Viruses are the smallest infectious agents that can only be seen using an electron microscope. They are obligatory intracellular parasites that contain either DNA or RNA, not both. Viruses are diagnosed through direct detection of the virus or its components using techniques like electron microscopy, immunoassays, and PCR. Indirect methods include serological diagnosis by detecting antibodies and skin tests. Viruses are cultivated inside living cells in tissue culture or laboratory animals since they cannot grow on artificial media.

1. GENERAL VIROLOGY

2. Viruses
The smallest infectious agents (20-300 nm)
only seen under
Electron Microscope
(except Poxviruses)

3. Electron Microscope
- The source of illumination is a beam of electrons
- Electromagnetic lenses
- Magnification is 100.000 or more

5. Electron Microscopy
Bacterial fimbriae

6. Structure of viruses
(Virus
particle (virion
Structure of Viruses

7. 7

8. 8

9. Viral Symmetry
Ico
1. Icosahedral symmetry:
e.g., all DNA viruses, except
poxviruses & some RNA viruses
2. Helical symmetry:
e.g., many RNA viruses (e.g. rabies virus)
3. Complex symmetry:
e.g., poxviruses (brick-shaped)

10. Icosahedral Symmetry

11. Icosahedral Symmetry
Rotavirus
Calicivirus
Astrovirus

12. Helical Symmetry

13. Complex symmetry

14. Differences between viruses & bacteria
1. They are obligatory intracellular parasites
They can not be cultivated on artificial culture
media.
 can only replicate inside living cells.


15. 2. Viruses contain only one type of nucleic acid
(DNA or RNA), never both.

17. 3. They can not be cultivated on artificial
culture media.

18. 4. They are not susceptible to antibacterial
agents.

19. Diagnosis of viral infections
Diagnosis of viral infections
A- Direct methods
A- Direct methods
I. Direct detection
I. Direct detection
of viruses and //or
of viruses and or
their components
their components
II. Isolation of
II. Isolation of
viruses
viruses
B- Indirect methods
B- Indirect methods
I. Serology
I. Serology
II. Skin tests
II. Skin tests

20. A- Direct methods
A- Direct methods
I. Direct detection of viruses & / or their
components:
1. Light microscopy:
Examination of large viruses
as Poxviruses
Detection of giant cells
in Herpes infection
Detection of inclusion bodies
e.g. Negri bodies in nerve
cells in rabies

21. 2. Electron microscopy (EM):
Large number of viruses in the sample.
Size and shape of viruses.

22. 3. Immunoelectron microscopy (IEM):
Sample (unknown virus) + known specific antibody
aggregation of unknown virus particles
e.g. hepatitis A virus in stools

23. 4. Fluorescent microscopy:
Direct immunofluorescent antibody technique (IF)
e.g. diagnosis of rabies in brain smears.

24. 5. Immunoassays :
For detection of the virus antigens by ELISA / RIA
e.g. hepatitis B antigens in blood

25. ELISA

26. 6. Nucleic acid hybridization:
 A highly sensitive and specific method.
 Viral nucleic acid in sample + Specific labeled probe
hybridization
fluorescence

27. 7. Polymerase chain reaction (PCR)
Amplification of a specific sequence of nucleic acid
Detection e.g. by gel electrophoresis.

28. A- Direct methods
A- Direct methods
II. Isolation of viruses:
• Obligatory intracellular parasites
• Replicate only in living susceptible
cells
a. Laboratory animals
b. Embryonated eggs
c. Cell culture (tissue culture)

29. Cultivation of viruses
a. Laboratory animals:
Used in:
• Isolation of coxsackie viruses by inoculation in
white suckling mice.
• Rabies virus inoculation in mice.
• For research.

30. Cultivation of viruses
b. Embryonated eggs

31. Cultivation of viruses
c. Cell culture (tissue culture)
The most widely used method

Tissues + trypsin → separate cells

Cells + growth media in
flat-sided bottles
monolayer culture

32. Virus inoculation → incubation at 37ºC (CO2 incubators)

33. Types of cell lines
Primary cell
lines
Diploid cell
lines
Continuous cell
lines
Prepared
from
Organ
fragments
Human fibroblasts
derived from
embryonic tissues
Tumor cells
Examples
Monkey kidney
Number of
Passages
(subcultures)
5-10
Human embryo lung
HeLa cells from
tissue
carcinoma of cervix
50-100
Unlimited

34. Detection of Virus Replication in Tissue Culture:
1- Cytopathic Effect (CPE):
i- Cell death or lysis

35. ii- Syncytial formation (multinucleated giant cells ):

36. 2- Transformation:
Viral nucleic acid + cellular DNA → cell transformation →
foci of malignant cells.

