Viruses are acellular organisms that replicate inside host cells using host cell machinery. They have either RNA or DNA genomes but not both. Viruses consist of a protein capsid that encloses and protects their nucleic acid. Inside host cells, viruses hijack the host's metabolic machinery and ribosomes to produce new viral components which then assemble and exit the cell. The viral life cycle consists of attachment, entry, uncoating, replication, assembly and release stages. Viruses are cultivated using techniques like cell culture, embryonated eggs and animal inoculation to study them and produce vaccines.

1. General Properties of Viruses
Virology
Virology is the bioscience for study of viral nature,and the relationship
between viruses and hosts
Definition of Virus
Viruses may be defined as acellular organisms whose genomes
consist of nucleic acid, and which obligately replicate inside host cells
using host metabolic machinery and ribosomes to form a pool of
components which assemble into particles called VIRIONS

2.  Smallpox was endemic in
China by 1000BC. In response,
the practice of variolation was
developed. Recognizing that
survivors of smallpox
outbreaks were protected from
subsequent infection,
variolation involved inhalation
of the dried crusts from
smallpox lesions like snuff, or
in later modifications,
inoculation of the pus from a
lesion into a scratch on the
forearm of a child.

3.  Viruses are inert (nucleoprotein ) filterable Agents
 Viruses are obligate intracellular parasites
 Viruses cannot make energy or proteins independent of a
host cell
 Viral genome are RNA or DNA but not both
 Viruses lack the enzymes necessary for protein and nucleic
acid synthesis
 Viruses do not have the genetic capability to multiply by
division
 Viruses occupy the twilight zone that separates the ‘living’
from the ‘nonliving’

4. Cellular
organisation
Growth
on
inanimate
media
Binary
fission
Both
DNA
and
RNA
Ribosome Sensitivity
to
antibiotics
Sensitivity
to
Interferons
Bacteria
+ + + + + + -
Mycoplasma
+ + + + + + -
Rickettsiae
+ - + + + + -
Chlamydiae
+ - + + + + +
Viruses
- - - - - - +
Properties of bacteria and viruses

5. Medical importance of viruses
 Viral diseases range from minor ailments such as the common cold
to terrifying diseases such as rabies or AIDS
 They may be sporadic like mumps, endemic like infectious hepatitis,
epidemic like dengue fever or pandemic like influenza
 They may be localised to circumscribed areas (as some
arbovirus diseases) or worldwide (as Herpes simplex)
 Viruses can cause cancer in animals and birds, as well as in
humans

6. Morphology
Size
Methods of analysis
1. Passing through collodion membrane filters of graded porosity
(gradocol membranes)
2. Ultracentrifuge
3. Electron microscope
4. X-ray crystallography

7. Size of Viruses
A small virus has a diameter of about 20 nm.
Example: Parvovirus
A large virus have a diameter of up to 400 nm.
Example: Poxviruses

10. Structure and shape
Virion
The complete infectious unit of virus particle
Capsid
The protein shell, or coat, that encloses the nucleic acid genome.
Functions:
a. Protect the viral nucleic acid.
b. Participate in the viral infection.
c. Antigenic and specific for each virus type
d. Provides structural symmetry to the virus particle
Nucleocapsid
The capsid with the enclosed nucleic acid

11. Capsid
Viral core
envelope

12. DNA RNA
Single Stranded
Double Stranded
Circular
+ or -
Segmented
Double Stranded Segmented

13. Symmetry of Nucleocapsid
1. Cubical/Icosahedral
(Adeno virus, Coxsackie virus, CMV, EBV, Hepatitis virus, HSV,
Polio virus, Rubella virus)
2. Helical
(Influenza, Rubeola, Mumps, Rabies, Hanta, Corona viruses)
3. Complex
(Bacteriophage, Pox viruses)

14. Cubic or icosahedral symmetry
An icosahedron is a polygon with 12 vertices or corners and
20 facets or sides
Two types of capsomers constitute the icosahedral capsid
1. Petagonal capsomers at the corners (pentons)
2. Hexagonal capsomers making up the sides (hexons)

15. Helical symmetry
Complex symmetry
T4 bacteriophage Pox virus

16. Envelope
 A lipid-containing membrane that surrounds some viral
particles
 It is derived from the plasma membrane of the host cell
during there release by budding from the cell surface
 Viruses-encoded glycoproteins are exposed on the
surface of the envelope (peplomers)
 A virus may have more than one type of peplomer
 Envelopes confer chemical, antigenic and biological
properties on viruses
 Not all viruses have the envelope, and viruses can be
divided into 2 kinds: enveloped virus and nonenveloped
(naked) virus

