The First I.M.

SECHENOV MOSCOW STATE MEDICAL UNIVERSITY

_______
MEDICAL FACULTY
Division for Foreign Students with Instruction Conducted in English
_______

Department of Microbiology, Virology and Immunology

GENERAL MICROBIOLOGY
THEORETICAL & LABORATORY MANUAL

GENERAL VIROLOGY

N. V. Khoroshko and E. V. Budanova

Edited by: Academician V.V. Zverev

Special English edited by E. V. Babchenko (Senior Teacher)
Computer Illustrations by: A.V. Budanov

M O S C O W - 2013

1
 SECTION 3. GENERAL VIROLOGY
The Objectives: To know the main principles of classification, taxonomy and
peculiarities of viruses; the principles and methods of their cultivation. To familiarize
oneself with the main epidemiological principles, pathogenesis and immunity of human
viral diseases. To study the methods for microbiological diagnosis of viral diseases. To
know immunobiological preparations for diagnostic purposes, therapy and
immunoprophylaxis of viral diseases.

Recommended reading:
1. Topical lectures
2. Textbook 1 (Mechanisms of Microbial Disease/edited by Moselio Schaechter, Gerald
Medoff, Barry I. Eisenstein. - 2nd ed.)
3. Textbook 2 (Medical Microbiology & Immunology. Examination and Board
Review./W. Levinson, E. Jawetz.-5th ed.)
4. Manual on Special Microbiology (Part III).

*GENERAL VIROLOGY
LESSON 11.
STRUCTURE and BIOLOGY of the VIRUSES. MICROBIOLOGICAL METHODS
for DIAGNOSIS of VIRAL DISEASES. CULTIVATION and INDICATION
(DETECTION) of VIRUSES
Prelab conference. Topics for discussion:
1. Viruses. Distinguishing characteristics.
2. Viruses. Structure and classification.
3. Types of viral replication. Stages of viral infection.
4. Principles of cultivation of the viruses. Virus isolation in animals, embryonated eggs
and in tissue cultures.
5. Detection (indication) of the viruses isolated in cell cultures, embryonated chicken eggs
and in susceptible animals.
6. Microbiological methods for the diagnosis of viral diseases.
7. Virus haemagglutination test. Mechanism, components, application.
8. Viruses. Identification in animals, embryonated chicken eggs and in tissue cultures.
9. Microbiological methods for the diagnosis of viral diseases. Rapid diagnosis.
10. Serological tests used to diagnose viral infections.
11. The antiviral immunity: host defense factors.

Viruses. Distinguishing Characteristics

The viruses are noncellular microbes which are composed solely of nucleic acid
(either DNA or RNA), surrounded by a protective protein shell, named the capsid. Some
of them may have an additional membrane layer (envelope) which surrounds the capsid.
This group of viruses is called enveloped (or complex) viruses.
Viruses have no cytoplasm, no internal organelles, and no cellular machinery to
synthesize their own proteins or to produce energy. Viruses are obligate intracellular
2
parasites - they require the biological machinery of a host cell to carry out the functions
needed for their reproduction and survival. The distinguishing characteristics of viruses are
listed in Table 13-1.
Table 11-1
The Distinguishing Features of Viruses
1. Viruses are obligate intracellular parasites
2. They are incapable of independent metabolism (unlike intracellular bacteria)
3. They have no cellular structure
4. They possess only a single type of nucleic acid, either DNA or RNA
5. They are filterable, i.e., they can pass through standard bacteriological membrane filters
6. They can be crystallized (virion) but remain infective;
7. They are characterized by a unique replicative cycle.

Viruses cause infectious diseases in humans, plants, bacteria, fungi and animals.
They are so small that they can be observed only with the electron microscope.

Classification of Viruses
Viruses are divided into major categories according to the range of hosts that can
be infected (they infect humans, animals, plants, fungi or prokaryotic cells). Further
classification is based primarily on the physical and chemical properties of the virus.
These properties are usually considered in the following order of importance:
1. Type of nucleic acid (RNA or DNA) (see Table 13-2).
2. Strategy of their genome (single or double strandedness of the nucleic acid).
3. Capsid morphology (shape and size; symmetry cubic, helical or combined; and
the number of capsomers).
4. Presence or absence of envelope (thus, sensitivity to inactivation by physical or
chemical agents, especially ether), etc.

