PHARMACEUTICAL MICROBIOLOGY (BP303T)Unit-IIIPart-2Study of morphology, classification, reproduction/replication and cultivation of Virus. Introduction, Def General characteristics of Viruses: small size characteristic shapes, obligate intracellular parasites no built-in metabolic machinery no ribosomes only one type of nucleic acid do not grow in size. Morphology of Virus: Helical, Polyhedral (Icosahedral) Viral Envelop, Complex virus, Classification of virus. Viral Replication LIFE CYCLE OF BACTIRIOPHAGES Lytic cycle: Attachment, Penetration, Biosynthesis, Maturation and Release of progeny Phage Particles. The Lysogenic Cycle, Cultivation of virus : Animal inoculation, Embryonated eggs or chick embryo method and Tissue culture or cell culture: Organ cultures Explant culture and Cell culture. Types of cell culture 1.Primary cell culture: 2. Diploid cell culture (Semi-continuous cell lines):3. Heteroploid cultures (Continuous cell lines): MULTIPLICATION OF HUMAN VIRUS:1. Attachment of Viral Particles 2. Penetration 3. Uncoating 4. Replication Of Viral Nucleic Acids And Translation Of The Genome 5) Maturation Or Assembly Of Virions. ) 6. Release Of Virions Into The Surrounding Environment

1. PHARMACEUTICAL MICROBIOLOGY (BP303T)
Unit-IiI
Part-2
Study of morphology, classification, reproduction/replication
and cultivation of Virus.
Name: Mrs. Pooja Deepak Bhandare
Assistant Professor
G H RAISONI UNIVERSITY
SCHOOL OF PHARMACY

3. Introduction :
• Viruses are acellular, meaning they are biological entities that
do not have a cellular structure.
• Therefore, they lack most of the components of cells, such as
organelles, ribosomes, and the plasma membrane.
• A virion consists of a nucleic acid core, an outer protein
coating or capsid, and sometimes an outer envelope made of
protein and phospholipid membranes derived from the host
cell.
• Virus are infectious agents so small that they can only be seen
at magnification provide by electron microscope.

4. • Def : Sub microscopic entity consisting of a single nucleic
acid surrounded by a protein coat and capable of replication
only within the living cells of bacteria, animals or plants

5. General characteristics of Viruses
1. small size: cannot be viewed with a light microscope, range of size =
30-400 nm
2. characteristic shapes - spherical (complex), helical, rod or
polyhedral, sometimes with tails or envelopes. Most common
polyhedron is the icosahedron which as 20 triangular faces.
3. obligate intracellular parasites: Viruses do not contain within their
coats the machinery for replication. For this they depend upon a host
cell and this accounts for their existence as obligate intracellular
parasites. Each virus can only infect certain species of cells. This refers
to the virus host range.
4. no built-in metabolic machinery: Viruses have no metabolic
enzymes and cannot generate their own energy.

6. 5. no ribosomes: Viruses cannot synthesize their own
proteins. For this they utilize host cell ribosomes during
replication.
6. only one type of nucleic acid: Viruses contain either
DNA or RNA (never both) as their genetic material. The
nucleic acid can be single-stranded or double stranded.
7. do not grow in size: Unlike cells, viruses do not grow in
size and mass leading to a division process. Rather viruses
grow by separate synthesis and assembly of their
components resulting in production of a "crop" of mature
viruses. General

7. Morphology of Virus
• Extracellular infectious viral particle called ‘Virion’.
• Virus are much smaller than bacteria. For a time they were known
as filterable agent as they pass through filters that can hold back
bacteria.
• They can not be seen under light microscope hence as a
ultramicroscopic.
• The virus particle seen in this manner are known as elementary
bodies.

8. 1. Helical
1. Helical -The protomeres are not
grouped in capsomeres, but are bound to
each other so as to form a ribbon-like
structure. This structure folds into a helix
because the protomeres are thicker at one
end than at the other. The diameter of the
helical capsid is determined by
characteristics of its protomeres, while its
length is determined by the length of the
nucleic acid it encloses.
Eg. Rabies virus, Tobacco Mosaic virus.

9. 2. Icosahedral (Polyhedral)-
The protomeres aggregate in groups of five
or six to form the capsomere. In electron
micrographs, capsomeres are recognized as
regularly spaced rings with a central hole.
The shape and dimensions of the icosahedron
depends on characteristics of its protomeres.
All icosahedral capsids have 12 corners each
occupied by a penton capsomere and 20
triangular faces, each containing the same
number of hexon capsomeres. Icosahedral
symmetry is identical to cubic symmetry.
Eg. Adenovirus, Poliovirus.

