«Viruses morphology and ultrastructure.

Viruses cultivation in chicken

embryo and laboratory animals organism»

STRUCTURE AND CONTENT OF THE TOPIC

Actuality.

Viruses are the etiologic cause of a large group of infectious diseases that are
characterized by various clinical manifestations and course, are highly contagious and
can cause epidemics and pandemics. Today Virology develops intensively as one of
biomedical sciences, although recently it has a narrow branch of science.
The importance of Virology is stipulanted for the leading role of viruses in
human infectious diseases. Their impotance increases due to decreasing morbidity of
bacterial, fungal and protozoal infections with limited ways of specific chemotherapy.
Secondly, models of viruses as the most simple organized form of life uses for solving
many fundamental questions of biology and molecular genetics (e.g., the doctrine of
nirones, splicing, oncogenes). In modern science one of the most important
contributions of Virology is the discovery of reverse transcriptase, the basis of genetic
engineering.
The knowledge of the topic is necessary for understanding the pathogenesis of
viral infections are studied at clinical departments. In physician’s practice this is an
important moment for the clinical and morphological differentiation of viral and other
infections. At the practical lesson the students are able to master the methods of
cultivation in cell culture, cultination with virus-containing cell material. This is
necessary for formation of the idea about methods of diagnose viral infections.

General information
Virology is the study of viruses and virus-like agents:
 their structure,
 classification and evolution,
 their ways to infect and exploit host cells for virus reproduction,
 their interaction with host organism physiology and immunity,
 the diseases they cause,
 the techniques to isolate and culture them, and their use in research and therapy.
Virology is considered to be a subfield of microbiology or of medicine.

Virology also studies subviral particles that are infectious entities notably smaller
and simpler than viruses:
  v i r o i d s - naked circular RNA molecules infecting plants,
 s a t e l l i t e s - nucleic acid molecules with or without a capsid that require a
helper virus for infection and reproduction), and
 p r i o n s - proteins that can exist in a pathological conformation that induces
other prion molecules to assume that same conformation.

A virus is a biological agent that reproduces inside the cells of living hosts. Over
2,000 species of viruses have been discovered.
Virus structure
A virus particle, known as a virion, consists of genes (DNA or RNA) which are
surrounded by a protective coat of protein called a capsid. Virion is a single, fully
functional virus particle outside its host cell.

A coat of protein around genes = a capsid

Association of viral capsid proteins + a viral nucleic acid = a nucleocapsid
A coat of lipid around a nucleocapsid = an envelope

The capsid is made of many smaller, identical protein molecules which are called
capsomers. Some viruses are surrounded by a bubble of lipid (fat) called an envelope.
The envelopes typically are derived from portions of the host cell membranes
(phospholipids and proteins), but include some viral glycoproteins. Functionally, viral
envelopes are used to help viruses enter host cells. Glycoproteins on the surface of the
envelope serve to identify and bind to receptor sites on the host's membrane. The
viral envelope then fuses with the host's membrane, allowing the capsid and viral
genome to enter and infect the host.

A virion structure
Classes of enveloped viruses that contain human pathogens:
DNA viruses RNA viruses Retroviruses
Herpesviruses Flavivirus Retroviruses
Poxviruses Togavirus Hepadnavirus
Hepadnaviruses Coronavirus
Hepatitis D
Orthomyxovirus
Paramyxovirus
Rhabdovirus
Bunyavirus
Filovirus

Arrangement of the capsomers

The arrangement of the capsomers can either be icosahedral (20-sided), helical
or more complex.
Helical
These viruses are composed of a single type of capsomer stacked around a central axis
to form a helical structure, which may have a central cavity, or hollow tube.