The Types of Viral infection. Human Viruses Replication

Unlike cellular organisms, viruses cannot reproduce by fission. Because they have
no independent metabolism, they must use a host cell to perform all the functions
necessary for producing new infectious virions. Sometimes the virus will replicate in the
host cell followed by the release of progeny virions. This is called a productive infection.
Often the progeny viruses escape from the host cell by lysing it (naked, non-enveloped
viruses). Other viruses are less damaging, and they produce progeny without killing the
host cells (complex enveloped viruses). Sometimes, viruses produce no virions when they
infect host cells. Instead, they integrate their genome into a host chromosome and
replicate along with the cell's chromosomes. This is called virogeny or integrative type of
viral replication. Even these viruses, however, are capable of productive infection.
NOTE: In bacteriophages (i.e., viruses of bacteria) this phenomenon is called lysogeny.

When for some reasons a virus cannot produce progeny virions, this phenomenon
is called abortive infection. Viruses can infect cells that are not fully permissive and even
non-permissive (i.e., such cells are nor sensitive to virus). Often, in such cells, viruses

3
cannot multiply, because some essential step of the replication cycle cannot proceed. The
infection of permissive cells with the presence of antiviral agents is also abortive.
Productive infections of all viruses consist of the following main steps: (1)the virus
attachment to the target cell; (2)penetration and uncoating; (3)the biosynthesis of viral
components; (4)the assembly of viral components, and (5)the release of progeny virions.
The accomplishment of these tasks depends on the type of a virus and on the type of the
cell infected (human, animal, plant, fungal or bacterial).
Table 11-2
Classification of Medically important Viruses (according to the Nucleic Acid Type)
Family of Viruses Genus (group) Representative Human Disease

The RNA-genome viruses:

The RNA (ds)* - viruses
1. Reoviridae Orthoreovirus Respiratory tract infections
Coltivirus Colorado tick-borne fever
Orbivirus Hemorrhagic fever
Rotavirus Infants viral diarrhea
The RNA (ss**, positive-sensed) - viruses
1. Astroviridae Astrovirus Diarrhea
2. Caliciviridae Calicivirus Human gastroenteritis Norwalk
3. Coronaviridae Coronavirus Common cold, SARS
4. Flavivridae Flavivirus Yellow fever; encephalitis
Hepacivirus Hepatitis C (HCV)
5. Hepeviridae Hepevirus Hepatitis E (HEV)
6. Picornaviridae Enterovirus Poliomyelitis; common cold
Hepatovirus Hepatitis A (HAV)
Rhinovirus Common cold
Cardiovirus Encephalitis; myocarditis
7. Togaviridae Alphavirus Encephalomyelitis
Rubivirus Rubella
The RNA (ss**, negative-sensed) - viruses
1. Arenaviridae Arenavirus Lassa fever
2. Bornaviridae Bornavirus(?) Neurological diseases
3. Bunyaviridae Bunyavirus Hemorrhagic fevers
Phlebovirus Mosquitous fever
Hantavirus Hantaan fever
4. (unknown) Deltavirus Hepatitis D (HDV)
5. Filoviridae Filovirus Ebola disease, Marburg disease (acute
hemorrhagic fevers)
6. Orthomyxoviridae Influenzavirus Influenza A, B, and C
7. Paramyxoviridae Subfamily Paramyxovirinae:
Morbillivirus Measles
Respirovirus (formerly - Parainfluenza; respiratory tract
Paramyxovirus) infections
Rubulavirus Mumps
Subfamily Pneumovirinae:
Pneumovirus (RS-virus) Respiratory tract infections
8. Rhabdoviridae Vesiculovirus Vesicular stomatitis
Lyssavirus Rabies
The RNA (ss**, retroviruses with reverse transcriptase) - viruses
4
 1. Retroviridae Alpharetrovirus Leukosis; possible role in tumor
Betaretrovirus induction
Gammaretrovirus Role in other tumor induction
Deltaretrovirus T-lymphotropic human viruses (HTLV-
I; HTLV-II)
Epsilonretrovirus Skin sarcoma
Lentivirus AIDS (HIV-I; HIV-II)
Spumavirus Human foamy virus disease