10. 3. Viral envelop
• The envelop or outer covering of virus containing lipid is derived from the
plasma membrane of the host cell during the release by budding from the
cell surface.
• The envelop is glycoprotein in nature.
• Envelop virus are susceptible to the action of lipid solvent such as ether,
chloroform and detergent.
• When helical or polyhedral virus are enclosed by envelopes, they are called
helical (e.g. Influenza Virus) and Polyhedral virus (e.g Herpes Simplex
virus).
• The viral envelop is responsible for protection of virus from enzyme and
certain chemical)
• e.g. Herpes virus, Hepatitis B virus, HIV virus.

11. 4. Complex or Head-tail Virus
• These are the combination of both helical
and icosahedral and may contain a complex
outer wall or head-tail morphology.
• These types of head-tail or complex viruses
are known as a bacteriophage, they mainly
infect the bacteria.
• The head portion of this virus appears in an
icosahedral shape whereas the tail portion
appears as helical shape.
• They use their tail portion to get attached to
the surface of the bacterial cell, then it starts
to create a hole in the cell wall and injects its
DNA within the cell using the tail as a
channel.

13. Classification of virus

16. Viral Replication
• Viruses require living cells for reproduction. The cell that may be
infected named host cell or target cell. Viruses are obligate
intracellular parasites that are dependent on cellular energy production
and cellular machinery for synthesis of their components. Viruses have
unique replication strategies – disjunctive mode of reproduction. This
mode consists of separable synthesis of viral components in a host cell
and spontaneous macromolecular interaction for their maturation.
• Every viral family has a different strategy of replication. Process of
viral replication may be short – 4-12 hours or very long – for all the
organism life.

17. LIFE CYCLE OF BACTIRIOPHAGES
• A bacteriophage is any one of a number of viruses that infect bacteria
• • Inject genetic material, which they carry enclosed in an outer protein
capsid.
• Bacteriophages exhibit two different types of life cycle
1. Lytic cycle or virulent cycle: In a virulent cycle, there is intracellular
multiplication of phages followed by lysis and release of progeny
virions. This called lytic cycle.
2. Lysogenic or Temperate or Avirulent Cycle: In lysogenic cycle the phage
DNA becomes integrated with bacterial genome, replicating
synchronously without cell lysis.

18. Lytic cycle
• During the lytic cycle of virulent phage, the bacteriophage takes over
the cell, reproduces new phages, and destroys the cell. T-even phage is
a good example of a well-characterized class of virulent phages. There
are five stages in the bacteriophage lytic cycle.
1. Attachment or absorption
2. Penetration
3. Biosynthesis or phage component.
4. Maturation
5. Release or progeny phase component.

20. 1.Attachment
Attachment is the first stage in the infection process in which the phage
interacts with specific bacterial surface receptors (e.g.,
lipopolysaccharides and OmpC protein on host surfaces). Most phages
have a narrow host range and may infect one species of bacteria or one
strain within a species. This unique recognition can be exploited for
targeted treatment of bacterial infection by phage therapy or for phage
typing to identify unique bacterial subspecies or strains.

21. 2. Penetration :
The second stage of infection is entry or penetration. This
occurs through contraction of the tail sheath, which acts
like a hypodermic needle to inject the viral genome through
the cell wall and membrane. The phage head and remaining
components remain outside the bacteria.

22. 3. Biosynthesis :
The third stage of infection is biosynthesis of new viral
components. After entering the host cell, the virus synthesizes
virus-encoded endonucleases to degrade the bacterial
chromosome. It then hijacks the host cell to replicate, transcribe,
and translate the necessary viral components (capsomeres, sheath,
base plates, tail fibers, and viral enzymes) for the assembly of
new viruses. Polymerase genes are usually expressed early in the
cycle, while capsid and tail proteins are expressed later.

23. 4. Maturation: During the maturation phase, new virions are
created. To liberate free phages, the bacterial cell wall is
disrupted by phage proteins such as holin or lysozyme.
5. Release of progeny Phage Particles : The final stage is
release. Mature viruses burst out of the host cell in a process
called lysis and the progeny viruses are liberated into the
environment to infect new cells.
Each cycle of phage reproduction may require 20 to 60 mins and
a single phage infection may produce 200 or more progeny.