37. 3- Inclusion bodies
Site of virus assembly or degenerative changes
Their location and appearance are diagnostic for a
particular virus.
a- Intracytoplasmic: e.g.
Rabies (Negri bodies)
. b- Intranuclear: e.g
Herpes viruses
c- Both: e.g. Measles virus and CMV

38. 4- Plaque formation:
Infected monolayer + vital dye → unstained areas (plaques)

39. 5- Haemadsorption:
Monolayer + hemagglutinating virus + RBCs →
hemadsorption (RBCs clumping) to infected cells

40. 6. Interference phenomenon:
 Monolayer + rubella virus → no change for
weeks
 Add CPE-producing virus → NO CPE
(due to interference)
7- Detection of viral antigens

41. 8- Direct fluorescent antibody staining of
infected cells (DFA):
:Neutralization test- 9
Monolayer + unknown virus + known specific Ab
NO CPE (due to neutralization)

42. Diagnosis of viral infections
Diagnosis of viral infections
A- Direct methods
A- Direct methods
I. Direct detection
I. Direct detection
of viruses and //or
of viruses and or
their components
their components
II. Isolation of
II. Isolation of
viruses
viruses
B- Indirect methods
B- Indirect methods
I. Serology
I. Serology
II. Skin tests
II. Skin tests

43. B- Indirect methods
B- Indirect methods
I. Serological diagnosis:
 Detection of antiviral antibodies
 2 serum samples
acute phase & 2-3 weeks later,
to demonstrate a rising titer
(4 fold increase or more is diagnostic).
 Only one sample may be used in the acute
stage to detect IgM

44. I. Serological diagnosis:
 Serological methods include:






Neutralization test
Complement fixation test
Haemagglutination inhibition test
Indirect IF
ELISA
RIA

45. II. Skin tests
Used as an indication of cell-mediated immunity (CMI)
in some viral infections, e.g. mumps.

46. MCQs

47. 1) Viruses differ from bacteria in all of the
following EXCEPT:
a) Viruses are very small in size.
b) Viruses are obligatory intracellular parasites.
c) Viruses contain both DNA and RNA.
d) Viruses cannot be cultivated on artificial
culture media.
e) Viruses are not susceptible to antibiotics.

48. 2) Sites of viral assembly in tissue culture are
called:
a)Plaques
b)Inclusion bodies
c) Viral capsid
d)CPE
e)Areas of transformation

49. 3) Regarding continuous cell lines, all of the
following are true EXCEPT:
a)They allow unlimited number of passages.
b)They are prepared from tumor cells.
c) After viral inoculation, they should be
sterilized by autoclaving.
d)They are used for isolation of viruses.
e)HeLa cell line is an example of continuous cell
lines.

50. 4) PCR may be used to diagnose viral infections
by detecting:
a)Antiviral IgM antibodies
b)Viral antigens
c) Rising titer of antiviral antibodies
d)Viral nucleic acid
e)CPE produced by the virus in tissue culture

51. 5) Direct detection of viruses and/or their
components can be done by all of the
following EXCEPT:
a)Fluorescent microscope
b)Electron microscope
c) Light microscope
d)PCR
e)Biochemical reactions

52. 6) Cultivation of viruses can be done on:
a)Blood agar
b)Tissue culture
c) MacConkey’s medium
d)Nutrient broth
e)Anaerobic media

53. THANK
YOU

54. 1) Viruses differ from bacteria in all the
following EXCEPT:
a) Viruses are very small in size.
b) Viruses are obligatory intracellular parasites.
c) Viruses contain both DNA and RNA.
d) Viruses cannot be cultivated on artificial
culture media.
e) Viruses are not susceptible to antibiotics.

55. 2) Sites of viral assembly in tissue culture are
called:
a)Plaques
b)Inclusion bodies
c) Viral capsid
d)CPE
e)Areas of transformation

56. 3) Regarding continuous cell lines, all of the
following are true EXCEPT:
a)They allow unlimited number of passages.
b)They are prepared from tumor cells.
c) After viral inoculation, they should be
sterilized by autoclaving.
d)They are used for isolation of viruses.
e)HeLa cell line is an example of continuous cell
lines.

57. 4) PCR may be used to diagnose viral infections
by detecting:
a)Antiviral IgM antibodies
b)Viral antigens
c) Rising titer of antiviral antibodies
d)Viral nucleic acid
e)CPE produced by the virus in tissue culture

58. 5) Direct detection of viruses and/or their
components can be done by all of the
following EXCEPT:
a)Fluorescent microscope
b)Electron microscope
c) Light microscope
d)PCR
e)Biochemical reactions

59. 6) Cultivation of viruses can be done on:
a)Blood agar
b)Tissue culture
c) MacConkey’s medium
d)Nutrient broth
e)Anaerobic media