The DNA-genome viruses:

The DNA (ds)* - viruses
1. Adenoviridae Mastadenovirus Respiratory tract infections
2. Herpetoviridae Subfamily HV-1 Herpes simplex; cold sores
Alphaherpesvirinae: HV-2 Genital herpes; meningoencephalitis
HV-3 Chickenpox; varicella-zoster
Subfamily HV-5 Cytomegalovirus (CMV) infection
Betaherpesvirinae: HV-6 Viral exanthema (roseola infantum,
HV-7 Duke disease, Fourth disease)
Subfamily HV-4 Infectious mononucleosis; Berkitt
Gammaherpesvirinae: lymphoma; nasopharyngeal carcinoma
HV-8 Kaposhi sarcoma (?)
3. Papillomaviridae Papillomavirus Warts; papillomatosis; cervical cancer
4. Polyomaviridae Polyomavirus Human encephalopathy
5. Poxviridae Orthopoxvirus Smallpox; cowpox (vaccinia)
The DNA (ss)* - viruses
1. Parvoviridae Erythrovirus Fifth disease
2. (unknown) Anellovirus Hepatitis TT (TTV and TTV-like
viruses)
The DNA (ds*) - viruses with reverse transcriptase
1. Hepadnaviridae Orthohepadnavirus Hepatitis B (HBV)
Non-classified viruses
Hepatitis G
*ds = the double-stranded nucleic acid;
**ss = the single-stranded nucleic acid
The Host-Virus Interaction
The final factor to be considered in the patterns of host-virus interactions is the
type of infection that results. The outcome of exposure to viruses depends on the agent
causing the infection (virus type), the routes and modes of its transmission, the infection
time and the immune response of the host.
The standard viral infection is acute and usually self-limited (e.g., respiratory tract and
gastrointestinal tract infections).
Damage due to acute infection may have other consequences, e.g., congenital
infections and malformations. Viruses such as Rubella Virus and cytomegalovirus
(CMV) are the well-known cause of intrauterine infections.
Many human viruses can cause persistent infections. A well-known example is
hepatitis B virus (HBV), which may persist in carriers in large amounts for years. The
results of this infection can be asymptomatic infection or symptomatic liver disease,
with cirrhosis and hepatocellular carcinoma as possible outcomes.
5
 A special example of persistent infection is latency. The herpes group of viruses
(Varicella-Zoster virus, Herpes Simplex Viruses) elicit a type of the virus - host
interaction in which the initial infection is followed by a variable period of time when
no demonstrable signs of infection are present. Subsequently, recurrent acute infection
occurs. In latent infection, the multiplication cycle of viruses is completely arrested at
some stage. In some cases, such arrest is permanent; but in others is not. The virus
multiplication cycle may be resumed after a period that may last from several weeks to
many years. The subacute spongioform encephalopathies (SSE) are slow viral
infections of the central nervous system.
The final example of this spectrum of viral illness is oncogenesis. Several human
viruses either cause, or are cofactors, in carcinogenesis. The Hepatitis B Virus (HBV) is
associated with hepatocellular carcinoma; Epstein-Barr virus (EBV) is a cofactor of
Burkitts lymphoma, etc.

The Viruses Culturing

Because viruses are obligate intracellular parasites, they cannot be cultivated on
inanimate media such as nutrient agar. They require a living cell system. Bacteriophages,
for example, can be propagated on susceptible bacterial cultures. Human and animal
viruses can be grown in laboratory animals, cell cultures or in fertile eggs (usually,
embryonated chicken eggs) under the strict controlled conditions. Research laboratories
cultivate viruses as the basis for detailed analyses of viral expression and replication.
Growth of virus in animals is still used for the primary isolation of certain viruses
and for studies of the pathogenesis of viral diseases and of viral oncogenesis. Diagnostic
laboratories attempt to recover viruses from clinical samples to establish disease
etiologies.
Viruses that infect human and animal cells can be detected in a similar fashion,
using susceptible animal or human cells as the indicator. Cell culture, the growth of animal
or human cells on artificial media, is the most common system for detection and
cultivation human viruses. These cells are grown in a monolayer, a uniform layer one cell
thick on the inner surface of a bottle or a test tube. The availability of cells grown in vitro
has facilitated the identification and cultivation of newly isolated viruses and the
characterization of previously known ones. There are three basic known types of cell
culture.
1) Primary cultures (trypsin-treated primary cell cultures) are made by dispersing
cells (usually with trypsin) from freshly removed host tissues. Since the cells of most
primary cultures remain viable for several generations, they may be repeatedly
subcultured (passaged). But, in general, they are unable to grow for more than a few
passages in culture, as secondary cultures.
2) Diploid cell lines are secondary cultures, which have undergone a change that
allows their limited culture (up to 50 passages) but which retain their normal
chromosome pattern. Human diploid cells are highly sensitive to numerous viruses and
are extensively used in virology.

