig. 2.18B, right).
•Instead of a single cycle, there is a stepwise increase in numbers of
new viruses with time.
•Each step represents one cycle of virus infection.
 One step growth cycle

Fig: Growth of a bacteriophage in E. coli under conditions when all cells are infected (left) and when
only a few cells are infected (right).

•Synchronous infection‐ key to one-

step growth cycle
•To achieve this, we need to infect all
the cells -­‐ but how do we know?
The time between infection by (or induction of) a
bacteriophage, or other virus, and the appearance
of mature virus within the cell is called the eclipse
phase
 The One-Step Growth Cycle
•The results of these experiments defined two new terms in
virology: the eclipse period, the phase in which infectivity is lost
when virions are disassembled after penetrating cells; and

•the latent period , the time it takes to replicate, assemble, and

release new virus particles before lysis, 20 to 25 min for E. coli
bacteriophages.
•Synchronous infection, the key to the one-step growth cycle, is
usually accomplished by infecting cells with a sufficient number
of virus particles to ensure that most of the cells are infected
rapidly.
 Application of One-Step Growth Cycle
•One-step growth curve analysis can provide quantitative
information about different virus-host systems.

•It is frequently employed to study mutant viruses to

determine what parts of the infectious cycle are affected by
a particular genetic lesion.

•It is also valuable for studying the multiplication of a new

virus or viral replication in a new virus-host cell combination.
 Multiplicity of infection (MOI)
∙ The MOI is the average number of viruses/cell
∙ Number of infectious particles added per cell

∙ The number of infectious particles each cell

Receives is not same
∙ Hence classic one-step growth experiments
usually use an MOI of 10 (10 viruses/cell)
∙ Add 107 virions to 106 cells

-­‐ MOI of 10
 MOI

∙ Infection depends on the random collision of

virions and cells
∙ When susceptible cells are mixed with virus,
some
cells are uninfected, some receive one, two, three
or more particles
∙ The distribution of virus particles per cell is best
described by the Poisson distribution
 P(k) = e-mmk/k!

P(k): fraction of cells infected by k virus particles

m: multiplicity of infection (moi)
uninfected cells: P(0) = e‐m
cells receiving 1 particle: P(1) = me-m
cells multiply infected: P(>1) = 1- e‐m(m+1)
[obtained by subtracting from 1 {the sum of all
probabilities for any value of k} the probabilities P(0) and
P(1)]
Examples: The fraction of cells receiving 0, 1, and 1 virus particle in a culture of 10 -6
cells infected with an MOI of 10 can be determined as follows.

If 106 cells are infected at MOI of 10:

45 cells are uninfected
450 cells receive 1 particle
the rest receive >1 particle

If 106 cells are infected at MOI of 1:

37% of the cells are uninfected
37% of the cells receive 1 particle
26% receive >1 particle

If 106 cells are infected at MOI of .001:

99.9% of the cells are uninfected
00.099% of the cells receive 1 particle (990)
00.0001% receive >1 particle

Page 50-BOX
 Transformation Assay
•The transformation assay is useful for determining the titers of
some retroviruses that do not form plaques.

•For example, when Rous sarcoma virus transforms chicken

embryo cells, the cells lose their contact inhibition (the property
that governs whether cultured cells grow as a single monolayer
and become heaped up on one another.

•The transformed cells form small piles, or foci, that can be

distinguished easily from the rest of the monolayer (Fig. 2.12).

•Infectivity is expressed in focus-forming units per milliliter.

 A

Figure 2.12 Transformation assay.

Chicken cells transformed by two
different strains of Rous sarcoma
virus are shown.
Loss of contact inhibition causes
cells to pile up rather than grow
as a monolayer. One focus is seen
in panel A,
and three foci are seen in panel B
at the same magnification.

