Introduction, Classification, Morphology and Methods for the detection of Viruses.ppt

1. Classification, Morphology and Methods for
the detection of Viruses
Dr. Rakesh Prasad Sah
Assistant Professor, Microbiology

2. Definition
• are the smallest known infective agents  containing only
one type of nucleic acid (DNA or RNA) as their genome.
• Do not possess cellular organization and they have no
metabolic activity.

3. Properties of Viruses
• Do not have cellular organization.
• Contain only one type of nucleic acid; either DNA or RNA but
never both.
• Obligate intracellular parasites.
• Lack enzymes necessary for protein and nucleic acid synthesis
and are dependent for replicaton on the machinery of host cells.
• Multiply by a complex process and not by binary fission.
• Unaffected by antibacterial antibiotics.

4. Differences between Virus and Bacteria
Properties Bacteria Viruses
Cell wall + _
Ribosome's & enzymes + _
DND & RNA Both present Only one
Binary fission + _
Growth in Inanimate media + _
Sensitivity to antibiotics + _
Sensitivity to interferon _ +

5. Morphology of Viruses
• Viriods: ssRNA that lacks protein coat.
• Prions: infectious proteins without any detectable N.A.

6. Morphology of Viruses
• Extracellular infectious viral particle is called ‘Virion’.
• Viruses are much smaller than bacteria. For a time, they were
known as ‘filterable agents’ as they can pass through filters that can
hold back bacteria.
• They can not be seen under light microscope hence called as
‘ultramicroscopic’.
• Size range: 20-300 nm
• Parvovirus: 20 nm (smallest virus)
• Pox virus: 300 nm (biggest virus and can be seen under light

8. Capsid
Nucleic Acid
Envelope
Peplomer
E
L
E
C
T
R
O
N
M
I
C
R
O
S
C
O
P
E

9. Viral Capsid
• Viruses consists of nucleic acid
core surrounded by a protein called capsid.
• Capsid is composed of large number of
capsomer which is made up of polypeptide
molecules.
• The capsid with theenclosed
nucleic acid is known as nucleocapsid.

10. Functions of Capsid
• It protects  viral genome from physical destruction & enzymatic
inactivation by nucleases in biological material.
• It provides  binding site which enable the virus to attach to
specific site on the host cell.
• It facilitates  assembly and packaging of viral genetic
information.
• It serves as a vehicle of transmission from host to another.
• It is antigenic and specific for each viruses
• It provides the structural symmetry to the virus particle.

11. Viral Symmetry
1. Icosahedral Symmetry
• An icosahedral (icosa, meaning 20 in greek) is a polgon with 12
vertices or corners and 20 facets or sides.
• Each facet is in the shape of an equilateral triangle.
• Eg. Adeno viruses
Adenovirus

12. 2. Helical Symmetry
• The nucleic acid and capsomers are wound
together in the form of helix or spiral.
• Eg. Influenza virus, parainfluenz virus,
rabies virus.
3. Complex Symmetry
• Viruses which don not show either icoshedral or
helical symmetry
due to complexity of their
structure are referred to
have complex symmetry.
• Eg. Pox viruses

14. Viral Envelope
• Virions may be enveloped and nonenveloped (naked).
1. Enveloped Virus
• 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.
• Enveloped viruses are susceptible to the action of lipid solvent
such as ether, chloroform and detergent.
• Eg. Herpes virus, Hepatitis B virus, HIV virus

15. 2. Non enveloped virus
• Viruses which does not have outer
covering.
• Naked viruses are more likely to be
resistant to lipid solvents like ether,
chloroform and detergent.
Peplomers
• In mature virus particle, the
glycoproteins often appear as
projecting spikes on the outer
surface of the envelop which are known
as peplomers.
• A virus may have more than one type of
peplomers. E.g the influenza virus
carries two types of peplomers, the
hemagglutinin and neuraminidase

16. Functions of Peplomers
• It helps for attachment of virus to the host cell receptors to
initiate the entrance of the virion into the cell.
• It attach to receptors on red blood cells, causing these cell to
agglutinate.
• It has enzymatic activity like neuraminidase which cleave neuraminic
acid from host cell glycoproteins.
• It has antigenic properties.

