MULUNGUSHI UNIVERSITY

SCHOOL OF NATURAL AND APPLIED SCIENCES

NAME: LUNGU TAMARA

STUDENT ID: 202108493

COURSE: VIROLOGY

COURSE CODE: BIO 342

LECTURER: MR DERRICK BANDA

DUE DATE: 24 April 2024

ASSIGNMENT NO#: TWO (2)

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 INTRODUCTION
Viruses are the smallest infectious agents containing only one kind of nucleic acid (RNA or
DNA) as their genome, the nucleic acid is encased in a protein shell, which may be
surrounded by a lipid-containing membrane. The entire infectious unit is termed a virion.

Viruses are everywhere. Viruses are the most abundant biological entities on our planet.
They are found in all of our surrounding: the air, the ocean, the soil, and in rivers, streams,
and ponds. They are present wherever life occurs, and it is thought that every living thing has
a virus that infect it. Some viruses cause diseases (e.g. Ebola, Avian flu and corona virus).
Viruses can cause serious epidemics and pandemics. Some viruses are useful (e.g. phage
typing of bacteria, sources of enzymes, gene vector for protein production and gene vector for
treatment of genetic diseases). Genetically modified strains of viruses, such as herpes simplex
virus and vaccinia virus, are being investigated for the treatment of cancer.

As obligate intracellular parasites, viruses require a cell to replicate. The cells must express
appropriate receptors and other proteins required by the virus. Cultured cells are often used to
study basic steps in virus replication. Viruses can be purified away from cellular proteins and
organelles using centrifugation techniques. Most viruses are cannot be seen using standard
light microscopes, but are often imaged using electron microscopy. Methods that combine
image collection and computationally demanding image processing can provide incredible
details about virus architecture. Another common way to visualize viruses is to use
fluorescence tags or dyes. In this write up, we will discuss the techniques used to visualize
and enumerate viral particles, measure biological activities of viruses and assays for
estimation and manipulation of viruses in details.

Visualization and enumeration of viral particles

Visualization and enumeration of viral particles involve techniques aimed at observing and
qualifying viruses, while measuring biological activity focuses on assessing the functional
aspects of viruses. Assays for virus estimation and manipulation refer to the methods used to
determine virus concentration and perform controlled experiments on viruses.

Visualization techniques enable scientist to observe viral particles using various tools such
as electron microscopes, fluorescence microscopy and cryo-electron microscopy. These
methods provide detailed images of viruses, allowing researchers to study their structure,
morphology and interactions with host cells.

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Enumeration refers to the process of counting viral particles. This can be done using methods
like plaque assays, where viral particles are cultured with host cells to form visible plaques or
using flow cytometry, which uses lasers and fluorescent dyes to count individual viral
particles in a sample.

The common methods used to visualize and enumerate viral particles include the following:

ELECTRON MICROSCOPY

This method provides high-resolution images of viral particles using a transmission electron
microscope (TEM) or a scanning electron microscope. The development of TEM in 1930s
allowed individual viruses to be seen for the first time. EM uses an electron beam of light,
and the beam is focused by electromagnets, rather than by glass lenses. EM has great
resolving power and can magnify objects by up to – 10,000,000 times. Disadvantages of EM
are that fixed and processed samples are dead, and biological samples ae heavily damaged by
the electron beam even as they are been imaged. However, the simple structures and
crystalline nature of viruses were first revealed by EM and it remains the common tool of
virologists.

CRYO- ELECTRON MICROSCOPY

This technique involves rapidly freezing samples to preserve their native structure. Cryo-EM
can capture high-resolution images of viral particles in their natural state, revealing details
about viral morphology and assembly. Cryo- EM uses ultralow temperatures to preserve
biological samples. Cryo-fixation of samples produces a glass like material rather than
crystalline ice. Cryo-EM allows the observation of specimens that have not been chemically
stained or fixed thus showing them in their native aqueous environment. Low temperatures
provide better images with less specimen damage, but cryo-EM micrographs have less
contrast than stained images. Cryo-EM images are more complex than negative stains
because the entire particle is seen in the image. Images generated by cryo-EM are often
enhanced by collecting and computationally averaging multiple images. This allows use of
lower and less damaging doses of electrons. Cryo-EM, while a powerful technique, requires
expensive and specialized equipment and highly trained personnel.