24. The Lysogenic Cycle
• In a lysogenic cycle, the phage genome also enters the cell through
attachment and penetration. A prime example of a phage with this type of
life cycle is the lambda phage. During the lysogenic cycle, instead of
killing the host, the phage genome integrates into the bacterial chromosome
and becomes part of the host. The integrated phage genome is called
a prophage.
• A bacterial host with a prophage is called a lysogen. The process in which a
bacterium is infected by a temperate phage is called lysogeny. It is typical
of temperate phages to be latent or inactive within the cell. As the
bacterium replicates its chromosome, it also replicates the phage’s DNA
and passes it on to new daughter cells during reproduction. The presence of
the phage may alter the phenotype of the bacterium, since it can bring in
extra genes (e.g., toxin genes that can increase bacterial virulence).

25. • This change in the host phenotype is called lysogenic conversion or phage
conversion. Some bacteria, such as Vibrio cholerae and Clostridium botulinum,
are less virulent in the absence of the prophage. The phages infecting these
bacteria carry the toxin genes in their genome and enhance the virulence of the
host when the toxin genes are expressed. In the case of V. cholera, phage encoded
toxin can cause severe diarrhea; in C. botulinum, the toxin can cause paralysis.
During lysogeny, the prophage will persist in the host chromosome
until induction, which results in the excision of the viral genome from the host
chromosome. After induction has occurred the temperate phage can proceed
through a lytic cycle and then undergo lysogeny in a newly infected cell

26. Lysogenic life cycle of bacteriophage

27. Cultivation of virus :
• As viruses are intracellular obligatory parasites, they always need
living cells for their growth.
• They cannot be grown on any artificial media.
• There are three methods employed for the cultivation of animal viruses
1. Animal inoculation
2. Embryonated eggs or chick embryo method.
3. Tissue culture or cell culture.

28. 1. Animal Inoculation
• Viruses which are cultivated in laboratory animals such as mice, guinea pig,
hamster, rabbits and primates are used.
• The selected animals should be healthy and free from any communicable
diseases.
• Suckling mice(less than 48 hours old) are most commonly used.
• Suckling mice are susceptible to togavirus and coxsackie virues, which are
inoculated by intracerebral and intranasal route.
• Viruses can also be inoculated by intraperitoneal and subcutaneous route.
• After inoculation, virus multiply in host and develops disease. The animals are
observed for symptoms of disease and death.
• Then the virus is isolated and purified from the tissue of these animals.
• Live inoculation was first used on human volunteers for the study of yellow fever
virus.

30. • Advantages of Animal Inoculation
1. Diagnosis, Pathogenesis and clinical symptoms are determined.
2. Production of antibodies can be identified.
3. Primary isolation of certain viruses.
4. Mice provide a reliable model for studying viral replication.
5. Used for the study of immune responses, epidemiology and oncogenesis.
• Disadvantages of Animal Inoculation
1. Expensive and difficulties in maintenance of animals.
2. Difficulty in choosing of animals for particular virus
3. Some human viruses cannot be grown in animals,or can be grown but do not
cause disease.
4. Mice do not provide models for vaccine development.
5. It will lead to generation of escape mutants
6. Issues related to animal welfare systems.

31. 2. Embryonate Eggs or Chick embryo method
• Good pasture (1931) was the first who used hen’s embryonated egg for
the cultivation of viruses. Embryonated egg provides several sites for
the cultivation of viruses.
• 1. Chorio- allantoic membrane 2. Allantoic cavity 3. Amniotic cavity
4. Yolk sac 5. Embryo
• Different site is used for growth of different viruses. Eg. Chorio-
allantoic membrance is used for the cultivation of pox virus. Allantoic
cavity is employed for the Influenza virus.
• There are several advantages, chick embryos are packed in their shells
and have natural resistant against bacterial contamination. Chick
embryo method is cheaper and easy to handle.

33. • Advantages of Inoculation into embryonated egg
1. Widely used method for the isolation of virus and growth.
2. Ideal substrate for the viral growth and replication.
3. Isolation and cultivation of many avian and few mammalian viruses.
4. Cost effective and maintenance is much easier.
5. Less labor is needed.
6. The embryonated eggs are readily available.
7. Sterile and wide range of tissues and fluids
8. They are free from contaminating bacteria and many latent viruses.
9. Specific and non specific factors of defense are not involved in embryonated eggs.
10. Widely used method to grow virus for some vaccine production.
• Disadvantages of Inoculation into embryonated egg
1. The site of inoculation for varies with different virus. That is, each virus have different sites for their
growth and replication.