6
3) Continuous cell lines are cultures capable of more prolonged, perhaps indefinite
growth that have been usually derived from malignant tissues. They invariably have
altered and irregular numbers of chromosomes.
The type of the cell culture used for viral cultivation depends on the sensitivity of
the cells to a particular virus.
Cell cultures are cultivated in glass or plastic bottles and tubes of various size and
shape, preferably disposable, with sterility strictly observed at all stages of cultivation.
Sterile nutrient media for cell cultures contain the whole range of amino acids, vitamins,
and growth factors. The Eagles medium, medium 199, lactic albumin hydrolysate are
commercial artificial culture media for cell lines which are most commonly used. Before
the medium is used, antibiotics are added to it in order to prevent bacterial contamination.
Using buffer systems, the pH of the medium is maintained at 7.2-7.6. An indicator
(commonly, phenol red) is also added to the culture media. It becomes orange-yellow in
acid medium or crimson in alkaline medium.
Although the cell culture is the preferred method of viral cultivation, some viruses
can only be grown in living experimental animals. Others are cultivated on embryonated
chicken eggs.
NOTE: Precautions must be taken by the laboratory personnel to prevent the
transmission of the pathogens. Specimens should be transported to the laboratory
in special doubling mailed containers with a secure lid to prevent leaking during
transport. The procedure of inoculation of living models and the further isolation
and identification of the pathogens must be performed only in Biohazard Cabinets
by highly qualified vaccinated personnel.

The Main Principles of Laboratory Diagnosis of Viral Infections

The laboratory diagnosis of a viral infection requires the detection (1) of a virus
itself, its genome or antigens in a patients specimens; or (2)an immune response to the
virus.
Specimen Collection and Transport. The crucial element in making the laboratory
diagnosis of a virus is the acquisition and processing of the appropriate clinical specimen.
Attempts at virus isolation, nucleic acid or antigen detection techniques, etc. will not be
successful unless the correct specimen is collected properly. In addition, the best
specimens are usually collected early in the illness when the virus is being excreted at
relatively high levels and has not yet been bound by antibody. Specimens obtained within
the first 72 h of an illness are more likely to yield a virus than specimens obtained later in
the course of disease.
Specimens should be obtained aseptically. The volume of the sample should be
sufficient to permit direct testing and virus isolation. Inoculation of tissue culture with
specimens within 2 to 4 hours is most suitable for virus isolation. Specimens should be
kept at 40C or on crushed ice until inoculated. Swabs of mucosal surfaces, skin scrapings,
and tissues should be placed in a transport medium. Tissue specimens from biopsies or an
autopsy material (postmortem examination) are suitable for attempted virus isolation if
fresh or frozen but not in formalin (=formaldehyde) or other fixative.
The typical sources (specimen) of viral culture include the nasopharyngeal swabs,
urine, stool, blood, cerebrospinal fluid, biopsy material and skin vesicles or exudate. In
7
addition, pleural fluid, pericardial fluid, bone marrow, and skin scrapings can be used for
viral culture.

