2. 20-06-2015 Dept of Oral Pathology, TNGDC, Chennai
Introduction
Types of Viral Lab
Diagnosis

3. What is a virus?
 It is a segment of either RNA or DNA protected
by a protein coat and in some families of viruses a
host derived envelope with attached viral proteins
 It lacks:
- Protein synthesizing machinery
- Energy producing system
- No mitochondria
- No stores of amino acids, nucleotides
energy rich molecules
 It is a compulsory intra cellular parasite

4. Diagnostic Methods in Virology
1. Direct Examination
2. Indirect Examination (Virus Isolation)
3. Serology

5. Direct Examination
1. Antigen Detection immunofluorescence, ELISA etc.
2. Electron Microscopy morphology of virus particles
immune electron microscopy
3. Light Microscopy histological appearance
inclusion bodies
4. Viral Genome Detection hybridization with specific
nucleic acid probes
polymerase chain reaction (PCR)

6. Indirect Examination
1. Cell Culture cytopathic effect (CPE)
haemadsorption
immunofluorescence
2. Eggs pocks on CAM
haemagglutination
inclusion bodies
3. Animals disease or death

7. Serology
Detection of rising titres of antibody between acute and
convalescent stages of infection, or the detection of IgM in
primary infection.
Classical Techniques Newer Techniques
1. Complement fixation tests (CFT) 1. Radioimmunoassay (RIA)
2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA)
3. Immunofluorescence techniques (IF) 3. Particle agglutination
4. Neutralization tests 4. Western Blot (WB)
5. Counter-immunoelectrophoresis 5. RIBA, Line immunoassay

8. Specimens for Routine Tests
Clinical Category Blood Throat
swab
Faeces CSF Other
1. Meningitis + + + +
2. Encephalitis + + + + Brain biopsy
3. Paralytic disease + + + +
4. Respiratory illness + + Nasopharyngeal aspirate
5. Hepatitis +
6. Gastroenteritis +
7. Congenital diseases + Urine, saliva
8. Skin lesions + + Lesion sample e.g. vesicle
fluid, skin scrapping
9. Eye lesions Eye swab
10.Myocarditis + Pericardial fluid
11.Myositis + +
12.Glandular fever +
13.Post Mortem + Autopsy
After use, swabs should be broken into a small bottle containing 2 ml of virus transport medium.
Swabs should be sent to the laboratory as soon as possible without freezing. Faeces, CSF, biopsy or
autopsy specimens should be put into a dry sterile container.

9. Electron Microscopy
106 virus particles per ml required for visualization,  50,000 - 60,000
magnification normally used. Viruses may be detected in the following
specimens.
Faeces Rotavirus, Adenovirus
Norwalk like viruses
Astrovirus, Calicivirus
Vesicle Fluid HSV
VZV
Skin scrapings papillomavirus

10. Electronmicrographs
Adenovirus Rotavirus
(courtesy of Linda Stannard, University of Cape Town, S.A.)

11. Inclusion bodies
Negri
bodies
Negri
bodies
neuron
Immunohistochemical staining of intra-cytoplasmic viral inclusions in the neuron of a human rabies patient. (Fields Vriology (2007) 5th edition,
Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 39.9)

12. Immune Electron Microscopy
The sensitivity and specificity of EM may be enhanced by
immune electron microscopy. There are two variants:-
Classical Immune electron microscopy (IEM) - the
sample is treated with specific anti-sera before being put up for
EM. Viral particles present will be agglutinated and thus
congregate together by the antibody.
Solid phase immune electron microscopy (SPIEM) -
the grid is coated with specific anti-sera. Virus particles present
in the sample will be absorbed onto the grid by the antibody.

13. Immunofluorescence
• Immunofluorescence is the labeling of antibodies or antigens
with fluorescent dyes.
• Immunofluorescent labeled tissue sections are studied using a
fluorescence microscope.
• Fluorescein is a dye which emits greenish fluorescence under
UV light. It can be tagged to immunoglobulin molecules.

14. Direct IF Indirect IF

16. ELISA

17. Embryonated Egg
Cell Lines/
Tissue cultures
Animal inoculation
Chorioallantioc membrane (CAM)
Allantoic cavity
Amniotic cavity
Yolk Sac
Primary
Diploid/ Secondary
Continuous
Suckling mice
Virus Culture

18. Embryonated Hen’s Egg
• Chorioallantoic membrane (CAM) – visible lesions called
pocks. Each infectious virus particle forms one pock. e.g.
Variola, Vaccinia virus
• Allantoic cavity – Influenza virus (vaccine production) &
paramyxoviruses
• Amniotic cavity – primary isolation of Influenza virus
• Yolk sac – Chlamyadia, Rickettsiae & some viruses

19. Growth of virus on embryonated eggs
Davis, Duylbecco, Eisen, Ginsberg “Microbiology” 4th ed, J.B. Lippincott 1990, Fig. 48-1