This arrangement results in rod-shaped or filamentous virions which can be short and
highly rigid, or long and very flexible. The genetic material, in general, single-stranded
RNA, but ssDNA in some cases, is bound into the protein helix by interactions between
the negatively charged nucleic acid and positive charges on the protein. Overall, the
length of a helical capsid is related to the length of the nucleic acid contained
within it and the diameter is dependent on the size and arrangement of
capsomers.
The well-studied tobacco mosaic virus is an example of a helical virus:

Structure of tobacoo mosaic virus

 Icosahedral
Most animal viruses are icosahedral or near-spherical with icosahedral symmetry
A regular icosahedron is the optimum way of forming a closed shell from identical
sub-units. The minimum number of identical capsomers required is twelve, each
composed of five identical sub-units. Many viruses, such as rotavirus, have more than
twelve capsomers and appear spherical but they retain this symmetry. Capsomers at
the apices are surrounded by five other capsomers and are called pentons. Capsomers
on the triangular faces are surrounded by six others and are called hexons. Hexons are
essentially flat and pentons, which form the 12 vertices, are curved. The same protein
may act as the subunit of both the pentamers and hexamers or they may be composed
of different proteins.

Electron micrograph of icosahedral adenovirus

Complex
These viruses possess a capsid that is neither purely helical nor purely icosahedral, and
that may possess extra structures such as protein tails or a complex outer wall.
Some bacteriophages, such as Enterobacteria phage T4, have a complex
structure consisting of an icosahedral head bound to a helical tail, which may have a
hexagonal base plate with protruding protein tail fibres. This tail structure acts like a
molecular syringe, attaching to the bacterial host and then injecting the viral genome
into the cell
 .
Enterobacteria phage
Classification of viruses
Currently there are two main schemes used for the classification of viruses:
 the ICTV system and
 Baltimore classification system, which places viruses into one of seven groups.

ICTV classification

The International Committee on Taxonomy of

Viruses began to devise and implement rules for the
naming and classification of viruses early in the
1970s, an effort that continues to the present day.
The ICTV is the only body charged by the
International Union of Microbiological Societies
(IUMS) with the task of developing, refining, and
maintaining a universal virus taxonomy. The system
shares many features with the classification system
of cellular organisms, such as taxon structure. Viral
classification starts at the level of order and follows
as thus, with the taxon suffixes given in italics:

- Order (-virales)
- Family (-viridae)
- Subfamily (-virinae)
- Genus (-virus)
- Species
 #. Family Genus Examples
Subfamily
Double-stranded DNA, enveloped virions
1. Poxviridae
Chordopoxvirinae Orthopoxvirus Smallpox (variola), vaccinia
Parapoxviruses Orf
Molluscipoxvirus Molluseum contagiosum viruses
2. Herpesviridae
Alphaherpesvirinae Simplex virus Herpes simplex virus 1 and 2
Varicellovirus Varicella-zoster virus
Betaherpesvirinae Cytomegalovirus Human cytomegalovirus
Roseolovirus Human herpesvirus 6
Gammaherpesvirina Lymphocryptovirus Epstein-Barr virus
e

Double-stranded DNA, non-enveloped virions

1. Adenoviridae Mastadenovirus Human adenoviruses
2. Papovaviridae Papillomavirus Human papillomavirus
Polyomavirus Human BK and JC virus

Partial double-stranded partial single-stranded DNA, non-enveloped

virions
1. Hepadnaviridae Orthohepadnavirus Human hepatitis B virus

Single-stranded DNA, non-enveloped virions

1. Parvoviridae
Chordoparvovirinae Erythrovirus Parvovirus B19

Double-stranded RNA, non-enveloped virions

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1. Reoviridae Reovirus Reovirus types 1, 2, 3
Rotavirus Human rotaviruses (A and B)
Orbivirus Orungovirus, Kemerovo virus
Coltivirus Colorado tick fever virus