B
 Physical measurements of virus
particles

∙ Hemagglutination

https://www.youtube.com/watch?v=HMg48a2W_6o
Hemagglutination - measurement of virus particles
GK Hirst, 1941 First rapid, quantitative assay for eukaryotic viruses

Sialic acid receptors on the surface of red blood

cells (RBCs) bind to the hemagglutinin
glycoprotein found on the surface of influenza
virus (and several other viruses) and create a
network, or lattice structure, of interconnected
RBC’s and virus particles. The agglutinated lattice
maintains the RBC’s in a suspended distribution,
typically viewed as a diffuse reddish solution.
 Hemagglutination

+
Influenza RBC
Virus
Suspension

Settling Pattern
Hemagglutination
 Hemagglutination Inhibition
assay

+ +
HA Virus Strain RBC
specific
antibody Suspension

Antibodies against viral proteins with hemagglutination activity can block

the ability of virus to bind red blood cells. The goal is to characterize the
concentration of antibodies in the antiserum or other samples containing No RBC settled
antibodies
Hemagglutination (HA) assay:
Recognized method for virus quantitation
Dilutio
n
Neat
2
4
8
16
32
64
128
Ref: Methods Used in Virology

Virus purification

Many virus purification procedures involve centrifugation;

partial purification can be achieved by differential
centrifugation and a higher degree of purity can be
achieved by some form of density gradient centrifugation.

Centrifugation is a technique used for the

separation of particles from a solution according to
their size, shape, density, viscosity of the medium
and rotor speed.
 Differential centrifugation
Differential centrifugation involves alternating cycles of low-speed
centrifugation, after which most of the virus is still in the supernatant, and
high-speed centrifugation, after which the virus is in the pellet
Figure shows the partial purification of virions by differential centrifugation. A crude
preparation of virus containing host debris is subjected to low-speed/short-time
centrifugation (e.g. 10000 g/20 minutes) followed by high-speed/longtime centrifugation
(e.g. 100000 g/2 hours). This cycle can be repeated to obtain a higher degree of purity. The
final pellet containing partly purified virus is resuspended in a small volume of fluid.
 Density gradient centrifugation
Density gradient centrifugation involves centrifuging particles
(such as virions) or molecules (such as nucleic acids) in a
solution of increasing concentration, and therefore densify.
The solutes used have high solubility: sucrose and cesium
chloride.
There are two major categories of density gradient centrifugation:
1) Rate zonal and 2) equilibrium (isopycnic) centrifugation.
⮚In rate zonal centrifugation a particle moves through the
gradient at a rate determined by its sedimentation coefficient, a
value that depends principally on its size.
⮚Homogeneous particles, such as identical virions, should move
as a sharp band that can be harvested after the band has moved
part-way through the gradient.
 Density gradient centrifugation
In equilibrium centrifugation a concentration of solute is
selected to ensure that the density at the bottom of the gradient
is greater than that of the particles/molecules to be purified.
A particle/molecule suspended in the gradient moves to a point
where the gradient density is the same as its own density.
This technique enables the determination of the buoyant densities
of nucleic acids and of virions.
Buoyant densities of virions determined in gradients of caesium
chloride are used as criteria in the characterization of viruses.

Figure shows the Purification of virions by density gradient centrifugation. A partly purified preparation
of virus is further purified in a density gradient. Rate zonal centrifugation involves layering the
preparation on top of a pre-formed gradient. Equilibrium centrifugation can often be done starting
with a suspension of the impure virus in a solution of the gradient material; the gradient is formed
during centrifugation.
 Questions
Lecture 2.1: The life cycle of Virus
1. Define the terms used in virology: susceptible, resistant,
permissive.
2. What is the cell culture for viral growth?
3. How can you identify viral growth in cell culture?
4. What is plaque assay? How can you quantify viruses by this
assay?
5. What is particle to PFU ration? Why this is high for some
viruses?
6. What is multiplicity of infection? How can you use this for
synchronized growth of virus?
7. Draw the infectious cycle of a virus and show the steps.
8. What would you expect to see at different time intervals
during viral replication cycle?
9. What are the assays you can use to physical measure of
viruses? What are the principles of these assays?