34. 3. Cell Culture (Tissue Culture)
• There are three types of tissue culture; organ culture, explant culture and cell
culture.
• Organ cultures are mainly done for highly specialized parasites of certain
organs e.g. tracheal ring culture is done for isolation of coronavirus.
• Explant culture is rarely done.
• Cell culture is mostly used for identification and cultivation of viruses.

35.  Cell culture
• Cell culture is the process by which cells are grown under controlled
conditions.
• Cells are grown in vitro on glass or a treated plastic surface in a
suitable growth medium.
• At first growth medium, usually balanced salt solution containing 13
amino acids, sugar, proteins, salts, calf serum, buffer, antibiotics and
phenol red are taken and the host tissue or cell is inoculated.
• On incubation the cell divide and spread out on the glass surface to
form a confluent monolayer.

36. Types of cell culture
1. Primary cell culture:
• These are normal cells derived from animal or human cells.
• They are able to grow only for limited time and cannot be
maintained in serial culture.
• They are used for the primary isolation of viruses and production
of vaccine.
• Examples: Monkey kidney cell culture, Human amnion cell
culture

37. 2. Diploid cell culture (Semi-continuous cell lines):
• They are diploid and contain the same number of chromosomes as the
parent cells.
• They can be sub-cultured up to 50 times by serial transfer following
senescence and the cell strain is lost.
• They are used for the isolation of some fastidious viruses and production
of viral vaccines.
• Examples: Human embryonic lung strain, Rhesus embryo cell strain

38. 3. Heteroploid cultures (Continuous cell lines):
• They are derived from cancer cells.
• They can be serially cultured indefinitely so named as continuous
cell lines
• They can be maintained either by serial subculture or by storing in
deep freeze at -70°c.
• Due to derivation from cancer cells they are not useful for vaccine
production.
• Examples: HeLa (Human Carcinoma of cervix cell line), HEP-2
(Humman Epithelioma of larynx cell line), Vero (Vervet monkey)
kidney cell lines, BHK-21 (Baby Hamster Kidney cell line).

39. 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.

40. MULTIPLICATION OF HUMAN VIRUS:

41. 1. Attachment of Viral Particles
All viruses possess receptors on their surface, usually in the form of glycoproteins embedded in
the viral envelope or protruding as spike from the viral capsid. These structures recognize and
bind receptors on the host cell and provide the virus with its high specificity although different
viruses might share the same receptor. The virus–cell recognition event is similar to any
protein–protein interaction in that it occurs through a stereospecific network of hydrogen bonds
and lipophilic associations. For example, the haemagglutinin receptor of influenza virus binds
the terminal glycoside residues of gangliosides (cell surface glycolipids) of the target cell
leading directly to the virus particle adhering to the cell. Similarly, the interaction between the
HIV receptor (i.e. gp120) and the Tlymphocyte receptor (i.e. CD4) has been intensively
studied.

42. 2. Penetration
• Following the irreversible attachment of the virus to the host cell, penetration of
the virus through the cell membrane is initiated following two energydependent
mechanisms, endocytosis or fusion. A third mechanism has been identified in
some bacteriophages that can inject their nucleic acid inside the bacterium (see
section 8.1). During endocytosis, the association between virus receptor and host
receptor triggers a number of mechanisms that draw the cell membrane to engulf
the virus particle forming a cytosolic vacuole. This process is widespread among
nonenveloped viruses, but is also used with some enveloped viruses such as
influenza (orthomyxoviruses). Certain enveloped viruses (e.g. herpes simplex
virus, HIV) can penetrate following fusion of their envelope with the host cell
membrane, liberating the viral capsid within the cell cytoplasm.

43. 3. Uncoating
• Following penetration of the virus in a vacuole or directly into the cell
cytoplasm, the viral nucleic acid then needs to be released from the
capsid/coat(s) to initiate viral replication. This is the uncoating process. For
viruses that penetrate by endocytoses, the acidification of the cytosolic
vacuoles following endosome fusion induces a conformational change in
the capsid and the release of viral nucleocapsid (some helper proteins are
associated with the viral nucleic acid) into the cytoplasm. For certain
viruses, such as reovirus, only a partial uncoating is necessary for the
expression of the viral genome. The release of the nucleocapsid from
vacuoles can occur in the cytoplasm, close to the nucleus or within the cell
nucleus.