17. Envelope
d Virus
Naked
Virus
Cubic Helical

18. Shape of viruses
Overall shape of the virus particle varies in different groups of viruses
Filament-shape Brick-shape Tadpole-shape
Spherical-shape Rod-shape Bullet-shape

19. Viral Hemagglutination
 A large number of viruses agglutinate erythrocytes from different
species
 Hemagglutination by the influenza virus is due to the presence of
hemagglutinin spikes on the surface of the virus
 In the haemagglutination test RBCs are added to serial dilutions of
viral suspension, the highest dilution that produces haemagglutination
provides the haemagglutination titer (HA units)
 The haemagglutination test can be carried out in test tubes or
special plastic trays
 The test is a convenient method for detection and assay of the
influenza virus and also serves to titrate killed influenza vaccine

20. Elution
In myxoviruses, neuraminidase (RDE) acts on the receptor and
destroys it and this leads to the reversal of hemagglutination and
the release of the virus from the red cell surface
 As hemagglutination is specifically inhibited by the antibody to
virus, hemagglutination inhibition assay is useful for determining
antiviral antibody

21. Replication of viruses
The viral multiplication cycle can be divided into six sequential phases,
though the phases may sometimes be overlapping
1. Adsorption or attachment
2. Penetration
3. Uncoating
4. Biosynthesis
5. Maturation
6. Release

22. Adsorption
 Virions may come into contact with cells by random collision but
adsorption takes place only if there is an affinity between the two
 Differences in susceptibility to viral infection are to a large extent
based on the presence or absence of receptors on cells
Penetration
 Virus particles may be engulfed by a mechanism resembling
phagocytosis, a process known as ‘viropexis’
 In the case of enveloped viruses, the viral envelope may fuse with
the plasma membrane of the host cell and release the nucleocapsid
into the cytoplasm

23. Uncoating
 Process of stripping the the virus of its outer layers and capsid
so that the nucleic acid is released into the cell
 With most viruses, uncoating is effected by the action of lysosomal
enzymes of the host cell
Biosynthesis
 During this phase, viral nucleic acid, capsid protein, enzymes
necessary in the various stages of viral synthesis, assembly
and release will be synthesised
 Certain ‘regulatory proteins’ which serve to shut down the normal
cellular metabolism and direct the sequential production of viral
components are also synthesised
 The site of viral synthesis depends on the type of virus

24. Biosynthesis consists essentially of the following steps
1.Transcription of mRNA from the viral nucleic acid
2. Translation of the mRNA into ‘early proteins’. These are enzymes
which initiate and maintain synthesis of virus components
3.Replication of viral nucleic acid
4. Synthesis of late or structural proteins, which are the components
of daughter virion capsids
The critical step in viral biosynthesis is the transcription of mRNA
from the viral nucleic acid
The mechanisms of nucleic acid synthesis differ in the different
type of viruses

25. Maturation
 Assembly of daughter virions follows the synthesis of nucleic acid & proteins
 Virion assembly may take place in the host cell nucleus (Herpes and
adenoviruses) or cytoplasm (picorna and poxviruses)
 Naked viruses are present intracellularly as fully developed virions but
in the case of enveloped viruses, only the nucleocapsid is complete
Release
 Progeny of bacterial viruses release by the lysis of the infected bacterium.
In the case of animal viruses, release usually occurs without cell lysis
 Myxoviruses are released by budding from the cell membrane
over a period of time
 In the case of some viruses (varicella), transmission occurs directly
from cell to cell and poliovirus causes profound damage to the host cell
and may be released by cell lysis

26. Stages in the infection of a host’s cell and replication of a virus

27. Abnormal replicative cycles
Incomplete viruses
 Incomplete viruses are seen in large proportions, when cells are
infected with a high dose (MOI) of the influenza virus
 This is the result of defective assembly
 The virus yield will have a high hemagglutinin titer but low infectivity
and this is known as the ‘von Magnus phenomenon’
Abortive infection
 In nonpermissive cells, viral components may be synthesised but
maturation or assembly is defective, and either no release occurs
or the progeny is noninfectious

28. Defective viruses
 Some viruses are genetically defective in that when they infect cells,
they are unable to give rise to fully formed progeny
 Yield of progeny virions occurs only if the cells are simultaneously
infected with a helper virus, which can supplement the genetic
deficiency
(eg; hepatitis D, adeno-associated satellite viruses, which replicate
only in the presence of their helper viruses – hepatitis B and
adenoviruses, respectively)