18. Viral Nucleic Acids
• Viruses contain a single kind of nucleic acid either DNA or
RNA which encodes the genetic information necessary for
replication of the virus.
• The genome may be single stranded or double stranded,
circular or linear, segmented or non segmented.
• According to nucleic acid present, viruses can be classified in to
DNA viruses and RNA viruses.

20. Classification of viruses
• Type of nucleic acid
• Number of strands of nucleic acid
• Polarity of viral genome
• Symmetry of nucleic acid
• Presence / absence of a lipid envelope

21. Family DNA type Envel
ope
Symmetry Size
(nm)
Representative
Viruses
DNA viruses DNA
Herpesviridae ds,linear Yes Icosahedron 150-200 Herpes simplex virus -
1
Herpes simplex virus- 2
Varicella-zoster virus
Epstein-Barr virus
Cytomegalovirus
Human herpes virus 6,7
& 8
Hepadnaviridae ds,
circular,
incomplete
Yes Icosahedron 40–48 Hepatitis B virus
Parvoviridae ss, linear Absent Icosahedron 18–26 Parvovirus B19
Classification of viruses

22. Family DNA type Envel
ope
Symmetry Size
(nm)
Representative
Viruses
DNA
viruses
DNA
Papovavirida
e
ds, circular Absent Icosahedron 45-55 Human papillomaviruses
JC virus and BK virus
Poxviridae ds, linear Yes Complex 230 x
400
Variola (smallpox)
Molluscum contagiosum
virus
Adenoviridae ds, linear Absent Icosahedron 70–90 Human adenoviruses
Classification of viruses

23. Family DNA type Envel
ope
Symmetry Size
(nm)
Representative
Viruses
RNA
viruses
RNA
Picornavirida
e
ss, +ve
sense
Absent Icosahedral 28–30 Poliovirus
Coxsackievirus
Echovirus
Enterovirus
Rhinovirus
Hepatitis A virus
Caliciviridae ss, +ve
sense
Absent Icosahedral 27-40 Norwalk agent
Hepatitis E virus
Togaviridae ss, +ve
sense
Yes Icosahedral 50-70 Rubella virus
Eastern equine
encephalitis virus
Western equine
encephalitis virus
Classification of viruses

25. Family DNA
type
Envel
ope
Symmetry Size
(nm)
Representative
Viruses
RNA viruses RNA
Flaviviridae ss, +ve
sense
Yes Icosahedral
(?)
40-60 Yellow fever virus
Dengue virus
St. Louis
encephalitis virus
West Nile virus
Hepatitis C virus
Coronaviridae ss, +ve
sense
Yes Helical 120-
160
Coronaviruses
Rhabdoviridae ss, -ve
sense
Yes Helical 75x180 Rabies virus
Vesicular stomatitis
virus
Filoviridae ss, -ve
sense
Yes Helical 80 x
1000
Marburg virus
Ebola virus
Classification of viruses

26. Family DNA type Envel
ope
Symmetry Size
(nm)
Representative
Viruses
RNA viruses RNA
Paramyxoviridae ss, -ve
sense
Yes Helical 150–
300
Parainfluenza virus
Mumps virus
Measles virus
Respiratory syncytial
virus
Newcastle disease virus
Metapneumovirus
Orthomyxoviridae ss, -ve
sense, 8
segments
Yes Helical 80–120 Influenza viruses- A, B,
and C
Bunyaviridae ss, -ve
sense,
3 circular
segments
Yes Helical 80–120 Hantavirus
California encephalitis
virus
Sandfly fever virus
Classification of viruses

27. Family DNA type Envel
ope
Symmetry Size
(nm)
Representative
Viruses
RNA
viruses
RNA
Arenaviridae ss, -ve
sense,
RNA,2
circular
segments
Yes Helical (?) 50-300 Lymphocytic
choriomeningitis virus
Lassa fever virus
South American
hemorrhagic fever virus
Reoviridae ds, 10–12
segments
Absent Icosahedral 60-80 Rotavirus
Reovirus
Colorado tick fever virus
Retroviridae 2 identical
copies of
+ve sense
ss RNA
Yes Icosahedral
(spherical)
80-110 HTLV (Human T
Lymphotropic virus)
HIV (Human
immunodeficiency virus)
Classification of viruses