The Diagnosis of viral diseases is based on a combination of clinical symptoms

and laboratory diagnostic tests. The particular method to be used depends on which virus
is suspected of being the etiologic agent.
The laboratory diagnosis of viral infections is based on either detecting a causative
agent in the patients specimens (rapid, virusoscopical or virusological diagnosis) or
demonstrating specific antibodies in the patients blood serum (serological diagnosis) -
see Table 13-3.
Rapid diagnostic techniques - immunoelectron microscopy, immunofluorescent (IF)
test, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), PCR,
etc. are increasingly gaining in importance.
Virus isolation is performed using cell cultures, chicken embryos, or experimental
animals. Isolated viruses are then indicated and identified with specific immunological
antigen-antibody tests. There are several disadvantages to viral cultivation. First, the
process is often slow, requiring days to weeks for identification. Therefore, virus
identification may not be available in time to affect patient care. It is also labor
intensive, very dangerous for staff, expensive, requires technical expertise and
experience.
Specific antiviral antibodies in the patients serum are studied over time, using paired
sera taken 10-14 days apart (in the acute and convalescent stage of the disease). An
increase in the serum antibody titer becomes diagnostically significant only when it is at
least a four-fold magnitude.
Table 11-3
Methods for Diagnosing Viral Infections
Method of Diagnostic laboratory tests. Purposes
diagnosis
Rapid diagnosis The main aim: to detect the viral genome or to identify the viral antigens in
the patients specimens directly.
Tests: Electron microscopy; immunoelectron microscopy; ELISA; RIA; IF-test;
PCR, etc.*
Virusoscopical The main aim: to detect the virus particles in a patients specimens or to
diagnosis reveal inclusion bodies in infected host cells**
Tests: Electron microscopy; brightfield microscopy
Virusological The main am: to isolate the virus from the patients specimens and identify it.
diagnosis Tests: Cultivation and isolation of the virus from the patients specimens in
living models, indication (clinical findings, pathomorphology, VHA-test, etc. in
animals; characteristic changes on the CAM, VHA-test with embryonic fluids
and tissues of chicken embryos; CPEs, hemadsorption, VHA-test, plaque
formation, color test in cell cultures) and identification of the isolated virus
with the neutralization test (NT), CFT, IF, RIA, ELISA, hemagglutination
inhibition test (HAI), etc. (see the text).
Serological The main am: Detection of specific anti-viral antibody titer in patients blood
diagnosis serum. The rise in specific anti-viral antibody level (at least four-fold
increasing) in paired patients sera, obtained at a 10 days interval should be
determined
8
 Tests: CFT; NT; HAI; RIA; ELISA, etc.
*NOTE: for abbreviations see below.
**NOTE: Smallpox virus virions (the so-called Pashens particles stained by the silvering stain
technique) can be observed under the bright field microscope.
By direct light microscopy, multinucleated giant cells, intranuclear inclusions, or both in a lesion
indicate an infection with either herpes simplex or herpes (varicella) zoster viruses.
The intracytoplasmic inclusion in nerve cells, the Babes-Negri body, is pathognomonic for rabies.

The Virus Demonstration (Indication) in Animals

Animals are used in virology for the following purposes:
(a) making diagnosis of viral infections (for virus isolation, accumulation, identification,
typing and titration);
(b) obtaining immune antiviral sera;
(c) modeling viral infections for the study of pathogenesis, immunity, pathomorphology,
etc.;
(d) testing of specific and nonspecific methods of the prophylaxis and treatment of viral
diseases.
Virus detection in infected animals can be made on the basis of specific clinical
findings in the course of a viral disease. Viral infection may result in the death of infected
animals, and specific and nonspecific pathologic changes in organs can be observed.
Viral infection may induce several morphological changes in organs: cell necrosis and cell
destruction, focal areas of swollen rounded cells, focal areas of fused cells, etc. The
supernatant of washed cell suspension from these organs may cause clumping of red blood
cells (virus hemagglutination test) due to the presence of viruses, which possess the
hemagglutinins.