29. Cultivation of viruses
Systems for the propagation of viruses
 Human volunteers
 Animal inoculation
 Embryonated eggs
 Tissue culture
a) Organ culture
b) Explant culture
c) Cell culture

30. Animal inoculation
 Mice, Infant (suckling) mice, rats, monkeys, chikens, guinea pigs,
rabbits, ferrets are used for inoculation
 Growth of the virus in inoculated animals may be indicated
by death, disease or visible lesions
 Animal inoculation is also used for the study of pathogenesis,
immune response, epidemiology and oncogenesis
 Disadvantages of animal inoculation are that immunity may interfere
with viral growth and that animals often harbour latent viruses

31. Embryonated eggs
 The embryonated egg offers several sites for the cultivation of viruses
 Inoculation on the chorioallantoic membrane (CAM) produces visible
lesions (pocks); pock-forming viruses eg: variola or vaccinia
 Inoculation into the allantoic cavity provides a rich yield of influenza
and some paramyxo viruses; hence employed for vaccine production
 Inoculation into the amniotic sac is employed for the primary isolation
of the influenza virus
 Yolk sac inoculation is used for the cultivation of some viruses,
chlamydiae and rickettsiae

32. Embryonated egg showing different routes of inoculation

33. Small pox (variola) virus pocks on CAM of embryonated egg

34. Tissue culture
Organ culture
 Small bits of organs can be maintained in vitro for days and weeks,
preserving their original architecture and function
 Useful for the isolation of some viruses which appear to be highly
specialised parasites of certain organs (eg: Tracheal ring culture –
corona virus, a respiratory pathogen)
Explant culture
 Fragments of minced tissue can be grown as ‘explants’ embedded in
plasma clots or may be cultivated in suspension (eg: adenoid tissue
explant cultures – adenoviruses)

35. Cell culture
Although embryonated eggs and laboratory animals are very useful for
isolation viruses, cell culture is the sole system for virus isolation in
most laboratories
Based on their origin, chromosomal characters and the number of
generations through which they can be maintained, cell cultures
are classified into three types
1. Primary cell cultures
2. Diploid cell cultures (semi-continuous)
3. Continuous cell lines

36. Primary cell cultures
Prepared directly from animal or human tissues and can be
subcultured only once or twice (eg: monkey kidney, human
embryonic kidney cell cultures)
 Useful for the isolation of viruses and their cultivation for vaccine
production
Diploid cell cultures
 Derived from human fetal tissue and can be subcultured 20-50 times
(human diploid fibroblasts – MRC-5, WI-38 (derived from normal
embryonic lung tissue)
 They are useful for the isolation of some fastidious pathogens
 They are also useful for the production of viral vaccines
(eg: poliomyelitis, rubella, rabies, CMV, VZV)

37. Continuous cell lines
 These are cells of a single type, usually derived from cancer cells
and capable of continuous serial cultivation indefinitely
(eg: HeLa, HEp-2, Vero, KB cell lines)
HeLa Human carcinoma of cervix cell line
HEp-2 Human epithelioma of larynx cell line
Vero Vervet monkey kidney cell line
KB Human carcinoma of nasopharynx cell line
McCoy Human synovial carcinoma cell line
BHK-21 Baby hamster kidney cell line
Some continuous cell lines in common use

38. Detection of virus growth in cell cultures
Cytopathic effect
Many viruses cause morphological changes in cultured cells, these
changes can be readily observed by microscopic examination and
these changes are known as ‘cytopathic effects’ (CPE)
Cytopathic effects may be cytocidal (cell death) or non-cytocidal
Non-cytocidal effects include acidophilic or basophilic inclusion bodies
in the nucleus or cytoplasm, or both; cell fusion, and transformation
Cytopathic effects can be so characteristic of individual viruses that
they can often be used to identify viruses
(Measles – syncytium formation; adenovirus – large granular clumps)

39. (a)Cytoplasmic inclusion body caused by rabies virus in brain tissue
(b)Syncytium formed by cell fusion due to infection by measles virus
Normal cell CPE

40. Metabolic inhibition
When viruses grow in cell cultures, cell metabolism is inhibited and
there is no acid production
Hemadsorption
When hemagglutinating viruses grow in cell culture, their presence
can be indicated by the addition of guinea pig erythrocytes to the
cultures
Interference
The growth of a non-cytopathogenic virus in cell culture can be tested
by the subsequent challenge with a known cytopathogenic virus
Transformation
Oncogenic viruses induce cell transformation and loss of contact
inhibition, so that growth appears in a piled-up fashion producing
microtumors
Immunofluorescence

