Viruses can be cultivated through several methods, including inoculation in animals, embryonated eggs, and tissue/cell culture. Inoculation in animals allows study of viral replication and immune responses but is expensive. Embryonated eggs are widely used as they are inexpensive and viruses can replicate, though not all human viruses grow well. Tissue/cell culture is now preferred, using primary cultures, continuous cell lines, or explant cultures. Growth is detected through cytopathic effects, staining, or metabolic changes in infected cells.

1. VIRIOLOGY

2. Introduction to viruses
Definition:
• An infective agent that typically consists of a nucleic acid molecule in
a protein coat, is too small to be seen by light microscopy, and is able
to multiply only within the living cells of a host.
• The are 10-100 times smaller(20-300nm) than most bacteria
• They can grow only in animals or plants or microorganisms (Obligate
intracellular parasites)
• Viruses do not have cells that divide; new viruses are assembled in the
infected host cell
• But unlike still simpler infectious agents, viruses contain genes, which
gives them the ability to mutate and evolve, contain a nucleic acid
either DNA or RNA which is enclosed by a protein coat called nucleo-capsid.
• Evolved from plasmids : pieces of DNA that can move between cells while others
may have evolved from bacteria.
• The structurally complete, matured and infectious virus is called Virion.
• In 1897, Martinus Beijerinck suggested that tiny particles in the juice caused the
disease, and he named these particles viruses, after the Latin word for “poison.”
• Over 5,000 species of viruses have been discovered.

3. • A virus consists of two or three parts: genes,
made from either DNA or RNA, long molecules
that carry genetic information
• Protein coat that protects the genes; and in
some viruses, an envelope of fat
• Viruses vary in shape from the simple helical
and icosahedral to more complex structures.

4. Classification of virus
International Committee on Taxonomy of Viruses(ICTV)
Basis on the host they lives.
1. Animal viruses:
• Have DNA and may have RNA also
• Viruses of animal host
• Rabies, Polio, Mumps, Chicken pox, Small pox, and
Influenza.
2. Plant Viruses:
• have RNA and infected potato, sugarcane, tabacco,
cucurbitis and other higher plants. Viruses which show
their live characteristics when attached to plants.
• Tobacco mosaic virus (TMV), Banana streak virus,
Carrot thin leaf virus
3.Bacterial Virus: Have DNA and are called
Bacteriophages(phages) ( T1, T2, T3, and T4.)

5. Classification of viruses
• Based on physicochemical properties/characteristics:
1. Primary characteristics
• Chemical nature of nucleic acid- RNA or DNA, single
stranded/ double stranded, single/ segmented genome,
• Structure of virion-Helical icosahedral or complex,
nacked or enveloped, etc.
• Site of replication-Nucleus or cytoplams
2. Secondary characteristics
• Host range- host specific, specific host tissues or cell type
• Specific surface structure- eg: Antigenic properties
• Mode of transmission- eg: feces

8. • Nomenclature of viruses is decided by Internation
committee on taxanomy of viruses
• Viruses are grouped into families named by suffix
viridae, subfamilies virinae and genera virus
Sizes of viruses

9. Morphology of viruses
• Virion: Complete, mature, fully developed viral particle
composed of nucleic acid surrounded by a coat that
protects it from the environment and serves as a vehicle of
transmission from one host cell to another host cell.
• Nucleic acid-either DNA or RNA
• Capsid or envelope- protein coat surrounding the NA is
called capsid composed of protein subunits called as
capsomeres. The capsomere might be of single type or
different types proteins depending on virus. Structure
might be covering capsid called as envelope which is
composed of proteins, lipids, carbohydrates.
• Viruses are not cellular and therefore do not have a
nucleus, cytoplasm membrane.

11. Structure based on capsid structure

12. T-even bacteriophage

13. T-even phages
• T-even phages, also known as the E. coli phages, are a
group of double-stranded DNA bacteriophages from the
family Myoviridae.
• T-even phages(T1,T2,T3,T4,T5,T6,T7)
• Tadpole shaped, with hexagonal head and cylindrical tail.
• Head consists of nucleic acids(ds DNA) surrounded by a
protein coat or capsid.
• The base of the tail portion have six pins and six tails
attached together that bind to specific receptor sites on
the bacterial surface.
• Phages T1, T5 and lambda of E. coli do not possess a
contractile sheath and tails of phages T3 and T7 of E.coli
and P22 Salmonella are short and non-contractile.

15. Life cycle/replication of Bacteriophages
• Bacteriophage, also called phage or bacterial virus, any of
a group of viruses that infect bacteria.
• Discovered by Frederick W. Twort in Great Britain (1915)
and Felix d’Herelle in France (1917).
• Bacteriophage, meaning “bacteria eater”
There are two different types of life cycle:
1. Lytic or Virulent cycle: In this cycle, there is intracellular
multiplication of phages followed by lysis and release of
progeny virions.
2. Temperate or avirulent or lysogenic cycle: In lysogenic
cycle, the phage DNA becomes integrated with the bacterial
genome, replicating synchronously without any cell lysis.

16. Steps involved in Lytic or Virulent cycle

17. Temperate or avirulent or lysogenic cycle

18. Cultivation of Viruses
• Viruses are obligate intracellular parasites, which
cannot be grown on inanimate culture media.
• Viruses are host specific and grow only in selective
hosts. Virologists use only a suitable host system for
cultivation of a virus.
• Viruses cannot grow in artificial media. They
cannot grown in non-living culture or on agar
plates alone, they must require living cells to
support their replication.
• There is no universal cell that will support all
viruses.