Single-stranded RNA, enveloped virions without DNA step in replication

cycle
(a) Positive-sense genome
1. Togaviridae Alphavirus Sindbis virus (arbovirus group A)
Rubivirus Rubellavirus
2. Flaviviridae Flavivirus Yellow fever virus (arbovirus
group B)
Unnamed Hepatitis C virus
3. Coronaviridae Coronavirus Human coronavirus
(b) Negative-sense, non-segmented genome
1. Paramyxoviridae
Paramyxovirinae Paramyxovirus Parainfluenzaviruses 1 and 3
Morbillivirus Measles virus
Rubulavirus Mumps virus,
parainfluenzaviruses 2 and 4
Pneumovirinae Pneumovirus Respiratory syncytial virus
2. Rhabdoviridae Lyssavirus Rabies virus
Vesiculovirus Vesicular stomatitis virus
3. Filoviridae Filovirus Marburg and Ebola viruses
(с) Negative-sense, segmented genome
1. Influenzavirus A, B Influenza A and B viruses
Orthomyxoviridae
Influenzavirus C Influenza C virus
2. Bunyaviridae Bunyavirus Bunyamwera virus, La Crosse
virus, California encephalitis
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 virus
Phlebovirus Sandfly fever virus, Sicilian virus,
Rift Valley fever virus, Uukuniemi
virus
Nairovirus Crimean-Congo haemorrhagic
fever virus
Hantavirus Hantaan virus, Seoul virus, Sin
Nombre virus, Puumala virus
3. Arenaviridae Arenavirus Lymphocytic choriomeningitis
virus, Lassa virus, Venezuelan
haemorrhagic fever virus

Single-stranded RNA, enveloped virions with DNA in the replication cycle

1. Retroviridae HTLV-BLV group Human T-cell
leukemia/lymphotropic virus
(HTLV-1 and HTLV-2)
Spumavirus Human foamy virus
Lentivirus Human immunodeficiency
viruses (HIV-1 and HIV-2)

Single-stranded RNA, positive-sense, non-enveloped virions

1. Picornaviridae Enterovirus Poliovirus 1-3, coxsackieviruses
A 1-22, A 24, B 1-6, echoviruses
1-7, 9, 11-27, 29-33,
enteroviruses 68-71
Hepatovirus Hepatitis A virus
Rhinovirus Rhinoviruses 1-100
2. Caliciviridae Calicivirus Norwalk agent, hepatitis E virus?

Baltimore classification
The Baltimore classification, developed by David Baltimore, is a virus
classification system that groups viruses into families, depending on their type of
genome

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  DNA,
 RNA,
 single-stranded (ss),
 double-stranded (ds), etc. and their method of replication.

In virology, the genome of an RNA virus can be said to be either positive-sense, also
known as a "plus-strand", or negative-sense, also known as a "minus-strand". In most
cases, the terms sense and strand are used interchangeably, making such terms as
positive-strand equivalent to positive-sense, and plus-strand equivalent to plus-sense.
Whether a virus genome is positive-sense or negative-sense can be used as a basis for
classifying viruses.
Groups:
Group/Class I: Double-stranded DNA viruses – dsDNA viruses - usually must
enter the host nucleus before it is able to replicate and require
 host cell polymerases to replicate the viral genome and,
 are highly dependent on the cell cycle.
Proper infection and production of progeny requires that the cell be in replication,
as it is during replication that the cell's polymerases are active.

Class II: Single-stranded DNA viruses - ssDNA viruses. Eukaryote-infecting

viruses replicate mostly within the nucleus - usually via a rolling circle mechanism,
forming double-stranded DNA intermediate in the process.

Class III: Double-stranded RNA viruses - dsRNA viruses. As with most RNA
viruses, this class replicates in the cytoplasm, not having to use the host replication
polymerases to as much a degree as DNA viruses.

Class IV: Single-stranded RNA viruses (positive-sense) - (+)ssRNA viruses. The

positive-sense RNA viruses and indeed all RNA defined as positive-sense can be
directly accessed by host ribosomes to immediately form proteins.
Positive-sense (5' to 3') viral RNA signifies that a particular viral RNA sequence
may be directly translated into the desired viral proteins. Therefore, in positive-
sense RNA viruses, the viral RNA genome can be considered viral mRNA, and can be
immediately translated by the host cell. Unlike negative-sense RNA, positive-sense
RNA is of the same sense as mRNA. Some viruses (e.g., Coronaviridae) have
positive-sense genomes that can act as mRNA and be used directly to synthesize
proteins without the help of a complementary RNA intermediate. Because of this,
these viruses do not need to have an RNA polymerase packaged into the virion.
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 Class V: Single-stranded RNA viruses (negative-sense) - (-)ssRNA viruses. The
negative-sense RNA viruses and indeed all genes defined as negative-sense cannot be
directly accessed by host ribosomes to immediately form proteins. Instead, they
must be transcribed by viral polymerases into a "readable" form, which is the
positive-sense reciprocal.