20. Embryonated Hen’s Egg

21. Types of Tissue Culture
Organ culture
Explant culture
Cell culture

22. Cell Culture
• Tissues Individual cells
trypsin & mechanical shaking
• Cells are washed, counted & suspended in a
growth medium.
• Growth medium – Minimum Essential Medium
(MEM): essential aminoacids, vitamins, salts,
glucose & bicarbonate in 5% CO2 with 5% fetal calf
or calf serum, antibiotics & phenol red indicator

23. Cell Culture
• Routinely used for growing viruses
• Classified into 3 types:
– Primary cell culture – normal cells freshly taken
from body & cultured, limited growth
1. Rhesus monkey kidney
2. Chick embryo fibroblast
3. Human amnion cell culture
– Diploid cell strains – cells of single type (fibroblast cells)
that can be subcultivated for limited number of times,
mostly 50
1. WI-38: human embryonic lung cell
2. HL-8: Rhesus embryo cell

24. – Continuous cell lines – malignant cells, indefinite
subcultivation
1. HeLa: Human Ca of cervix cell line
2. HEP-2: Human epithelioma of larynx
3. Vero: Vervet monkey kidney
4. McCoy, Detroit-6, BHK-21, KB

25. Cell Culture Bottles / Tubes

26. Detection of virus growth in cell cultures
1. Cytopathic effects (CPE) – morphological changes
in cultured cells, seen under microscope,
characteristic CPE for different groups of viruses
2. Metabolic Inhibition – no acid production in
presence of virus
3. Hemadsorption – influenza & parainfluenza
viruses, by adding guinea pig erythrocytes to the
culture, erythocytes are absorbed on surface of
cells

27. Cytopathic Effect
Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells.
(Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town)

28. Hemadsorbtion
add red blood cells

29. Haemadsorption
Syncytial formation caused by mumps virus and
haemadsorption of erythrocytes onto the surface of the cell
sheet.
(courtesy of Linda Stannard, University of Cape Town, S.A.)

30. 4. Interference – growth of a non cytopathogenic
virus can be tested by inoculating a known
cytopathogenic virus: growth of first virus will
inhibit the infection by second
5. Transformation – oncogenic viruses induce
transformation & loss of contact inhibition –
growth appear in a pile-up fashion producing
microtumors
6. Immunofluorescence – test for viral Ag in cells
from viral infected cultures.

31. Transformation
loss of contact inhibition

32. Problems with cell culture
• Long period (up to 4 weeks) required for result.
• Often very poor sensitivity, sensitivity depends on a
large extent on the condition of the specimen.
• Susceptible to bacterial contamination.
• Susceptible to toxic substances which may be present in
the specimen.
• Many viruses will not grow in cell culture e.g. Hepatitis
B, Diarrhoeal viruses, parvovirus, papillomavirus.

33. Rapid Culture Techniques
Rapid culture techniques are available whereby viral antigens
are detected 2 to 4 days after inoculation. The CMV DEAFF
test is the best example, whereby
• The cell sheet is grown on individual cover slips in a plastic
bottle.
• Following inoculation, the bottle then is spun at a low speed
for one hour (to speed up the adsorption of the virus) and
then incubated for 2 to 4 days.
• The cover slip is then taken out and examined for the
presence of CMV early antigens by immunofluorescence.

34. DEAFF test for CMV
(Virology Laboratory, Yale-New Haven Hospital)

35. Animal inoculation
• Useful for study of pathogenesis, immune response,
epidemilogy and oncogenesis
• Earlier human volunteers and monkey were used
• Suckling mice are used (arbo, coxsackie)
• Mice is inoculated (intracerebral, subcutaneous,
intraperitoneal, intranasal)
• Growth of virus is indicated by death, disease or
viable lesion

37. Serology
Criteria for diagnosing Primary Infection
• 4 fold or more increase in titre of IgG or total antibody
between acute and convalescent sera
• Presence of IgM
• Seroconversion
• A single high titre of IgG (or total antibody) - very
unreliable
Criteria for diagnosing Reinfection
• fold or more increase in titre of IgG or total antibody
between acute and convalescent sera
• Absence or slight increase in IgM

38. – Primary and secondary responses to viral infections
• IgM (1st exposure)
• IgG (2nd exposure)
Figure 5.18: Primary (1 degree) and secondary (2 degree) antibody responses toward a viral pathogen.

39. Serological Test
Solid-Phase Immunoassays
Enzyme immunoassay Passive latex agglutination
Immunofluorescence immunoassay Passive
hemagglutination
Chemiluminescence immunoassay
Immunoflourescence
Indirect immunofluorescence Neutralization
Anticomplement immunofluorescence

40. Agglutination Assays
Passive latex agglutination
Passive hemagglutination
Hemagglutination inhibition
Complement fixation
Neutralization
Immunoblotting

41. Viral Hemagglutination
• Hemagglutination
– Originally seen with the Influenza virus by Hirst in 1941.
– A convenient method of detection & assay of Influenza
virus.
– Due to the presence of Hemagglutinin spikes on the
surface.
• Reversal of hemagglutination – Elution
– Due to the presence of Neuraminidase enzyme, Receptor
Destroying Enzyme (RDE)
– Destruction of receptor – reversal of hemagglutination –
release of virus from the red cell surface
– Found only in Myxoviuses.