19. Main purpose of virus cultivation
• To isolate and identify viruses
in clinical samples.
• To prepare viruses for vaccine
production.
• To do research on viral
structure, replication ,
genetics and effects on host
cells.

20. METHODS FOR CULTIVATION OF VIRUSES

21. 1). Inoculation of Virus in
Animals
• Viruses which are not cultivated in embryonated
egg and tissue culture are cultivated in laboratory
animals. e.g: mice, guinea pig , hamester , rabbits
and primates are used.
• The selected animals should be healthy and free
from any communicable diseases.
• Suckling mice (less than 48- 300 hours old) are
most commonly used.
• Different ways of inoculation in mice are:
1). Intracerebral.
2). Subcutaneous.
3). Intraperitoneal.
4). Intranasal.

22. Observation
•The growth of the virus
in inoculation animals
may be indicated by
death, diseases or
visible lesions.

23. Advantages and disadvantages
of animal inoculation :
Advantages:
• Production of antibodies can be identified.
• Diagnosis , pathogenesis and clinical symptoms
are determined.
• Primary isolation of certain viruses.
• Mice provide a reliable model for studying viral
replication.
• Used for the study of immune responses,
epidemology and oncogenesis.

24. Disadvantages:
• Expensive and difficulties in maintainance of
animals.
• Difficulty in choosing of animals for particular
virus.
• Some human viruses cannot be grown in animals
or can be grown but do not cause diseases.
• Mice do not provide models for vaccine
development.

25. 2). Inoculation of virus into
embryonated egg
• The process of cultivation of viruses in
embryonated eggs.
• Depend upon the type of egg being used.
• First cultivated by Goodpasture(1931), further
developed by Burnet.
• Egg provide a suitable means for :
i. The primary isolation and identification of
viruses.
ii. The production of vaccines.
iii. The maintenance of stock culture.

27. The egg is broken and virus is isolated from tissue of egg
Incubation can be done at 36°C for respective incubation time of the
virus
Eggs are selected (7-12 days old), Sterilization of egg’s cell is done by
using iodine(disinfectant) and penetrated with a small sterile drill

28. Observation
• Virus growth and multiplication in
the egg embryo is indicated by the
death of the embryo , by embryo
cell damage , or by the formation
of typical pocks or lesions on the
egg membrane.

29. Advantages and disadvantages of inoculation into
embryonated egg
• Widely used method for the isolation of virus and
growth.
• Cost effective and maintenance is much easier.
• The embryonated eggs are readily available.
• They are free from contaminating bacteria and many
latent viruses.
• Ideal substrate for the viral growth and replication.
• less labour is needed.
• Widely used method to grow virus for some vaccine
production.
• Defence mechanisms are not involved in
embryonated eggs.

30. Disadvantages
• The site of inoculation for varies with different
virus . That is , each virus have different sites
for growth and replication.

31. 3) Tissue culture
• Cultivation of bits of tissues and organs in vitro had been used by physiologists
and surgeons for the study of morphogenesis and wound healing.
• The first application of tissue culture in virology was done by Steinhardt and
colleagues (1913), who maintained the vaccinia virus in fragments in rabbit
cornea.
• Maitland (1928) used chopped tissue in nutrient media for cultivation of
vaccinia viruses.
• Before the advent of cell culture, animal viruses could be propagated only on
whole animals or embryonated chicken eggs.
• Cell cultures have replaced embryonated eggs as preferred type of growth
medium for many viruses.
• Cell culture consists of cells grown in culture media in the laboratory.
• There are three types of tissue culture:
• 1). Organ culture.
• 2). Explants culture.
• 3). Cell culture.

32. 1) Organ culture

34. 2). Explants culture

35. Explants culture

36. 3) Cell culture

39. Types of cell culture

41. 1) Primary cell culture

44. 2. Secondary cell culture

46. 3. Continuous cell lines
• Animal cells capable of indefinite growth are
called continuous cell lines or cell lines.
• These are the cells of a single type, usually
derived from cancer cells, that are capable of
continuous serial cultivation indefinitely.
• Standard cell lines derived from human cancers,
such as HeLa , HEp–2 and KB cell lines have been
used in laboratories throughout the world for
many years.

47. • These cell lines may be maintained by serial
sub-cultivation or stored in the cold (-70⁰C )
for use when necessary.
• Some cell lines are now permitted to be used
for vaccine manufacture, for example: Vero
cells for rabies vaccine.

49. Advantages of cell culture
• Relative ease, broad spectrum, cheaper and
sensitivity
Disadvantage of cell culture
• The process requires trained technicians with
experience in working on a full time basis.
• State health laboratories and hospital
laboratories do not isolate and identify viruses
in clinical work.
• Tissue or serum for analysis is sent to central
laboratories to identify virus.

50. Detection of virus growth
• The following methods are available to detect
the virus growth in the cell or tissue cultures.
a). Cytopathic effect.
b). Haemadsorption.
c). Interference.
d). Transformation.
e). Immuno-fluorescence.
f). Metabolic inhibition.

51. a). Cytopathic effect.
• Many viruses cause morphological changes in cultured cells in
which they grow. These changes can be readily observed by
microscopic examination of the cultures.
• These changes are known as ‘cytopathic effects’ (CPE) and the
viruses causing CPE are called ‘cytopathogenic viruses’.
• The CPE produced by different groups of viruses are characteristic
and help in presumptive identification of virus isolates.
• For example, enteroviruses produce rapid CPE with crenation of
cells and degeneration entire cell sheet ; measles virus produce
syncytium formation; herpes virus causes discrete focal
degeneration; adenovirus produce large granular clumps of
grapes.