41. Viral assay
The virus content of a specimen can be assayed in two ways
1. Total virus particles: Electron microscopy and haemagglutination
2. Assay of infectivity (with reference to the infectious virions only)
a)Quantal assays
Only indicate the presence or absence of infectious viruses but using
serial dilutions of virus suspensions and with the aid of statistical
methods, reasonably accurate estimates of infectivity can be obtained
 End points used for infectivity titration are the death of the animal,
production of hemagglutinin in allantoic fluid or the appearance of
CPE in cell cultures
 The titers are expressed as the ‘50 percent infectious dose’ (ID50)
or LD50

42. b) Quantitative assays
 Similar to the estimation of bacterial viable counts by colony
counting
Two methods are available;
1.Plaque assay
A viral suspension is added to a monolayer of cultured cells in a bottle
or petri dish, and after adsorption, the medium is removed and
replaced with a solid agar gel
Each infectious viral particle gives rise to a localised focus of infected
cells that can be seen with the naked eye. Such foci are known
as ‘plaques’
2. Pock assay

43. Plaque assay Pock assay

44. Viral Genetics
Two main mechanisms for genetic modification in viruses are mutation
and recombination
Mutation
 The frequency of mutation in viruses is about 10-4
to 10-8
 Mutations may be lethal or sub lethal, may occur spontaneously
or induced by mutagens
 Mutations affecting virulence, host range, antigenicity and pock
plaque morphology are of clinical and laboratory interest
 Mutants which are able to grow under certain conditions (permissive
conditions) and can not grow or lethal under some specific conditions
(restrictive conditions) are called as ‘conditional lethal mutants’

45. Interaction between viruses (Genetic)
Reassortment
 Found in viruses which have segmented genomes (eg: influenza,
bunya and arena viruses)
 In a single cell infected with two related viruses, there is an exchange
of segments with production of reassortants
 It is probably one of the ways by which the pandemic strains of
the influenza virus originate in nature
Reactivation
when a cell is infected with an active virus and a different but related
inactive virus, recombination may occur and as result of this the
progeny possessing one or more genetic traits of inactive virus
may be produced. This process is called cross reactivation or
marker rescue

46. Interaction between viral gene products (nongenetic)
1. Phenotypic mixing
 When two different viruses infect the same cell, some mix up may
take place during assembly, so that progeny genome of one virus
may be surrounded by a capsid belonging partially or entirely to
other virus
 This altered phenotype is not a stable variation
 When the genome of one virus is surrounded by the entire capsid of
the other virus, it is called ‘transcapsidation’

47. 2. Genotypic mixing
 It occurs when more than one complete genomes are accidentally
surrounded by a single virus capsid
 There is no recombination between the different genomes so that the
two kinds of viral progeny are formed on passage
3. Complementation
 One virus provides the gene products to the second virus in which
the latter is defective, this allows the second virus to replicate

48. 4. Interference
 Infection of a cell by one virus inhibits simultaneous or subsequent
infection by other virus
 The most important mediator of interference is interferon
 Interference may also be produced by destruction of cell receptors
by an active or inactive virus
 Viral interference has been applied in the field in controlling
poliomyelitis outbreaks by introducing vaccine strain into
the population
5. Enhancement
 Mixed infection of cells may sometimes lead to increased virus yield
or greater CPE

49. Classification and nomenclature of viruses
 Viruses are classified on the basis of biological, physical and
chemical properties
 Viruses are broadly classified into DNA and RNA viruses and then
further divided into families, subfamilies, genera and species

52. DNA viruses
1. Poxviridae
2. Herpesviridae
3. Adenoviridae
4. Papovaviridae
5. Parvoviridae
6. Hepadnaviridae

53. RNA viruses
1. Picornaviridae
2. Orthomyxoviridae
3. Paramyxoviridae
4. Togaviridae
5. Flaviviridae
6. Bunyaviridae
7. Arenaviridae
8. Rhabdoviridae
9. Reoviridae
10.Coronaviridae
11.Retroviridae
12.Caliciviridae
13. Filoviridae

54. Viroids
 Viroids are single-stranded circular RNA molecules that lacks a
protein coat and they are mainly plant pathogens
Prions
 Prions are infectious proteins without any detectable nucleic acid
 They are highly resistant to physical and chemical agents
 They produce slow infections with very long incubation period
 Diseases caused by prions in animals include, scrapie of sheep and
goats,mink encephalopathy, bovine spongiform encephalopathy
 Responsible for Kuru, Creutzfeldt-Jakob disease and some other
chronic neurological degenerative diseases of humans

55. Thank you for your attention !!!