Negative-sense (3' to 5') viral RNA is complementary (opposite) to the viral

mRNA and thus must be converted to positive-sense RNA by an RNA polymerase
prior to translation. Negative-sense RNA (like DNA) has a nucleotide sequence
complementary to the mRNA that it encodes. Like DNA, this RNA cannot be
translated into protein directly. Instead, it must first be transcribed into a
positive-sense RNA that acts as an mRNA. Some viruses (Influenza, for example)
have negative-sense genomes and so must carry an RNA polymerase inside the
virion.

Class VI: Positive-sense single-stranded RNA viruses that replicate through a

DNA intermediate – ss RNA-RT viruses. A well-studied family of this class of viruses
include the retroviruses. One defining feature is the use of reverse transcriptase to
convert the positive-sense RNA into DNA. Instead of using the RNA for templates
of proteins, they use DNA to create the templates, which is spliced into the host
genome using integrase. Replication can then commence with the help of the host
cell's polymerases. A well-studied example includes HIV.

Class VII: Double-stranded DNA viruses that replicate through a single-

stranded RNA intermediate - dsDNA-RT viruses. This small group of viruses,
exemplified by the Hepatitis B virus (which is in the Hepadnaviridae family), have a
double-stranded, gapped genome that is subsequently filled in to form a covalently
closed circle (ccc DNA) that serves as a template for production of viral mRNAs and a
subgenomic RNA. The pregenome RNA serves as template for the viral reverse
transcriptase and for production of the DNA genome.

Viral life-cycle
When a virus infects a cell, the virus forces it to make thousands more viruses.
It does this by making the cell copy the virus's DNA or RNA, making viral proteins,
which all assemble to form new virus particles.

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 There are six basic, overlapping stages in the life cycle of viruses in living cells:
1. Attachment is the binding of the virus to specific molecules on the
surface of the cell. This specificity restricts the virus to a very limited type of cell. For
example, the human immunodeficiency virus (HIV) infects only human T cells,
because its surface protein, gp120, can only react with CD4 and other molecules on
the T cell's surface. Plant viruses can only attach to plant cells and cannot infect
animals. This mechanism has evolved to favour those viruses that only infect cells in
which they are capable of reproducing.

2. Penetration follows attachment; viruses penetrate the host cell by

endocytosis or by fusion with the cell.

3. Uncoating happens inside the cell when the viral capsid is removed and
destroyed by viral enzymes or host enzymes, thereby exposing the viral nucleic acid.

4. Replication of virus particles is the stage where a cell uses viral

messenger RNA in its protein synthesis systems to produce viral proteins. The RNA
or DNA synthesis abilities of the cell produce the virus's DNA or RNA.

5. Assembly takes place in the cell when the newly created viral proteins
and nucleic acid combine to form hundreds of new virus particles.

6. Release occurs when the new viruses escape or are released from the
cell.
 Most viruses achieve this by making the cells burst, a process called lysis.
 Other viruses such as HIV are released more gently by a process called
budding.

Entry and uncoating of different viruses

1) Bacteriophage entry
- The processes of
penetration and
uncoating are
simultaneous for all
bacteriophages.
- Only the nucleic
acid genome enters
the cell
Bacteriophage entry

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2) Enveloped viruses
Entry by direct fusion Viropexis (receptor-mediated endocytosis)

Viropexis

Entry by direct fusion

3) Naked capsid viruses enter a cell by voripexis

Virus Cultivation- Purposes and Methods

 Viruses are obligate intracellular parasites so they depend on host for their
survival.
 They cannot be grown in non-living culture media or on agar plates alone, they
must require living cells to support their replication.
 The primary purposes of virus cultivation is:
 To isolate and identify viruses in clinical samples. Demonstration of virus in
appropriate clinical specimens by culture establishes diagnosis of viral
diseases.
 To do research on viral structure, replication, genetics and effects on host
cell.
 To prepare viruses for vaccine production.
 Isolation of virus is always considered as a gold standard for establishing
viral etiology of a disease.
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  Most of the viruses can be cultivated in
 Experimental animals
 Embryonated eggs or
 Tissue culture.