28. Pathogenesis of Viral Infections
• Progress in following steps inside the human body
– Transmission (entry into the body)
– Primary site replication
– Spread to secondary site
– Manifestations of the disease.
Mode of
transmission
Produce Local infection at the
portal of entry
Spread to distant sites from the
portal of entry
Respiratory route
(probably the
most common
route)
Produce Respiratory infection-
1. Influenza virus
2. Parainfluenzavirus
3. Respiratory syncytial virus
4. Rhinovirus
5. Adenovirus
6. Coronavirus such as SARS-COV2
7. Herpes simplex virus
Measles virus
Mumps virus
Rubella virus
Varicella-zoster virus
Cytomegalovirus
Parvovirus
Small pox virus
Oral route Produce gastroenteritis
1. Rotavirus
2. Adenovirus-40,41
3. Calicivirus
4. Astrovirus
Poliovirus
Coxsackie virus
Hepatitis Virus – A & E
Cytomegalovirus
Epstein-Barr virus (EBV)

29. Mode of
transmission
Produce Local infection
at the portal of entry
Spread to distant sites
from the portal of entry
Cutaneous route Produce skin lesions
Herpes simplex virus
Human papilloma virus
Molluscum contagiosum virus
1. Herpes simplex virus
Vector bite - Arboviruses such as-
1. Dengue virus (Aedes)
2. Chikungunya virus(Aedes)
3. Japanese encephalitis virus
(Culex)
4. Yellow fever and Zika
virus(Aedes)
5. Kyasanur Forest disease
virus (Tick)
Animal bite - 1. Rabies virus

30. Mode of transmission Produce Local infection at
the portal of entry
Spread to distant sites from
the portal of entry
Sexual route Produce genital lesions-
1. Herpes simplex virus
2. Human papilloma virus
Hepatitis B, C& rarely D
HIV
Blood transfusion - 1. Hepatitis B, C & rarely D
2. HIV
3. Parvovirus
Injection - Hepatitis B, C & rarely D
HIV

31. Mode of transmission Produce Local infection at
the portal of entry
Spread to distant sites from
the portal of entry
Transplacental
route
Produce congenital
manifestations in fetus
1. Rubella virus
2. Cytomegalovirus (CMV)
3. Herpes simplex virus
4. Varicella-zoster virus
5. Parvovirus
Transmitted through placenta to
fetus, without congenital
manifestations
1. Measles virus
2. Mumps virus
3. Hepatitis B virus
4. Hepatitis C virus
5. Hepatitis D virus
6. HIV
Conjunctival route 1. Adenovirus
2. Enterovirus70
3. Coxsackie virus A-24
4. Herpes simplex virus

33. Cell tropism of human viruses
Cell Type Associated DNA Viruses RNA Viruses
Lymphocytes Epstein-Barr virus
Cytomegalovirus
Hepatitis B
JC virus,
BK virus
Mumps,
Measles,
Rubella,
HIV
Monocytes-
macrophages
Cytomegalovirus Poliovirus
HIV
Measles
Neutrophils - Influenza virus
Red blood cells Parvovirus B19 Colorado tick fever virus
None (free in plasma) - Togavirus, Picornavirus
Viruses for specific organs  determines pattern of illness (e.g. hepatitis
viruses have tropism for hepatocytes  produces hepatitis as primary ds)

34. Virus Shedding
• Shedding of viruses  important to maintain viruses in the
hosts. It occurs at various levels :-
Stages Explanation Examples
Portal of entry For those viruses that produce
local infection.
Influenza virus is shed in
respiratory secretions
Blood Viruses that spread through
vector bite or blood transfusion or
needle pricks
Arboviruses, hepatitis B
Near the target tissue
/organ
Skin, salivary gland and kidney Varicella zoster, mumps,
cytomegalovirus
No shedding Humans are the dead end for
certain viruses infecting CNS,
such as
Rabies.