The Virus Indication in Embryonated Chicken Eggs

Viruses are cultivated in 5- to 10-day-old chicken embryos. The material to be
tested is introduced with a sterile syringe onto the chorioallantoic membrane (CAM), into
the yolk sac, or into the amniotic or allantoic cavity. Before the embryo inoculation, the
eggshell is treated with iodine and alcohol solutions. The inoculation procedures of
chicken embryos are as follows:
A. For inoculation into the allantoic cavity, the tested material is introduced through the
lateral opening of the shell above the air sac 10 to 15 mm deep. The damaged site of the
shell is then coated with heated paraffin.
B. When the amniotic cavity is to be inoculated, the virus-containing specimen is injected
through the opening at the obtuse end of the egg. The needle should be directed toward the
embryonic body so as to ensure penetration of the virus into different organs and tissues of
the embryo. One should control the procedure of inoculating the amniotic cavity with the
ovoscope. Puncture sites are sealed with paraffin.
C. To infect the CAM, the eggshell is treated with iodine and alcohol solutions as usual,
punctured above the air cavity (sac) on the obtuse end of egg, and a 2x2 mm opening is
made laterally at the place of the greatest vascular ramification. Without destroying the
shell-underlying membrane, 1-2 drops of virus-containing material are placed onto the

9
CAM with a short thin needle. The damaged sites of the shell are coated with sterile
paraffin. The embryo is then put into an incubator horizontally.
The infected embryos are stored in an incubator at 35-37 oC for 48-72 h, depending
on the species of the virus suspected. The eggs are then cooled at 4 oC for 18 h for
maximum construction of the embryonic blood vessels and opened under sterile
conditions. The amniotic and allantoic fluids are aspirated with a syringe and the
membranes and the embryo are transferred into sterile Petri dishes.
Viral growth in an embryonated chicken egg may result in the death of the
embryo (e.g., encephalitis viruses), production of pocks or plaques on the
chorioallantoic membrane (e.g., herpes, smallpox, and vaccinia viruses), the development
of hemagglutinins in the embryonic fluids or tissues (e.g., influenza virus), or the
development of a infective virus. To detect the presence of hemagglutinin-positive viruses
in the allantoic and amniotic fluids of the embryo or in its tissues, the virus
hemagglutination test (VHA) should be performed (see below). Human or animal
erythrocytes are incubated with suspected virus-containing material from embryo and the
maximum dilution of virus-containing fluid which causes hemagglutination (clumping of
red blood cells) is then determined. A positive VHA-test results from the viral reproduction
in tested fluids and tissues.

The Indication of Viruses in the Cell Cultures

The cell tropism of the virus and the specific morphologic alterations provide
enough information for the preliminary identification of many viruses. Multiplication of a
virus in cell cultures can be monitored in a variety of ways:
1) Development of cytopathic effects (CPEs), i.e., morphologic changes in the cells. The
types of virus-induced cytopathic effects include cell lysis or necrosis, inclusion body
formation (intracytoplasmic or intranuclear), giant cell formation (syncytia), and
cytoplasmic vacuolization. Most CPEs can be readily observed in unfixed and
unstained cell cultures under low power of the light microscope. Several viruses
produce some obvious cytopathic effects in infected cells that are generally
characteristic of the viral group. A trained virologist can distinguish several types of
CPEs of different viruses.
In the course of viral multiplication within cells, virus-specific structures called
inclusion bodies can develop. They become far larger than the individual viral particle
and often have an affinity for acid dyes (e.g., eosin). They may be situated in the nucleus
(herpes virus), in the cytoplasm (pox virus), or in both (measles virus). In many viral
infections, the inclusion bodies are the site of development of the virions (the "viral
factories"). In some infections (pox viruses, reoviruses), the inclusion body consists of
masses of viral particles in the process of replication. In others (as in the intranuclear
inclusion body of the herpes virus), the virus multiplies earlier in the infection, and the
inclusion body appears to be a remnant of viral multiplication. Variations in the
appearance of inclusion material depend largely upon the tissue fixative used. The
presence of inclusion bodies may be of considerable diagnostic aid.
Several viruses, particularly paramyxoviruses and herpes viruses, cause cells to
fuse with one another, which results in the formation of giant cells (syncytia), masses of

10
cytoplasm bounded by one membrane and containing hundreds and even thousands of
nuclei.
Some viruses (lytic viruses, e.g., poliomyelitis virus) kill the cells, in which they
multiply, and produce plaques the areas of killed cells. This cytopathic effect can readily
be observed both macroscopically and microscopically.
2) Adsorption of erythrocytes onto infected cells, called hemadsorption, due to the
presence of virus-encoded hemagglutinin (influenza virus, parainfluenza virus) in
cellular membranes. In this test erythrocytes adhere to those cells in the cell culture
monolayer that are infected by a virus that causes hemagglutinin to be incorporated into
the plasma membrane. This reaction becomes positive before cytopathic changes are
visible and in some cases occurs in the absence of cytopathic effects.
NOTE: do not confuse hemadsorption and virus hemagglutination (VHA) tests!