Specimens for Culture of Virus

Collection of appropriate clinical specimens depends on type of the viral disease. For
example, cerebrospinal fluid (CSF) is the specimen of choice for diagnosis of viral
infections of the central nervous system (CNS) caused by arboviruses, picornavirus,
or rabies virus.
Animal inoculation

 Mouse is most frequently used for isolation of viruses by animal inoculation.

 In addition, rabbits, hamsters, newborn or suckling rodents are also used.
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  Experimental animals are rarely used for cultivation of viruses but play an
essential role in study of pathogenesis of viral infections and that of viral
oncogenesis.
 Intracerebral, subcutaneous, intraperitoneal, or intranasal routes are various
routes of inoculation.
 After inoculation, the animals are observed for signs of disease or death.
 The infected animals are then sacrificed and infected tissues are examined for
the presence of viruses by various tests, and also for inclusion bodies in
infected tissues.
 Furthermore, infant (suckling) mice are used for isolation of coxsackie virus
and rabies virus.

Embryonated Eggs

 Embryonated chick egg was used first for cultivation of viruses by Goodpasture
in 1931.
 The method further developed by Burnet was used for cultivation of viruses in
different sites of the embryonated egg.
 Usually, 8–11 days’ old chick eggs are used for culture of viruses.
 The viruses are isolated in different sites of the egg, such as yolk sac, amniotic
cavity, and allantoic cavity, and chorioallantoic membrane (CAM).
 Many of these viruses cause well-defined and characteristic foci, providing a
method for identification, quantification, or assessing virus pathogenicity.
 The embryonated egg is also used for growing higher titre stocks of some
viruses in research laboratories and for vaccine production.
 Yolk sac: Yolk sac inoculation is used for cultivation of Japanese
encephalitis, Saint Louis encephalitis, and West Nile virus. It is also used for
growth of chlamydia and rickettsia.
 Amniotic cavity: Inoculation in the amniotic cavity is used mainly for
primary isolation of influenza virus.
 Allantoic cavity: Inoculation in the allantoic cavity is used for serial
passages and for obtaining large quantities of virus, such as influenza virus,
yellow fever (17D strain), and rabies (Flury strain) viruses for preparation
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 of vaccines. For production of rabies virus, duck eggs were used due to their
bigger size than that of hen’s egg. This helped in production of large
quantities of rabies virus, which are used for preparation of the inactivated
non-neural rabies vaccine.
 Chorioallantoic membrane: Inoculation of some viruses on CAM produced
visible lesions known as pocks. Each infectious virus particle produces one
pock. The pox viruses, such as variola or vaccinia are identified by
demonstration of typical pocks on the CAM inoculated with the pox virus.
Nowadays, in a virology laboratory, chick embryo inoculation has been
replaced by cell cultures for routine isolation of viruses.
Tissue Culture
 Cell culture is most widely used in diagnostic virology for cultivation and
assays of viruses.
 The tissue culture was first applied in diagnostic virology by Steinhardt and
colleagues in 1913.
 They maintained the vaccinia virus by culture in tissues of rabbit cornea.
Subsequently, Maitland (1928) used cut tissues in nutrient media for
cultivation of vaccine viruses.
 Enders, Weller, and Robins (1949) were the first to culture poliovirus in tissue
cultures of nonneural origin. Since then, most of the virus had been grown in
tissue culture for diagnosis of viral diseases.
 Different types of tissue cultures are used to grow viruses. Tissue culture can
be of three different types as follows:
Organ Culture
 This was used earlier for the isolation of some viruses, which appear to show
affinity for certain tissue organs. For example, coronavirus, a respiratory
pathogen, was isolated in the tracheal ring organ culture.
 In this method, small bits of the organs are maintained in vitro for days and
weeks preserving their original morphology and function.
 Nowadays, organ culture is not used.
Explant Culture
 In this method, components of minced tissue are grown as explants embedded
in plasma clots.
 Earlier, adenoid tissue explant cultures were used for isolation of adenoviruses.
This method is now seldom used in virology.

