This document discusses several methods for identifying viruses, including cell culture-based identification using cytopathic effects, hemagglutination assays, nucleic acid amplification tests like PCR, enzyme immunoassays, immunofluorescence assays, molecular methods, viral plaque assays, and quantal assays like TCID50 that determine infectious viral titers. It provides details on how each method works and what types of information can be obtained about viruses using these techniques.

1. Methods: Identification of Viruses
Vivek Kumar
Department of Bioscienes
Swami Rama Himalayan University
Jolly Grant, Dehradun

2. Background
• The first methods for detection of bacterial infections were available
around 1880.
• After staining, bacterial pathogens were recognized in the light
microscope because of their size and could be cultivated in culture
media.
• Viruses evaded this approach, as they are significantly smaller, and as
obligate parasites are not able to multiply in cell culture media.
• Some viral infections could be associated with specific cellular
changes.
• Eg. certain depositions in the infected tissue - Negri inclusion bodies
in nerve cells during rabies.

3. Introduction
• In 1954, J. F. Enders was first to propose the adoption of a
cell culture-based system to classify animal viruses into four
distinct categories consisting
• viruses that caused cell degeneration,
• those that caused formation of inclusion bodies
• and cell degeneration,
• those that caused the formation of multinucleated cells
(syncytia),
• and those that caused no cytopathic effect or
cytopathogenic effect (CPE).

4. • Since then, cell cultures have been successfully tested
routinely for in vitro isolation of viruses.
• Presumptive identification of virus types can be made by
observing morphological changes produced in host cells
(CPE), caused by cytopathogenic viruses.
• Common damage to the host cells includes rounding of cells,
a change in texture (granular or hyaline—glassy), and
formation of a syncytium (fusion of infected cells).

5. • The extent of visible damage to cells caused by viral
infection varies with strain,
• type of host cells,
• and multiplicity of infection (MOI);
• Consequently CPEs becomes evident after as little as
two days to as much as four weeks.

6. • Although most laboratories combine traditional and
advanced laboratory approaches to optimize viral
diagnostics.
• Virus isolation from cell cultures still remains a primary
method, particularly when viable and nonviable virus need
to be differentiated.
• If a viable virus has to be isolated, and when infection is
not characteristic of any single virus.

7. Detection of a Virus
• Regardless of the method of cultivation, once a virus has been
introduced into a whole host organism, embryo, or tissue-culture cell,
a sample can be prepared from the infected host, embryo, or cell line
for further analysis under a brightfield, electron, or fluorescent
microscope.
• Cytopathic effects (CPEs) are distinct observable cell abnormalities
due to viral infection.
• CPEs can include loss of adherence to the surface of the container,
changes in cell shape from flat to round, shrinkage of the nucleus,
vacuoles in the cytoplasm, fusion of cytoplasmic membranes and the
formation of multinucleated syncytia, inclusion bodies in the nucleus
or cytoplasm, and complete cell lysis.

8. Paramyxovirus
Syncytium and faint
basophilic cytoplasmic
inclusion bodies
Poxyvirus
Pink eosinophilic
cytoplasmic inclusion
bodies (arrows) and cell
swelling

9. Herpesvirus
Cytoplasmic stranding (arrows)
and nuclear inclusion bodies
(dashed arrow)
Adenovirus
Cell enlargement, rounding,
and distinctive grape-like
clusters

10. Hemagglutination Assay
• A serological assay is used to detect the presence of certain
types of viruses in patient serum.
• Serum is the straw-colored liquid fraction of blood plasma
from which clotting factors have been removed.
• Serum can be used in a direct assay called a
hemagglutination assay to detect specific types of viruses in
the patient’s sample.
• Hemagglutination is the agglutination (clumping) together
of erythrocytes (red blood cells).

11. • Many viruses produce surface proteins or spikes
called hemagglutinins that can bind to receptors on
the membranes of erythrocytes and cause the cells to
agglutinate.
• Hemagglutination is observable without using the
microscope, but this method does not always
differentiate between infectious and noninfectious
viral particles, since both can agglutinate erythrocytes.

12. • To identify a specific pathogenic virus using
hemagglutination, we must use an indirect approach.
• Proteins called antibodies, generated by the patient’s
immune system to fight a specific virus, can be used to bind
to components such as hemagglutinins that are uniquely
associated with specific types of viruses.
• The binding of the antibodies with the hemagglutinins found
on the virus subsequently prevent erythrocytes from directly
interacting with the virus.

13. • So when erythrocytes are added to the antibody-coated
viruses, there is no appearance of agglutination;
agglutination has been inhibited.
• We call these types of indirect assays for virus-specific
antibodies hemagglutination inhibition (HAI) assays.
• HAI can be used to detect the presence of antibodies specific
to many types of viruses that may be causing or have caused
an infection in a patient even months or years after infection.

16. Nucleic Acid Amplification Test
• Nucleic acid amplification tests (NAAT) are used in molecular
biology to detect unique nucleic acid sequences of viruses in
patient samples.
• Polymerase chain reaction (PCR) is an NAAT used to detect
the presence of viral DNA in a patient’s tissue or body fluid
sample.
• PCR is a technique that amplifies (i.e., synthesizes many
copies) of a viral DNA segment of interest.
• Using PCR, short nucleotide sequences called primers bind
to specific sequences of viral DNA, enabling identification of
the virus.

17. • Reverse transcriptase-PCR (RT-PCR) is an NAAT used to
detect the presence of RNA viruses.
• RT-PCR differs from PCR in that the enzyme reverse
transcriptase (RT) is used to make a cDNA from the small
amount of viral RNA in the specimen.
• The cDNA can then be amplified by PCR.
• Both PCR and RT-PCR are used to detect and confirm the
presence of the viral nucleic acid in patient specimens.