3) The plaque formation test (plaque assay) is the most widely used assay for an
infectious virus. The plagues are the sites of virus-destroyed cells in the agar-coated
monolayer. To obtain the plaques, monolayers of host cells (cell culture) are inoculated
with suitable dilutions of a virus and after virus adsorption are overlaid with medium
containing a vital dye (neutral red) and agar to prevent the virus spreading throughout
the culture. After several days, the initially infected cells have produced a virus that
spreads only to surrounding cells, producing a small area of infection, or plaque.
Therefore, as the cytopathic effect of the virus becomes manifest, the cells lose their
vital stain and clear areas appear in the culture (plaques are recognized as light spots
on a pink-red cell monolayer). For example, enteroviruses, which grow rapidly, often
show detectable plaques after 24 to 48 hours, destroying the monolayer completely at 3
days.
4) Certain viruses contain a protein (surface antigen called hemagglutinin) that is able to
agglutinate red blood cells of humans or some animals. The hemagglutination assay
(virus hemagglutination test, VHA) can be used to detect the infected cells with these
types of viruses. The hemagglutination assay is an easy and rapid method for
quantitating hemagglutinin-producing viruses. A positive virus hemagglutination test
with culture fluid indicates the presence of virus in it and, therefore, in cell culture.
NOTE: The Virus Hemagglutination Test (VHA-test) is a non-immune test, because specific
antibodies are not employed.

5) The color test is a simple diagnostic test for virus detection in the infected cells of cell
cultures. The cell sheet is covered with highly enriched specific culture medium
containing amino acids, proteins, minerals, electrolytes, and vital dyes (usually, phenol
red) as an indicator (see above). The non-infected cells normally produce acid products
due to the normal metabolic processes, and the color of the indicator becomes yellow in
acid pH. The virus-infected cells fail to produce acid products, and the indicator color in
culture medium cannot be changed. Therefore, if the cell culture is initially infected
with the virus, no changes in color are observed. This fact verifies the presence of some
(unknown) virus in the sample tested.

11
 According to the nature of the material to be tested and the procedures utilized, the
methods for diagnosing viral infections may be categorized into rapid, virusological, and
serological (see Table 13-3).
The choice of laboratory diagnostic methods is primarily determined by the nature
of the disease and the casual virus, being dependent in each case on the disease stage and
the possibilities of a laboratory. It is important that specimens for the rapid diagnosis and
virus isolation be obtained from the patient as early as possible. The materials to be
examined include vesicular and pustule contents, scraped epithelium, cerebrospinal fluid,
feces, smears, and washings off the upper respiratory tract, etc. Specimens must be
obtained aseptically so as to avoid their microbial contamination. Specimens are stored
moist and cold and transported in containers with dry ice.

The Methods Used for Diagnosing of Viral Infections

Where a single virus is largely responsible for a clinical syndrome, rapid
diagnosis is now possible by detection of viral antigen in specimen, either by
immunofluorescence using a fluorescein-labelled specific antibody, or by enzyme
immunoassay (ELISA), and may virtually replace tissue culture.
Direct examination of the sample by electron microscopy (EM) or immune
electron microscopy (IEM) may demonstrate virus particles (virusoscopical
examination). The light microscopy may also allow seeing the viral cytopathic effects
(CPE) in the damaged tissues or other specimens directly.
IEM demonstrates specific binding of antibodies to virus antigens. Clusters of viral
particles and antibodies are visualized by electron microscopy. The advantage of this
method is a simultaneous concentration of the virus and its identification by means of a
specific antiserum.
Other methods are also available for more rapid and specific identification
techniques, including monoclonal antibodies specific to HSV-1 and -2 antigens and DNA-
typing methods (PCR, etc). The PCR can be used to amplify a specific DNA sequence to
produce millions of copies within a few hours. The specificity of PCR is determined by the
careful choice of primers.
Viruses may be identifiable by their cytopathic effect (CPE) in cell culture and
their morphology in electron microscopic preparations, but confirmation is often made by
virusological examination, i.e. virus isolation, detecting, and identification in the
"sensitive systems". For viral identification immune reactions are usually used (there is
some antigenic variation within many viral groups so that subgroups can be defined).
Serology is useful for diagnosing many infections, but generally it is less common
in babies and infants. For antibody testing, a 15 to 20 ml blood sample is aseptically
obtained with the syringe without adding anticoagulants or preservatives, since they can
affect the results of the serological study.
An increase in the antibody titers is determined by examining paired (acute and
convalescent) sera. Acute and convalescent sera taken at least 10 to 14 days apart are
needed for the detection of at least a four-fold rise in the antibody titer. This method can
be used for the retrospective diagnosis of primary infection and for epidemiological
surveys.