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 Cell Culture

 Cell culture is now routinely used for growing viruses.

 In this method, tissues are dissociated into component cells by treatment with
proteolytic enzymes (trypsin or collagenase) followed by mechanical shaking.
 The cells are then washed, counted, and suspended in a growth medium
containing essential amino acids and vitamins, salts, glucose, and a buffering
system. This medium is supplemented by up to 5% of fetal calf serum and
antibiotics.
 The cell suspension is dispensed in glass or plastic bottles, tables, or Petri
dishes.
 On incubation, the cells adhere to the glass surfaces and divide to form a
confluent monolayer sheet of cells covering the surface within a week.
 The cell culture may be incubated either as a stationery culture or as a roller
drum culture. The latter is useful for growth of some fastidious viruses due to
better aeration by rolling of theculture bottle in special roller drums.
 The cell cultures are classified into three different types based on their origin,
chromosomal characters, and number of generations for which they can be
maintained.

Primary cell culture:

 These are a culture of normal cells obtained freshly from the original tissues
that have been cultivated in vitro for the first time and that have not been
subcultured.
 These cell cultures can be established from whole animal embryo or from
selected tissues from adult, newborn, or embryos.
 These cells have the normal diploid chromosomal number and are capable of
only limited growth (5–10 divisions) in culture.

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  They cannot be maintained in serial culture, but can be subcultured to obtain
large number of cells.
 Monkey kidney cell culture, human embryonic kidney cell culture, and chick
embryo cell culture are the common examples of primary cell culture.
 Primary monkey kidney cell cultures are highly useful for the primary isolation
of myxovirus, paramyxovirus, many enteroviruses, and some adenoviruses.

Diploid cell strains:

 Diploid cell strains are of a single cell type that retains their original diploid
chromosome number and karyotype. However, they have specific
characteristics and compositions and are usually composed of one basic cell
type.
 They are usually fibroblasts and can be cultured for maximum 50 serial
passages before they undergo senescence (die off) or undergo a significant
change in their characteristics.
 Diploid cells derived from human fibroblasts are useful for isolation of some
fastidious viruses.
 They are also used for production of vaccines; for example, WI-38 human
embryonic, lung cell stem is used for the cultivation of fixed rabies virus, and
human fetal diploid cells for isolation of adenovirus, picornaviruses, HSV, CMV,
and VZV.

Continuous cell lines:

 Continuous or immortal cell lines are cells of a single type, which are derived
from cancerous tissue and are capable of continuous serial cultivation
indefinitely without senescing.
 The cells are usually derived from diploid cell lines or from malignant tissues
and have altered and irregular number of chromosomes.
 Immortalization may occur spontaneously or can be induced by chemical
mutagens, tumorigenic viruses, or oncogens. Hep-2, HeLa, and KB derived from
human carcinoma cervix, human epithelioma of larynx, and human carcinoma
of nasopharynx and other cell lines are excellent for recovery of a large number
of viruses.
 These cell lines have been used extensively for the growth of a number of
viruses. These cell lines are usually stored at -70°C for use when necessary or
are maintained by serial subculture.
 The type of cell line used for virus culture depends on the sensitivity of the cells
to a particular virus; for example, Hep-2 cell line is excellent for the recovery of
respiratory syncytial viruses, adenoviruses, and HSV.
 Most of the viruses can be isolated by using one of these cell lines.
 Growth of viruses in cell cultures can be detected by the following methods

Cytopathic effect:
 Many viruses can be detected and initially identified by observation of the
morphological changes in the cultured cells in which they replicate.