18. Enzyme Immunoassay
• Enzyme immunoassays (EIAs) rely on the ability of antibodies to
detect and attach to specific biomolecules called antigens.
• The detecting antibody attaches to the target antigen with a high
degree of specificity in what might be a complex mixture of
biomolecules.
• Also included in this type of assay is a colorless enzyme attached to
the detecting antibody.
• The enzyme acts as a tag on the detecting antibody and can interact
with a colorless substrate, leading to the production of a colored end
product.

19. • EIAs often rely on layers of antibodies to capture and react
with antigens, all of which are attached to a membrane filter.
• EIAs for viral antigens are often used as preliminary
screening tests.
• If the results are positive, further confirmation will require
tests with even greater sensitivity, such as a western blot or
an NAAT.

23. Immunofluorescence (IF) Assay
• Immunofluorescence (IF) technique is widely used for rapid
detection of virus infections by identifying virus antigens in
clinical specimens.
• IF staining is usually considered very rapid (about 1 to 2 hr)
and overall gives a sensitive and specific viral identification.
• Unfortunately, IF technique may not able to confirm the
identity of all virus strains, for instance viruses of the
“enterovirus” group.

24. • Since most monoclonal antibodies (MAbs) for
enteroviral identification have been shown to lack
sensitivity, while cross-reactivity with rhinoviruses is
extremely common.
• In contrast, IF has been successfully used for better
management of influenza virus infection and
surveillance of influenza virus activity.

25. Molecular Methods
• The development of molecular methods for the direct identification
of a specific viral genome from the clinical sample is one of the
greatest achievements of the 21st century.
• Clearly nucleic acid amplification techniques including PCR, nucleic
acid sequence-based amplification (NASBA) and Lawrence Livermore
Microbial Detection Array (LMDA) are proven technology leaders for
rapid detection and molecular identification for most known human
viruses.
• As such, PCR allows the in vitro amplification of a specific region of
DNA sequences by a factor of 106 and thus considered as an
extremely sensitive detection technique.

26. Viral Plaque Assay
• Viral plaque assay is one of the most widely used methods in
virology to purify a clonal population of virus or to determine
viral titer.
• Renato Dulbecco was first to develop this procedure in 1952
to calculate the infectivity of bacteriophages stocks.
• A plaque usually is the result of infection of the cell by a
single virion on the host cell monolayer.
• As such, to perform a plaque assay, susceptible host cell
monolayer is infected with 10-fold serial dilutions of virus.

27. Viral Plaque Assay Determination

28. • After an incubation period, monolayers are then covered
with a semi-solid nutrient medium, (most commonly agar) to
prevent the virus from spreading from host cells to other
nearby uninfected cells.
• As a result, each infectious particle produces a small circular
zone in the monolayer called a plaque, while uninfected cells
surround the plaques.
• Eventually the plaque becomes large enough to be visible to
the naked eye or with light microscopy.

29. Quantal Assays - TCID50, LD50, EID50
• Although plaque assay is an extremely useful method for
determining viral titers, however there are several virus
types which do not form plaques in culture.
• Alternative procedures such as
• TCID50, (Tissue culture infectious dose)
• LD50, (Lethal dose)
• EID50 (Embryo Infectious Dose )

30. • Assays are being used
to determine the
infectious titer of any
such virus types,
which can cause
cytopathic effects
(CPE) in tissue culture
over a reasonable
period of 5 to 20 days
while cells in culture
remain viable.

31. • However, since not all virus types cause CPE in tissue
culture, and therefore cell line and virus must be
carefully matched in order to see a cytopathic effect.
• TCID50 is the tissue culture infectious dose defined as
that dilution of virus required to infect 50% of the cell
monolayers.
• The Infectivity Titre is established by carrying out a
titration.

32. • The unit of measurement of infectivity of avirulent
Newcastle disease virus is the 50 percent Embryo
Infectious Dose or EID50.
• One EID50 unit is the amount of virus that will infect
50 percent of inoculated eggs.
• *Lethal Dose, 50%" or median lethal dose, it is the
amount of the substance required to kill 50% of the
test population.

33. Immunofluorescence Foci Assay (IFA)
• The IFA (also known as fluorescent foci assay FFA) is a rapid
method of virus titration.
• This technique allows the quantification of virus in cell lines,
which does not support plaque formation or do not exhibit
detectable CPE (to perform TCID50).
• For example, hepatitis A virus (HAV) does not produce visible
CPE without multiple passages in cell culture.

34. • In contrast to other infectivity-
based assays, IFA is not dependent
on the ability of induction of cell
death, but it utilizes an antibody-
based staining methods to detect
virally infected cells as early as two
days.
• Overall IFA is considered more
sensitive and faster than traditional
plaque assays or TCID50, however
it sometimes could be quite
expensive, due to the cost of
antibodies used.

35. X-Ray Crystallography
• A method that is used to find out the arrangement of atoms within a
crystal is known as X-ray crystallography.
• A beam of X-rays scatters into many directions when it strikes a
crystal.
• According to the angles and intensities of these scattered beams, a
three dimensional picture of the density of electrons within a crystal
is produced from a crystallography.
• The mean positions of the atoms in the crystal, their chemical
bonding, their disorder and other information can be determined
from the electron density.

37. Summary
• A general method for obtaining and culturing cells.
• Primary cultures contain cells obtained from an animal and they have
a limited lifespan.
• A basic method for virus purification using centrifugation.
• Methods to count infectious virus particles (plaque assays and
endpoint dilution).
• The basic features of common techniques used to detect viruses and
antibodies.