35. Manifestations of Viral infections
• Incubation Period
– Time interval between the entry  appearance of first clinical
manifestation.
– Duration depends upon
• Site of entry
• Multiplications
• Site of lesions
• Develop either an inapparent (subclinical) infection or apparent
(clinical) infection.
• They may be
– Acute
– Subacute
– Chronic

36. Viral Pathogenesis at Cellular Level
Produces 3 types of infections
• Failed infection  non-permissive (i.e. absence of surface receptors
or machineries to support viral replication).
• Cell death (Cytocidal or Lytic infection)
• Inhibition of host cell DNA (herpesvirus)
• Inhibition of host cell protein synthesis
(Poliovirus)
• Fusion (Syncytia Formation)
– Some enveloped viruses  expressed on host cell surface 
tiggers the fusion of neighboring cells to form multinucleated giant
cells  Called Syncytia  allows the virus to spread from cell to
cell and escape Antibody neutralization.

37. Viral Pathogenesis at Cellular Level
• Immune mediated lysis
– Expression of viral Ags on host cell surface  binding of Abs 
complement activation or NK cells  Lysis.

38. Infection without Cell Death
Steady state infection -
• The virus and host cell enter into a peaceful coexistence, both
replicating independently without any cellular injury.
Persistent viral infection
• Latent infection with periodic exacerbations (Herpesviridiae family).
• Cell Transformation (Oncogenic viruses –HBV, EBV, HPV)
• Latency seen in HIV infection.
• Latency seen in slow virus infection.
• Persistent tolerant infection (lymphocytic choriomeningitis virus
infecting mice)
an increase in symptom
intensity occurring after a
certain period of time since the
last exacerbation.

39. Morphological Changes in the Host Cells
• Certain viruses induce  characteristic changes in the host cells
(e.g. inclusion body),  which can be detected by histopathological
staining.
• Inclusion Bodies
– Are virus specific intracellular globular masses  produced during
replication of virus in host cells.
– Can be demonstrated in virus infected cells under light microscope after
fixation and staining.

40. Inclusion Bodies
Location
Nucleus
Cytoplasm Both
Acidophilic in nature  seen
as pink structure (stained with
Geimsa or eosin methylene
blue stain)
Basophilic in nature
Cowdry classified into:
Cowdry type B
inclusions
Cowdry type A
inclusions
Variable in size &
granular appearance
Amorphous or
hyaline spheres;
multiple in number

43. Laboratory Diagnosis of Viral Infections
• For the proper management of certain diseases
– Rubella  first trimester  abortion is recommended.
– Baby born  HbsAg positive mother immunization at
birth (mandatory)
• Diagnosis
– E.g. Herpes viruses antiviral chemotherapy is available.
• Screening of blood donors (for HIV & Hep-B & others)
• Early detection of epidemics
– E.g. influenza, encephalitis, poliomyelitis etc  control
measures to prevent spread of inf.
• PEP (Post-exp prophylaxis)
– HIV, Hep-B

44. Lab Diagnosis of Viral
Infection
Direct demonstration
of Virus or it’s
components
Isolation of Virus
Detection of specific
Antibodies

45. Direct Demonstration of Virus and It’s
components
• Electron Microscopy
• Immunoelectron Microscopy
– Sensitivity of electron microscopy can be increased by adding specific
antibody to the specimen to aggregates can be observed under
electron microscopy.
Specimen Viruses
Faeces Rotavirus, Hepatitis A virus, adenovirus, Norwalk
virus, astrovirus
Vesicular Fluid Herpes simplex, Varicella-zoster
CSF Enterovirus, Varicella-zoster
Urine Cytomegaloviurs (CMV)

46. • Fluorescent Microscopy
• Light Microscopy

47. • Viral Antigens
• Nucleic Acid Probes

48. • PCR (Polymerase Chain Reaction)

49. • Isolation of the Virus
– Animal inoculation
– Egg inoculation
– Cell cultures

50. Animal Inoculation
• Because of ethical issues  use of animals is restricted for
research purpose only.
Animal Use
Research Diagnostic
To Study
•Viral pathogenesis
•Viral oncogenesis
•Viral vaccine trials
Difficult to cultivate

51. Egg Inoculation

52. Tissue Culture
• Organ Culture
– Used for certain fastidious viruses  have affinity to specific organs;
– Tracheal ring culture  corona viruses
• Explant Culture
– Fragments of minced tissue  grown as “explants”
– e.g. adenoid explants
– Not used nowadays
• Cell Culture
– Routinely employed for diagnostic virology.