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  The CPE produced by different types of viruses are characteristic and help in
the initial identification of virus isolates.
 Nuclear shrinking, vacuoles in the cytoplasm, syncytia formation, rounding up,
and detachment are the examples ofalteration of morphology of the cells.
 Most CPEs can be demonstrated in unfixed and unstained monolayer of cells
under low power of microscope.
 For example, adenoviruses produce large granular changes resembling
bunches of grapes, SV-14 produces well-defined cytoplasmic vacuolation,
measles virus produces syncytium formation, herpes virus produces discrete
focal degeneration, and enteroviruses cause crenation of cells and
degeneration of the entire cell sheet.

Hemadsorption:
 Hemadsorption is the process of adsorption of erythrocytes to the surfaces of
infected cells which serves as an indirect measurement of viral protein
synthesis.
 This property is made use of to detect infection with noncytocidal viruses as
well as the early stage of cytocidal viruses.
 Viruses, such as influenza virus, parainfluenza virus, mumps virus, and
togavirus, when infect cell lines code for the expression of red cell agglutinins,
which are expressed on the infected cell membrane during infections.
 These hemagglutinins bind some erythrocytes to the infected cell surface.
 Sometimes, viruses can be detected by agglutination of erythrocytes in the
culture medium.

Heterologous interference:
 This property is used to detect viruses that do not produce classic CPEs in the
cell lines.
 In this method, the growth of non-CPE-producing virus in cell culture can be
tested by subsequent challenge with a virus known to produce CPEs.
 The growth of the first virus will inhibit infection by the cytopathic challenge
virus by interference.
 For example, rubella virus usually does not produce any CPE, but prevents the
replication of picornaviruses, which is inoculated as a cytopathic challenge
virus.

Transformation:
 Oncogenic viruses that are associated with formation of tumors induce cell
transformation and loss of contact inhibition in the infected cell lines.
 This leads to surface growth that appears in a piled-up fashion producing
microtumors.
 Examples of such oncogenic viruses that produce transformation in cell lines
are some herpes viruses, adenoviruses, hepadanoviruses, papovavirus, and
retroviruses.

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 Classification of cell cultures and tissues that used in virology
1. Tissue or organ culture

Embryonic tissue organ or part of it that are supported in vitro and retain the cell
differentiation and their functions
2. Cell cultures

Descriptions primary transplanted cell Diploid cell

trypsinized cultures cultures
cultures
Morphology of cells Don”t differ Differ Differ
compared with the
original tissue
Set of chromosomes Diploid heteroploid diploid
Lifetime Limited by 1-3 Unlimited by the Limited by 20-
passages amount of passages 100 passages
Growth in impossible possible impossible
suspension
Signs of malignancy Absent always present absent

Period of 3-7 days 2/3-1 days 1-15 days

generation
Contact inhibition Present absent present
by growning on
glass
Examples 1. Culture of 1.HeLa (carcinoma of Fibroblast cell
monkey kidney cells the cervix) lines of human
embryo (WI-
2. Fibroblast cell 2. KB (oral humah
38, MRC-5,
culture of human carcinoma)
MRC-9, IMR-
embryos
3.HEp-2 (human 90), cows, pigs,
3. Cell culture of larynx carcinoma) sheeps
chicken embryo
4. Vero (green
fibroblasts
monkey kidney)

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 Classification of culture media for cell cultures

Natural enzymatic Synthetic

hydrolysates
Enders medium: 85- Aminopeptide 1. Balanced saline solutions:
90% cow amniotic Earle”s solution, Hank”s
Hemohidrolizate
fluid, 5-10% of cow solution, Dulbekko and Vogt
embryo extract, 5% lactalbumin solution
horse serum hydrolyzate 2. Supported medium: 199
medium, Igl medium, IMMD
(Igl medium modified by
Dulbekko), MMI (Miller-Igl
medium)

Used in the virology media for cell culture are divided into two main categories
- growth and support.

Growth media (GM), due to high content of serum are favourable for rapid cell
growth. After the formation of a monolayer and before inoculation of virus growth
the medium is removed and replaced with support medium. For preparing of the
growth medium bovine serum (BS) or embrio calf serum (ECS) and antibiotics
(penicillin and streptomycin) are added to the culture medium (e.g. IMMD), 5-10%.

Support media (SM) with low serum content can save the cell culture in the
state of persistent slow metabolism during viral replication.

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