FLUORESCENCE MICROSCOPY

Fluorescence microscopy techniques such as confocal microscopy or super- resolution

microscopy can be used to visualize fluorescently labelled viral particles viral proteins within

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cells. These techniques provide detailed spatial information about viral infection and
replication processes. Fluorescence is the emission of light by a substance that has absorbed
light. The basic function of fluorescence microscope is to irradiate a specimen with high
energy excitation light and detect the much weaker emitted fluorescence. The microscope is
designed so that only the emission light reaches the eye or detector, allowing fluorescent
structures to be seen with high contrast against a dark back ground. This is achieved by use of
the filters of specific wavelength. The advantage of fluorescence microscopy is that a single
fluorescein molecule can emit as many as 300,000 photons before it is destroyed thus making
it theoretically possible to detect a single molecule in a field of view.

MOST-PROBABLE-NUMBER ASSAYS (MPNs)

MPNs are usually done to determine the concentration of lytic viruses that infect hosts
which cannot be grown on solid medium but which can be grown in liquid. This is
particularly a problem with delicate phytoplankton. The assay is simple but labour intensive.
It is also very sensitive, a detection limit of 0.01virusesml-1is not unreasonable. However,
MPNs lack accuracy and precision of plaque assay. Traditionally, they have been done using
a series of 10-fold dilutions with 3-10 replicates at each dilution. The replicates in which no
growth or growth followed by cell lysis occurs are assumed to contain at least one infective
virus. By comparing the number of replicates that contain viruses to an MPN table, the
number of infective unit infective units can be estimated. With the wide availability of
computers and other technologies such as microplate readers, it is not necessary to be
constrained be a specific number of replicates or order-of-magnitude dilution regimes. The
first step is to determine the detection limit desired. For example, to achieve a detection limit
of 1 virus ml-1, several ml of water needs to be screened. In lieu of information of viral titer
it is necessary to do a broad dilution series. A series of 10-fold dilutions starting with 1 ml of
sample should span the range of concentrations typically found, although larger volumes can
be screened if desired. Changes in the phytoplankton biomass are monitored by measuring in
vivo chlorophyll fluorescence, which allows hundreds of cultures to be non-destructively
monitored on a daily basis. The culture tubes specified fit directly into a fluorometer. The
MPN, standard error and confidence interval are calculated using a program written in the
basis.

PLAQUE ASSAYS:

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 Plaque assays are routinely used to estimate titres of viruses that cause lysis of bacteria,
cyanobacteria and algae that can be grown on solid media. The basis of the method is that
each infective unit will form a clearing (plaque) on a lawn of host cells. Lawns are typically
made by mixing host cells and viruses in molten agar and quickly and evenly pouring this
mix over a bottom layer made with a higher percentage of agar. The number of plaque-
forming units (PFUs) in a given volume of water can be estimated using this method. As a
number of aquatic bacteria will form plaques on lawns of bacteria and algae (e.g. Adams
1959, Stewart and Daft 1977, Sakata et al. 1983). Filtration or pretreatment of water sample
with chloroform are the methods traditionally used to distinguish viral and bacterial
pathogens. However, as some algal viruses may be 0.4 micrometer or large in diameter, and
some are chloroform sensitive, these approaches may be selective.

Advantages of plaque assay method includes: It is relatively sensitive with a practical

detection of about 5 PFUs ml. However, in naturally seawater samples viruses infecting
specific host are often present even at lower concentrations. Accurate results can be achieved
with modest amount of effort. Plaque counts can even be done by image analysis. Pathogens
can be easily purified by cloning individual plaques picked from a plate. Only infective virus
are enumerated.