12
 PRACTICAL WORK and PRACTICAL TASKS
1. Study microscopically the demonstration preparations. Draw the pictures of what you
have observed in your protocol notebook.
Primary Cell Culture (Chicken Fibroblasts) obtained from chicken embryos
(Romanowsky-Giemsa Stain)
Continuous Cell Line HeLa obtained from the human cervical cancer cells
(Romanowsky-Giemsa Stain)

Cytopathic Effect (CPE) produced in the monolayer of a cell culture by viruses.

NOTE: You can observe cell rounding progressing to complete cell destruction, because
Poliovirus causes cell damage

Hemadsorption test for Influenza virus detection.

NOTE: erythrocytes adhere to those cells in the monolayer that are infected by a virus that
cause a hemagglutinin to be incorporated into the plasma membrane. You can see
red blood cells adsorbed to the cell culture surface.

The Cytopathic Effect of Smallpox virus. Intracytoplasmic inclusion bodies

(Guarnieris spots) in epithelial cells.
NOTE: You can see virus-specific round eosinophil structures (red color) in the cytoplasm
beside the nucleus. These structures are formed by virus. They are virus fabrics.

The Cytopathic Effect of Adenovirus. Intranuclear inclusion bodies.

NOTE: The dark-blue or purple structures resembling the aster flower are located in the
center of the nuclei.

2. Observe yourself the picture of Smallpox virus virions (Pashens particles) in your
classroom. One can see small black or dark brown dots (smallpox virions) against
yellow background (Morozovs Silvering Stain).

3. Familiarize yourself with the chicken embryo structure and different methods of its
inoculation listed in the diagram in your classroom.

4. Study the scheme and classification of methods for viral diseases diagnosing in your
classroom and in the manual (see above).

5. Perform the virus hemagglutination test (VHA-test) to detect the virus in allantoic fluid
of chicken embryo. The sterile allantoic fluid obtained from the previously infected
chicken embryo with virus-containing material will be supplied. While performing the
test follow Table 11-4:
The procedure of the VHA-test:
A. Prepare two-fold dilutions of allantoic fluid with suspected virus in isotonic
solution.
B. Add 0.5 ml of 1% suspension of erythrocytes.
13
 C. Mix the erythrocytes with the isotonic solution in the control well ("Erythrocytes
control").
D. Shake the inoculated plate carefully and allow it to stand at room temperature for
30 min.
E. Read the results of the test after incubation. (The results are assessed by the
presence of hemagglutination. Agglutinated red cells form a granular film (mat) at
the bottom of the well. The non-agglutinated cells roll into a small circular red
button. It is important to check the "control well" for the absence of spontaneous
agglutination or hemolysis).

6. Draw and fill in Table 11-4 in your protocol notebook. Make a conclusion.
Table 11-4
The Scheme of VHA-test for Virus Indication in the Chicken Embryo Allantoic Fluid
Ingredients Dilutions of tested virus-containing material Erythrocytes
1:2 1:4 1:8 1:16 1:32 1:64 control
Isotonic solution, ml 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Allantoic fluid (virus- 0.5 --
containing material), ml pour out
1% suspension of human 0.5 0.5 0.5 0.5 0.5 0.5 0.5
erythrocytes, ml
Shake carefully. Incubate at room temperature for 30 min.
Results:
Conclusion: the presence of virus in allantoic fluid tested is detected/not detected.
The virus titer is ______.

14