53. Cell Culture
• Preparation Tissues
Dissociated into components of cells by mechanical shaking
Proteolytic enzyme Trypsin
Dissociated cells are washed, counted and suspended in a viral
growth medium
The cell suspension is distributed in glass
or plastic bottles, tubes or petri dishes.
On incubation, the cells adhere to glass or plastic surface (wall of
test tube) and divide to form a confluent monolayer sheet of cells
within a period of one week.
•Balanced Salt solutions
•Essential amino acids
•Vitamins
•Fetal calf serum
•Antibiotics

54. Tissue Culture
• Types of cell lines
– Classified into 3 types based on their
• Origin
• Chromosomal characters and
• Maximum number of cell division that they can undergo.

55. Primary Cell Culture
• Derived from normal cells.
• Freshly taken from the organs and cultured.
• Capable of very limited growth in culture, maximum up to 5-10
divisions.
• maintain a diploid karyosome.
• Useful for both primary isolation as well as growth of the viruses for
vaccine production.
• Examples
– Monkey kidney cell line- useful for isolation of myxoviruses, enteroviruses&
adenoviruses
– Human amnion cell line
– Chick embryo cell line

56. Diploid Cell Strains
• Can divide maximum up to 10-50 divisions before they undergo
senescence (death).
• Also derived from the normal host cells.
• Maintain the diploid karyosome.
• Examples
– Human fibroblast cell line- excellent for the recovery of cytomegalovirus.
– MRC-5 &WI-38 (human embryonic lung cell strain)
• Karyosome:- dense bundle of chromatin inside the nucleus of a cell within an
organism.
• Haploid refers to the presence of a single set of chromosomes in an
organism's cells. Sexually reproducing organisms are diploid (having two sets
of chromosomes, one from each parent). In humans, only the egg and sperm

57. Continuous Cell line
• Derived from cancerous cell lines, hence are immortal.
• They also possess altered haploid chromosome.
• Easy to maintain  by serial subculturing  for indefinite divisions  so
most widely used cell lines.
• Examples:-
– HeLa cell line (Human carcinoma of cervix cell line)
– Hep-2 cell line (Human epithelioma of larynx cell line)- widely used for respiratory
syncytial viruses, adenoviruses and HSV
– KB cell line (Human carcinoma of nasopharynx cell line)
– McCoy cell line (Human synovial carcinoma cell line)- useful for isolation of
viruses as well as Chlamydia
– Vero cell line (Vervet monkey kidney cell line)-used for rabies vaccine production.

58. Types of cell lines
Human lung fibroblast cell line (Normal)
HeLa cell line
(normal,
uninfected)
Vero cell line (normal,
uninfected)
HEp-2 cell line (normal,
uninfected)

59. Detection of virus growth in cell cultures
• Can be detected by following methods
– Cytopathic effect
– Haemadsorption
– Interference
– Transformation
– Direct immunofluorescence
– Electron microscopy
– Detection of enzymes (Reverse transcriptase enzymes)

60. Cytopathic Effect
• The morphological changes produced by the viruses in the cell line in
which they grow are called as Cytopathic effects and viruses are
called Cytopathogenic viruses.
Type of Cytopathic effect (CPE) Virus
Rapid crenation (Leaf like) and
degeneration of the entire cell sheet
Enteroviruses e.g. Polio
virus
Syncytium or multinucleated giant cell
formation
Measles,
RSV, HSV
Diffuse roundening and ballooning of
the cell line
HSV
Cytoplasmic vacuolations SV 40 (Simian
vacuolating virus-40)
Large granular clumps resembling
bunches of grapes
Adenovirus

61. • Detection of Specific Antibodies
– Neutralization test

62. Haemagglutination test

63. – Complement fixation test
Positive Test
All the available complement is fixed by the
Ag-Ab rxn: no hemolysis occurs, so the test
is positive for the presence of Abs.
Negative Test
No Ag-Ab rxn occurs. The complement
remains and the RBCs are lysed in the
indicator stage. So the test is negative.

64. – ELISA
– ICT (Immunochromatographic tests)