Disadvantages includes that: the host must be culturable on solid medium. Different
pathogens cannot easily be distinguished. Natural bacteria can interfere with lawn-formation,
particularly of slower-growing hosts. Filtration may remove some viruses.

MEASURING BIOLOGICL ACTIVITIES OF VIRUSES

Measuring of biological activities of viruses involves several techniques and methods that
allow for the understanding of their behaviour, replication mechanism and effects on host
organisms. These methods are used to quantify the functional properties of viruses. This can
include measuring the following activities:

➢ Ability of the virus to infect cells

➢ Replication
➢ Ability of the virus to cause disease
➢ Interaction of the virus with the host immune system
➢ Response of viruses to antiviral drugs

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The goal of these measurements is to understand how viruses function and cause disease, and
to develop strategies for controlling and treating viral infections.

CYTOPATHIC EFFECT (CPE) ASSAY:

Is a method used to evaluate the biological activity of viruses by observing the changes they
induce in the infected host cells. Here’s how it works:

The assay begins by preparing a monolayer of susceptible cells in a culture dish or plate.
These cells are often derived from tissues relevant to the virus being studied and are cultured
under appropriate conditions to maintain their viability and growth. The cultured cells are
then exposed to the virus interest at a specific multiplicity of infection (MOI), which refers to
the ratio of infectious virus particles to target cells. The virus is allowed to infect the cells for
a predetermined period, typically ranging from hours to days, depending on the virus and the
desired endpoint of the assays.

Following viral infection, the cells are periodically examined under a light microscope
to observe any visible changes induced by the virus. These changes collectively known as
cytopathic effects (CPE), can include cell rounding, enlargement, detachment from the
culture surface (cytolysis), formation of syncytia (multinucleated giant cells), inclusion
bodies, or cell death.

The extent and nature of CPE are assessed quantitatively by visual observation and
quantitatively by scoring the percentage of affected cells or the severity of the observed

ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISA):

Measures the presence and or concentration of antibodies generated by an infection or

viral antigens in serological samples. As antibodies persist in the blood stream even for many
years after infection, a positive test is not an indicator of active infection but determines
patient immunity resulting from exposure to the virus, reinfection, or reactivation state.
ELISA are invaluable for studying the epidemiology of disease as they study present and past
disease prevalence in different population.

ELISAs are also used in the competitive way, measuring the concentration of an
antigen by detection of signal interference. The sample antigen competes with a reference
antigen for binding to a specific labelled antibody. The reference antigen for binding to a
specific labelled antibody. The reference antigen is pre-coated on a multiple-well plate. The

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sample is pre-incubated with labelled antibody and added to the wells. Depending on the
amount of antigen in the sample, the free antibodies will be available to bind the reference
antigen. This means the more antigen there is in the sample, the less reference antigen will be
detected and the weaker the signal. This method can also be adapted to detect antibodies in
samples.

HEAMAGGLUTININ ASSAY AND HAEMAGGLUTINATION INHIBITION ASSAY

(HIA):

Haemagglutinin is a protein on the envelope of arboviruses, influenza and parainfluenza

virus subtypes which binds red blood cells (RBCs) to form a lattice of agglutinated cells. In
the hemagglutination assay, serial dilutions of the virus are added to the red blood cells.
Samples are then screened for agglutinated cells. A variation of this virus assay used to
measure specific antibodies in the serum. If antibodies are present in the serum at a sufficient
concentration, they will interfere with viral attachment to the red blood cells, resulting in
inhibition of hemagglutination. Both virus assays provide a relative virus quantitation.

POLYMERASE CHAIN REACTION (PCR)

PCR was inverted by K.B. Mullis and F.A. Mullis in the year 1985 and since then
commonly used for nucleic acid amplification. PCR enables replication of millions of
identical DNA copies from a little quantity of the pathogen genome in the clinical sample.