44. 4) Replication Of Viral Nucleic Acids And
Translation Of The Genome
• The viral genome is replicated. The structure, size and nature of the viral
genome are extremely diverse and thus this stage of the viral multiplication
cycle reflects this diversity. Three main mechanisms are, however, common
to all viruses: the transcription of viral genes into viral mRNA, the translation
of the viral genome into proteins, and the replication of the viral genome.
Early transcription and translation usually occurring immediately after the
release of the nucleocapsid in the cytoplasm is also common, and ensures the
production of early proteins such as viral polymerases, and the hijacking of
the cell synthesis machinery. In addition, some viruses can encode for genes
the products of which regulate the host synthetic processes according to the
needs of the virus (e.g. tat gene in HIV).

45. • The replication of the viral genome depends on the type of nucleic acid carried by the virus. The
positive strand RNA in viruses such as the poliovirus can be used directly as mRNA following the
acquisition of a terminal sequence from the host cell. Negative strand RNA (e.g. in influenza virus)
is transcribed into a positive RNA complementary in base sequence to the parent RNA using an
RNAdependent RNA polymerase carried by the virus. In ds DNA viruses (e.g. adenoviruses), the
nucleic acid passes into the nucleus where it is usually transcribed by a host DNAdependent RNA
polymerase. In some viruses (e.g. poxvirus), this enzyme is contained within the virus and released
during uncoating, allowing the viral genome to be replicated in the cell cytoplasm. In retroviruses
(e.g. HIV), a single stranded proviral DNA is produced from the viral ss RNA by a viral enzyme
called reverse transcriptase. This unique enzyme acts both as an RNA and DNA directed DNA
polymerase, and has associated RNAase activity. The proviral DNA can be transported to the cell
nucleus where it can be integrated within the cell host genome by a viral integrase.
• One important difference between the host cell and the virus is in the nature of their mRNA. Host
cell mRNA encodes directly for functional proteins, whereas viral mRNA is polycistronic, which
means several distinct proteins are encoded within a single piece of mRNA. This implies that the
virus needs to use a virus-specific protease to cut at the correct place the polyprotein produced by
translation to restore the functionality of viral proteins.

46. 5) Maturation Or Assembly Of Virions.
• Towards the end of the multiplication process, large amounts of viral materials accumulate within the host. Viral
capsid starts to form from individual structural proteins. In certain viruses (e.g. poliovirus) the capsid self-
assembles. The replicated viral genome and some viral proteins become packaged within the capsid. Most
nonenveloped viruses accumulate within the cytoplasm or nucleus and are only released when the cell lyses.
Packaging of viral components can occur within the cytoplasm or in the cell nucleus. For example, with influenza
virus, the capsomeres are transported to the cell nucleus where they combined with the viral RNA and assemble
into helical capsids. The envelope of enveloped viruses originates from the host. With the influenza virus, viral
proteins such as neuraminidase and haemagglutinin migrate to the cell membrane, displacing cell protein. The
assembled nucleocapsids pass out from the nucleus to the cytoplasm and as they impinge on the altered
cytoplasmic membrane they cause it to bulge and bud off completed enveloped particles from the cell. In the
herpesvirus, the envelope originates from the nucleus membrane. The nucleocapsid assembles into the nucleus and
it acquires its envelope as it passes through the inner nuclear membrane. The complete virus is then incorporated
into a vesicle which migrates to the cell surface.

47. 6) Release Of Virions Into The Surrounding Environment
• At the end of the multiplication process, the mature virions are released
from the host cell. This can occur in a number of different ways. For most
nonenveloped viruses, the virus progeny accumulates within the host cell
cytoplasm and is released following cell lysis. Some viruses (e.g.
bacteriophages) produce a lytic enzyme (peptide) or proteases to lyse the
host enabling the release of infectious particles, although the host often
selfdisintegrates as it cannot maintain normal housekeeping functions during
a viral infection. Enveloped viruses are usually released by a budding
process over a period of hours. Ultimately the host cell will die following
damage to its metabolism and housekeeping functions during viral
replication.

48. THANK YOU