42. Hemagglutination (HA) Test
+
RBC Suspension HA Virus Settling Pattern

44. Hemagglutination-Inhibition (HI) Test
+
RBC Suspension HA Virus Settling Pattern
+
Antibody

45. ELISA
 (ELISA) can detect unknown Ag or Ab by
direct or indirect means.
 A positive result is visualized when a
colored product is released by an enzyme-
substrate reaction.
• Uses an enzyme as the label
– Reaction of the enzyme with
its substrate produces a
colored product indicative of
a positive test
• Most common form of ELISA is
used to detect the presence of
antibodies in serum

46. ELISA
Procedures
Figure 5-19a
Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third
Edition . ASM Press, 2000.
Figure 5-19b

47. © Hank Morgan/Science Photo Library/Photo Researchers, Inc.
HIV ELISA test.

48. Western Blot
HIV-1 Western Blot
• Lane1: Positive Control
• Lane 2: Negative Control
• Sample A: Negative
• Sample B: Indeterminate
• Sample C: Positive
Dr.T.V.Rao MD 48

49. Figure 5.21b: The structure of HIV-1.
Image courtesy of Bio-Rad Laboratories
Figure 5.21c: The typical results of a Western
blot testing patient serum for HIV-1 antibodies.
(c)(b)

50. Figure 5.21a: The basic principles behind the Western blotting procedure.
Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third Edition. ASM Press, 2000.

51. Neutralization Test
1 2 3
B
Following virus isolation:
1.Divide culture yield into small volume in a set of test tubes
2. Prepare the panel of antisera against which the virus isolate is to be
challenged
3. To each test tube add one antisera and leave one as a virus control and one
as serum control

52. • Incubate for one hour then inoculate each into
cell culture tubes, incubate and observe daily.

53. 2 31
Principle of Neutralization test

54. Complement fixation: Principle
54
 Complement fixation tests detect
lysins- Ab that fix complement and
can lyse target cells.
 Involves mixing test Ag and Ab
with complement and then with
sensitized sheep RBCs.
 If complement is fixed by the Ag-
Ab, the RBCs remain intact and the
test is positive.
 If RBCs are hemolyzed, specific Ab
are lacking and the test is negative.

55. Results and Interpretations:
• No haemolysis is considered as a positive test.
• haemolysis of erythrocytes indicative of a negative test.
1 2 3 4
A
B
• Microtiter plate showing Haemolysis (Well A2, A3, A4 and
B4) and No Haemolysis (Well A1, B1, B2 and B3)

57. Polymerase Chain Reaction
• PCR allows the in vitro amplification of specific target
DNA sequences by a factor of 106 and is thus an extremely
sensitive technique.
• It is based on an enzymatic reaction involving the use of
synthetic oligonucleotides flanking the target nucleic
sequence of interest.
• These oligonucleotides act as primers for the thermostable
Taq polymerase. Repeated cycles (usually 25 to 40) of
denaturation of the template DNA (at 94oC), annealing
of primers to their complementary sequences (50oC), and
primer extension (72oC) result in the exponential
production of the specific target fragment.

58. • Further sensitivity and specificity may be obtained by the
nested PCR
• Detection and identification of the PCR product is usually
carried out by
agarose gel electrophoresis,
hybridization with a specific oligonucleotide probe,
restriction enzyme analysis, or DNA sequencing.

59. • Advantages of PCR:
– Extremely high sensitivity, may detect down to one viral
genome per sample volume
– Easy to set up
– Fast turnaround time
• Disadvantages of PCR
– Extremely liable to contamination
– High degree of operator skill required
– Not easy to set up a quantitative assay.

60. Schematic of PCR
Each cycle doubles the copy number of the target

61. Other Newer Molecular Techniques
• Commercial proprietary techniques such as LCR,
NASBA, TMA depend on exponential amplification
of the signal or the target.
• PCR and related techniques are bound to play an
increasingly important role in the diagnosis of viral
infections.
• DNA chip is another promising technology where it
would be possible to detect a large number of
viruses, their pathogenic potential, and their drug
sensitivity at the same time.

62. References
• BAILEY & SCOTT`S DIAGNOSTIC MICROBILOGY-
12th Edition
• TOPLEY & WILSION`S Textbook of
Microbiology
• Ananthanarayan and Paniker`s Textbook of
Microbiology- 8th Edition
• Koneman`s Color Atlas and Textbook of
Diagnostic Microbiology- 6th Edition