In theory, firstly the target DNA was extracted, followed by heating and denaturation.
Specific oligonucleotide primers are annealed to the DNA at the lower temperature after that
the DNA polymerase enzyme replicated the template strand known as the extension phase.
This cycle frequently repeated by 30-40 times, producing millions of identical copies.

Since the discovery of the PCR it has been used to identify viral infections with sensitivity of
more than 77% and a clinical specificity of more than 85%. The PCR is used to diagnose
viruses, bacteria and fungi. PCR cannot be used to directly to detect RNA viruses and so a
Reverse Transcriptional PCR of the genome into the DNA is required prior to performing the
amplification reaction.

ASSAYS FOR VIRUS ESTIMATION AND MANIPULATION

Assays for virus estimation and manipulation refer to the techniques and methods used to
measure the amount of virus sample or to modify the properties of the virus in some way.

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Virus estimation assays determine the concentration of viruses in a sample, these assays
include quantitative PCR which amplifies and qualifies viral nucleic acids or plaques assays
mentioned earlier, which count infectious viral particles.

Virus manipulation assays involve experiments aimed at studying virus behaviour, replication
and response to treatments. This can include techniques like reverse genetics, where specific
mutations are introduced into viral genomes to study their effects on viral properties.

PLAQUE REDUCTION ASSAY

This assay measures the ability of antiviral agents to reduce plaque formation. Its used to
determine the effectiveness of antiviral drugs.

The advantage of this assay is that is directly assesses antiviral activity. Its quantitative and
widely used in antiviral drug development.

The disadvantage is that it requires culturing cells and may not reflect the vivo situation
accurately.

PLAQUE ASSAY

This technique, virus particles are serially diluted and added to a monolayer of susceptible
cells. After incubation, the cells are stained to visualize plaques, indicating virus presence.

The advantage of this technique is, quantitative allows for determination of viral titers.

The disadvantage is that it requires time (days to weeks), labour -intensive and limited to
viruses that produce visible plaques.

REAL-TIME PCR (RT-PCR)

This technique detects and quantifies viral RNA/DNA using specific primers and fluorescent
probes. Amplifications is monitored in real-time.

The advantage is that its highly sensitive, quantitative and gives rapid results.

The disadvantage is that it requires specialized equipment and expertise, expensive reagents.

REVERSE GENETICS

This is the technique that allows for the generation of recombinant viruses by manipulating
viral genomes. This can involve introducing mutations, deletions or substitutions.

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The technique enables precise control over virus modifications, and is useful for studying
viral pathogenesis.

The technique is technically challenging, time consuming and requires specific expertise
resources.

The method is highly specific as it involves the targeted modifications to the viral genome.

SITE-DIRECTED MUTAGENESIS

This method introduces specific mutations at desired locations in the viral genome using
PCR- based methods or gene editing techniques like CRISPR-Cas.

The technique allows for the studying of the impact of specific mutations on viral properties.

On the other hand, the technique requires molecular biology expertise and validation of
mutants.

VIRUS TITRATION ASSAYS

This method measures the infectious virus particles in a sample, often through endpoint
dilution or TCID50 (median tissue culture infectious dose) assays.

The advantages include, quantitative assessment of virus infectivity. However, its time
consuming, requires cell culture and subjective interpretation in some cases.

The method is specific to infectious viral particles in the samples.

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REFERENCE

➢ Flint., Enquist L. Racaniello V. Skalka A. principles of virology: molecular biology,

pathogenesis, and control of animal viruses. 2nd ed.
➢ Stephenson B. Kepler’s physical astronomy. Springer:
➢ Virology journal ; https:// virologyj. Biomedcenter by P Tripathi 2023
➢ BMG Labtech: https:// www.bmglabtech.com
➢ National Institutes of Health; Methods to study viruses-PMC by S Payne.2017

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